National Institute of Immunology

New Delhi

Scientific Reports

for the Annual Meeting of

Research Area Panel &

Scientific Advisory

Committee

April 7 & 8, 2015

(for limited circulation only)

Contents Page

Program 4

Publications and Patents 7

Research Reports

1. Plasmodium proteins involved in virulence and host

modulation: Host-parasite interactions in Plasmodium

liver stages

Agam P Singh 219

2. Genetic and functional analyses of host and HIV-1

genes that affect progression of HIV-1 and development

of nucleic acid based antiviral approaches

Akhil C Banerjea 188

3. Understanding the regulation of intracellular transport:

Role of GTPases Amitabha

Mukhopadhyay

21

4. Studies on immune response from antigen loaded

biodegradable polymer particles and protein refolding

from inclusion bodies

Amulya K Panda 57

5. Study of mucosal immune response Anna George 16

6. Analysis of Salmonella typhi-host cell interaction Ayub Qadri 107

7. Molecular basis of B cell responses Devinder Sehgal 112

8. Understanding the role of IRFs in dendritic cell

development and innate immunity

Prafullakumar

Tailor

210

9. Disorders of proliferation: Analysis of novel pathways

and targets

Rahul Pal 129

10. Study of genetic and immune factors associated with

autoimmune disorders: Type 1 diabetes and vitiligo

Rajni Rani 134

11. Study of immunotherapeutic potential of MIP and the

underlying mechanisms in animal models of

tuberculosis and tumor

Sangeeta Bhaskar 157

12. Analysis of antigen processing and presentation Satyajit Rath 63

13. Fine tunings of NF-κΒ signaling Soumen Basak 31

14. Biology of T lymphocytes Vineeta Bal 99

15. Cellular and molecular biology of human cancer Anil Suri 91

16. Study on expansion and plasticity of bone marrow stem

cells

Asok

Mukhopadhyay

26

17. Cell death regulation Chandrima Shaha 72

18. Cellular and molecular aspects of reproduction and viral

infections

Satish K Gupta 84

19. Studies of sertoli cells and spermatogonial stem cells of

the testis and other endocrinology related research

Subeer S

Majumdar

162

20. Molecular mechanism of enzymatic reactions and

enzyme-ligand interactions

Apurba K Sau 197

21. Structural and functional biology of mycobacterial

proteins

Bichitra K Biswal 214

22. Molecular modelling of proteins and protein-ligand

complexes using knowledge-based approaches and all

atom simulations

Debasisa Mohanty 44

23. Structure, interaction and design studies involving

regulatory peptides and proteins

Dinakar M Salunke 50

24. Ribonucleases and heat shock proteins: involvement in

host defense

Janendra K Batra 38

25. Role of carbohydrates in modulating the structure and

function of glycopeptides

Kanwal J Kaur 152

26. Structural studies on proteins, dynamics and ligand

interactions using NMR

Monica Sundd 183

27. To develop strategies for making sensors and actuators

for biological processes

Pramod K

Upadhyay

117

28. Protease-catalyzed splicing of peptide bond Rajendra P Roy 68

29. Therapeutic interventions in chronic diseases Sarika Gupta 192

30. Chemical Glycobiology : Glycoform modulation,

carbohydrate-based drug design, and glycomics S Gopalan

Sampathkumar

229

31. Biophysical and biochemical characterization of

Leishmania phosphoglycerate kinase: An enzyme in the

glycolytic pathway of parasitic protozoa

Vidya Raghunathan 226

32. Elucidating the molecular mechanisms of aging and

innate immunity using Caenorhabditis elegans as a

model system

Arnab

Mukhopadhyay

178

33. Molecular biology of infectious diseases Lalit C Garg 206

34. Epigenetic regulation of the eukaryotic genome: Role of

transcriptional insulators in organizing chromatin

Madhulika

Srivastava

104

35. Role of cell signaling in eukaryotic development Pushkar Sharma 124

36. Reconstructing the chemico-cellular trestle to decipher

biology of tuberculosis and vitiligo

Rajesh S Gokhale 54

37. Determining the signaling and repair pathways that are

altered in human cancer

Sagar Sengupta 147

38. Understanding the regulation of DNA replication Sandeep Saxena 140

39. The role of tumor suppressors in stress response Sanjeev Das 172

40. Characterization of Minisatellite tagged mRNA

transcripts from the human sperm genome

Sher Ali 77

41. Deciphering the role of cell signaling in M. tuberculosis

biology

Vinay K

Nandicoori

201

42. Production of transgenic and other animal models for

biomedical research (Ancillary activity)

Subeer S Majumdar 167

4

Program (April 7-8, 2015) April 7 (Seminar I)

09:15 Opening remarks Prof M Vijayan

09:30 Director‟s address Dr Chandrima Shaha

09:50 Tea (Central foyer)

10:15 Presentation by Principal Investigators

The presentations are divided in two groups and will run as parallel sessions in Seminar

I and Board Room respectively.

Group I (Seminar Room I)

Group II (Board Room)

Prof M Vijayan, Prof G Padmanaban, Prof

Anand Bachhawat, Prof V Nagaraja, Prof

Umesh Varshney, Dr R Sankaranarayanan,

Dr R Nagaraj, Dr Uttam Surana, Prof DN

Rao

Prof Ashok Venkitaraman, Dr Ranjan Sen,

Prof Subrata Sinha, Prof Saumitra Das, Dr

Apurva Sarin, Prof Gagandeep Kang,

Prof K Natarajan, Dr Shubhada

Chiplunkar, Prof Ramesh NK Bamezai

10:15

Arnab Mukhopadhyay 10:15

Amitabha Mukhopadhyay

11:00

Debasisa Mohanty 11:00

Vineeta Bal

11:45 Bichitra K Biswal

11:45 Sagar Sengupta

12:30 Janendra K Batra

12:30 Sanjeev Das

1:15 - 2:15 PM Lunch

02:15 Vinay K Nandicoori

02:15 Ayub Qadri

03:00

Agam P Singh 03:00

Akhil C Banerjea

03:45

Lalit C Garg 03:45

Asok Mukhopadhyay

04:30

Pramod K Upadhyay 04:30

Soumen Basak

05:15 Amulya K Panda 05:15 Sher Ali

Dinner, 6:30 PM onwards (Director's Residence Lawn)

April 8 (New Guest House)

10:00 Meeting with RAP/SAC Members, Director and Coordinators

12:00 Lunch and conclusion of the meeting

5

Review of reports by designated RAP/SAC members

Reviewer Principal Investigators Page

Prof M Vijayan S Gopalan Sampathkumar

Dinakar M Salunke

Vidya Raghunathan

229

50

226

Prof G Padmanaban

Chandrima Shaha

Apurba K Sau

Pushkar Sharma

Sandeep Saxena

72

197

124

140

Prof Ashok Venkitaraman

Prafullakumar B Tailor

Rahul Pal

Sarika Gupta

Subeer Majumdar

Anil Suri

210

129

192

162, 167

91

Dr Ranjan Sen

Anna George

Devinder Sehgal

Madhulika Srivastava

Sandeep Saxena

Satyajit Rath

16

112

104

140

63

Dr Uttam Surana

Kanwaljit Kaur

Sarika Gupta

Subeer Majumdar

Anil Suri

Rajesh S Gokhale

152

192

162, 167

91

54

Prof Subrata Sinha

Pushkar Sharma

Rahul Pal

Satish K Gupta

Anil Suri

124

129

84

91

Dr Anand Bachhawat Monica Sundd

RP Roy

Rajesh S Gokhale

183

68

54

Prof V Nagaraja

Chandrima Shaha

Kanwal J Kaur

Pushkar Sharma

Sangeeta Bhaskar

72

152

124

157

6

Reviewer Principal Investigators Page

Prof Umesh Varshney Chandrima Shaha

Madhulika Srivastava

Sangeeta Bhaskar

72

104

157

Prof Saumitra Das Sarika Gupta

Satish K Gupta

Satyajit Rath

192

84

63

Prof DN Rao Apurba K Sau

RP Roy

S Gopalan Sampathkumar

197

68

229

Dr Apurva Sarin

Anna George

Devinder Sehgal

Prafullakumar Tailor

Satyajit Rath

16

112

210

63

Dr Shubhada Chiplunkar Rahul Pal

Rajni Rani

Subeer Majumdar

129

134

162, 167

Dr R Nagaraj

RP Roy

Kanwal J Kaur

Monica Sundd

Vidya Raghunathan

68

152

183

226

Dr Rajan

Sankaranarayanan

Apurba K Sau

S Gopalan Sampatkumar

Monica Sundd

Vidya Raghunathan

Dinakar M Salunke

197

229

183

226

50

Prof RNK Bamezai Madhulika Srivastava

Rajni Rani

Sandeep Saxena

104

134

140

Prof K Natarajan

Anna George

Devinder Sehgal

Prafullakumar Tailor

Dinakar M Salunke

16

112

210

50

Prof Gagandeep Kang Rajni Rani

Sangeeta Bhaskar

Satish K Gupta

134

157

84

7

Publications and Patents

Original peer-reviewed articles

1. Agarwal S, Parashar D, Gupta N, Jagadish N, Thakar A, Suri V, Kumar R, Gupta A,

Ansari AS, Lohiya NK, Suri A (2014) Sperm associated antigen 9 (SPAG9) expression

and humoral response in benign and malignant salivary gland tumors. Oncoimmunol

3:12, e974382.

2. Ahmad J, Mir SR, Hohli K, Chuttani K, Mishra AK, Panda, AK, Amin S (2014) Solid

nano emulsion preconcentrate for oral delivery of paclitaxel: Formulation design, bio-

distribution and scintigraphy imaging. Biomed Res Int 2014: 984756.

3. Almaden JV, Tsui R, Liu Y-C, Birnbaum H, Shokhirev MN, Ngo K, Davis-Turak J,

Otero D, Basak S, Rickert RC, Hoffmann A (2014) A pathway switch directs BAFF

signaling to distinct NFB transcription factors in maturing and proliferating B cells.

Cell Rep 9: 2098–2111.

4. Anand R, Shanka, J, Tiwary BN, Singh AP (2015) Aspergillus flavus induces

granulomatous cerebral aspergillosis in mice with display of distinct cytokine profile.

Cytokine 72: 166-172.

5. Anish C, Upadhyay AK, Sehgal D, Panda AK (2014) Influences of process and

formulation parameters on powder flow properties and immunogenicity of spray dried

polymer particles entrapping recombinant Pneumococcal surface protein A. Int J

Pharm 466: 198-210.

6. Arora S, Verma S, Banerjea AC (2014) HIV-1 Vpr redirects host ubiquitination

pathway. J Virol 88: 9141-9152.

7. Ash D, Subramanian M, Surolia A, Shaha C (2015) Nitric oxide is the key mediator of

death induced by fisetin in human acute monocytic leukemia cells. Am J Cancer Res

5:481-497.

8. Bhatia B, Ponia SK, Solanki AK, Dixit A, Garg LC (2014) Identification of glutamate

ABC-Transporter component in Clostridium perfringens as a putative drug target.

Bioinformation 10: 401-405.

9. Bhatia B, Solanki AK, Kaushik H, Dixit A, Garg LC (2014) B-cell epitope of beta toxin

of Clostridium perfringens genetically conjugated to a carrier protein: Expression,

purification and characterization of the chimeric protein. Prot Expr Purif 102: 38-44.

10. Bhatnagar P, Patnaik S, Srivastava AK, Mudiam MKR, Shukla Y, Panda AK, Pant AB,

Kumar P, Gupta KC (2014) Anti-cancer acitivity of Bromelain nanoparticles by oral

administration. J Biomed Nanotech 10: 3558-3578.

11. Bhattacharya I, Gautam M, Majumdar SS (2015) The effect of IBMX and hormones on

gene expression by rat Sertoli cells, J Reprod Health Med 1:29-40.

12. Bhattacharya I, Basu S, Sarda K, Gautam M, Nagarajan P, Pradhan BS, Sarkar H,

Devi YS, Majumdar SS (2015) Low levels of Gs and Ric8b in testicular sertoli cells

8

may underlie restricted FSH action during infancy in primates Endocrinology 156:

1143-55.

13. Bhattacharya K, Bag AK, Tripathi R, Samanta, SK, Pal BC, Shaha C, Mandal C (2014)

Mahanine, a novel mitochondrial complex-III inhibitor induces G0/G1 arrest through

redox alteration-mediated DNA damage response and regresses glioblastoma

multiforme. Am J Cancer Res 4: 5-22.

14. Biswas T, Pawale VS, Choudhury D, Roy RP (2014) Sorting of LPXTG peptides by

archetypal Sortase A: Role of invariant substrate residues in modulating the enzyme

dynamics and conformational signature of a productive substrate. Biochemistry 53: 2515-

24.

15. Bradfute SB, Anthony SM, Stuthman KS, Ayithan N, Tailor P, Shaia CI, Bray M, Ozato

K, Bavari S (2015) Mechanisms of immunity in post-infection vaccination against

Ebola virus infection. PLoS One (in press).

16. *Chamoli M, Singh A, Malik Y, Mukhopadhyay A (2014) A novel kinase regulates

dietary restriction-mediated longevity in C. elegans. Aging Cell 13: 641-55.

17. Chawla Y, Upadhyay S, Khan S, Nagarajan SN, Forti F, Nandicoori VK (2014) Protein

Kinase B (PknB) of Mycobacterium tuberculosis is essential for growth of the pathogen

in vitro as well as for survival within the host. J Biol Chem 289: 13858 – 13875.

18. Chemmannur SV, Badhwar AJ, Mirlekar B, Malonia SK, Gupta M, Wadhwa N,

Bopanna R, Mabalirajan U, Majumdar S, Ghosh B, Chattopadhyay S (2015) Nuclear

matrix binding protein SMAR1 regulates T cell differentiation and allergic airway

disease. Mucosal Imunol doi:10.1038/mi.2015.11

19. Damle NP, Mohanty D (2014) Mechanism of autophosphorylation of mycobacterial

PknB Explored by molecular dynamics simulations. Biochemistry 53: 4715-26.

20. Dash P, Sahoo PK, Gupta PK, Garg LC, Dixit A (2014) Immune responses and

protective efficacy of recombinant outer membrane protein R (rOmpR)-based vaccine of

Aeromonas hydrophila with a modified adjuvant formulation in rohu (Labeo rohita).

Fish Shellfish Immunol 39: 512-523.

21. Ganguli N, Ganguli N, Usmani A, Majumdar SS (2015) Isolation and functional

characterization of buffalo (Bubalus bubalis) β-casein promoter for driving mammary

epithelial cell-specific gene expression. J Biotechnol 198:53-59

22. *Gill J, Jayaswal P, Salunke DM (2014) Antigen exposure leads to rigidification of

germline antibody combining site. J Bioinform Comput Biol 12:1450006.

23. Golegaonkar S, Tabrez SS, Pandit A, Sethurathinam S, Jagadeeshaprasad MG, Bansode

S, Sampathkumar SG, Kulkarni MJ, Mukhopadhyay A (2015) Rifampicin reduced

advanced glycation end products and activates DAF-16 to increase lifespan in

Caenorhabditis elegans. Aging Cell doi: 10.1111/acel.12327.

24. Haque AS, Patel KD, Deshmukh MV, Chhabra A, Gokhale RS, Sankaranarayanan R

(2014) Delineating the reaction mechanism of reductase domains of Nonribosomal

Peptide Synthetases from mycobacteria. J Struct Biol 187: 207-14.

9

25. Haque MA, Shah U, Zaidi S, Hassan MI, Islam A, Batra JK, Ahmad F (2015)

Characterization of pre-molten globule state of yeast iso-1-cytochrome c and its

deletants at pH 6.0 and 25°C. Int J Biol Macromol 72: 1406-1418.

26. Haque MA, Zaidi S, Shah U, Prakash A, Hassan MI, Islam A, Batra JK, Ahmad F

(2014) In vitro and in silico studies of urea-induced denaturation of yeast iso-1-

cytochrome c and its deletants at pH 6.0 and 25°C. J Biomol Struct Dyn 23: 1-10.

27. Iyer S, Arindkar S, Mishra A, Manglani K, Kumar JM, Majumdar SS, Upadhyay P,

Nagarajan P (2015) Development and evaluation of transgenic nude mice expressing

ubiquitous green fluorescent protein. Mol Imaging Biol PMID: 25595814 (in press)

28. Jacot D, Frénal K, Marq J, Sharma P, Soldati-Favre D (2014) Assessment of

phosphorylation in Toxoplasma glideosome assembly and function. Cell Microbiol 16:

1518-1532.

29. Jain A, Salunke DM (2015) Purification, identification and preliminary crystallographic

studies of an allergenic protein from Solanum melongena. Acta Cryst F Struct Biol

Commun 71: 221-225.

30. Kaiser K, Camargo N, Kappe SHI, Singh AP (2014) Plasmodium axenically developed

exo-erythrocytic forms immunization confer strong protection against infectious

sporozoite challenge. Austin J Vaccines Immunother 1: 6.

31. Kant R, Pasi S, Surolia A (2015) Homo-β-amino acid containing MBP(85-99) analogs

alleviate experimental autoimmune encephalomyelitis. Sci Rep 5: 8205.

32. Kapoor R, Harde H, Jain S, Panda AK, Panda BP (2015) Downstream processing,

formulation developments and antithromobotic evaluation of microbial nattokinases. J

Biomed Nanontech 11: 1213-1224.

33. Karan S, Kaushik H, Saini N, Sahoo PK, Dixit A, Garg LC (2014) Genomic cloning and

sequence analysis of Interleukin-10 from Labeo rohita. Bioinformation 10: 623-629.

34. Khan N, Qadri RA, Sehgal D (2015) Correlation between in vitro complement

deposition and passive mouse protection of anti-Pneumococcal surface protein A

monoclonal antibodies. Clin Vaccine Immunol 22: 99-107.

35. Khan T, Salunke DM (2014) Adjustable locks and flexible keys: plasticity of epitope-

paratope interactions in germline antibodies. J Immunol 192:5398-5405.

36. Khater S, Mohanty D (2014) Genome-wide search for eliminylating domains reveals

novel function for BLES03-like proteins. Genome Biol Evol 6: 2017-33.

37. Khuroo T, Verma D, Talegaonkar S, Padhi S, Panda AK, Iqbal Z (2014) Topotecan-

tamoxifen duple PLGA polymeric nanoparticles: Investigation of in vitro, in vivo and

cellular uptake potential. Int J Pharm 473: 384-394.

38. Kumar N, Damle NP, Mohanty D (2015) Getting phosphorylated: Is it necessary to be

solvent accessible? Proc Ind Natl Sci Acad (in press).

10

39. Kumar P, John V, Marathe S, Das G, Bhaskar S (2015) Mycobacterium indicus pranii

induces dendritic cell activation, survival and Th1/Th17 polarization potential in a TLR-

dependent manner. J Leukoc Biol 97: 511-20.

40. Kumar P, Tripathi A, Ranjan R, Halbert J, Gilberger T, Doerig C, Sharma P (2014)

Regulation of Plasmodium falciparum development by calcium-dependent protein

kinase 7 (PfCDPK7). J Biol Chem 289: 20386-20395.

41. Kumar P, Tyagi R, Das G, Bhaskar S (2014) Mycobacterium indicus pranii and

Mycobacterium bovis BCG lead to differential macrophage activation in Toll-like

receptor-dependent manner. Immunology 143: 258-268.

42. Kumar R, Majumdar DK, Panda AK and Pathak DP (2014) Eudragit coated

microparticulate delivery of bovine insulin for oral delivery. Int J Res Pharm Chem 4:

698-712.

43. Kushwaha RN, Debnath U, Singh P, Saxena R, Gupta SK, Tripathi RK, Siddiqui HH,

Katti SB (2015) New piperazine-derived NNRTIs as anti-HIV agent: synthesis,

biological evaluation and molecular docking studies. Indo American Journal of Pharm

Research 2015:5(01).

44. Lele DS, Talat S, Kumari S, Srivastava N, Kaur KJ (2015) Understanding the

importance of glycosylated threonine and stereospecific action of Drososcin, a proline

rich antimicrobial peptide Eur J Med Chem 92: 637-647.

45. Nagarajan SN, Upadhyay S, Chawla Y, Khan S, Naz S, Subramanian J, Gandotra S,

Nandicoori, VK (2015) Protein kinase A (PknA) of Mycobacterium tuberculosis is

independently activated and is critical for growth in vitro and survival of the pathogen in

the host. J Biol Chem doi:10.1074/jbc.M114.611822.

46. Nand Kripa N, Gupta JC, Panda AK, Jain SK (2015) Development of a recombinant

hCG-specific single chain immunotoxin cytotoxic to hCG expressing cancer cells.

Protein Expr and Purif 106: 10-17.

47. Oetropolis DB, Faust DM, Deep Jhingan G, Guillen (2014) A new human 3D-liver

model unravels the role of galectins in liver infection by parasite Entamoeba histolytica.

PLoS Pathog 10 (9):e1004381.

48. Pandharkar T, Zhu X, Mathur M, Jiang J, Schmittgen T, Shaha C, Werbovetz K (2014)

Studies on the antileishmanial mechanism of action of the arylimidamide DB766: azole

interactions and role of CYP5122A1, Antimicrob Agents Chemother 58:4682-4689.

49. Pandit A, Jain V, Kumar N, Mukhopadhyay A (2014) PHA-4/FOXA-regulated

microRNA feed forward loops during Caenorhabditis elegans dietary restriction. Aging

6: 835-55.

50. Panwar D, Rawal L, Ali S (2014) Molecular docking uncovers TSPY binds more

efficiently with eEF1A2 compared to eEF1A1. J Biomol Struct Dyn 21: 1-12.

51. Pasi S, Kant R, Gupta S, Surolia A (2015) Novel multimeric IL-1 receptor antagonist for

the treatment of rheumatoid arthritis. Biomaterials 42:121-33.

11

52. Patel TK, Anand R, Singh AP, Shankar J, Tiwary BN (2014) Evaluation of Aflatoxin B1

biosynthesis in A. flavus isolates from central India and identification of atoxigenic

isolates. Biotech Bioproc Engn 19:1105-13.

53. Perdomo D, Aït-Ammar N, Syan S, Sachse M, Jhingan GD, Guillén N (2015) Cellular

and proteomics analysis of the endomembrane system from the unicellular Entamoeba

histolytica. J Proteomics 112: 125-40.

54. Rajanala K, Sarkar A, Jhingan GD, Priyadarshini R, Jalan M, Sengupta S, Nandicoori

VK (2014) Phosphorylation of nucleoporin Tpr governs its differential localization and

is required for its mitotic functions. J Cell Sci 127: 3505-20.

55. Rajmohan G, Admane P, Anish CK, Panda AK (2014) Fusion and self-assembly of

biodegradable polymer particles into scaffoldlike and membrane like structures at room

temperature for regenerative medicine. Mol Pharm 11: 2190-2202.

56. Rane S, Das R, Ranganathan V, Prabhu S, Das A, Mattoo H, Durdik JM, George A, Rath

S, Bal V (2014) Peripheral residence of naïve CD4 T cells induces MHC class II-

dependent alterations in phenotype and function. BMC Biol 12: 106-125.

57. Rani R, Israni N, Kumar A, Vasudevan S, Singh J (2014) Association of protein

tyrosine phosphatase non-receptor, type 22 (PTPN22) C1858T polymorphism with

Type 1 diabetes in north India : A replication study. J Diabetes Metab 5: 342.

58. Rathore DK, Nair D, Raza S, Saini, Singh S, Kumar A, Tripathi R, Ramji S, Batra A,

Aggarwal KC, Chellani HK, Arya S, Bhatla N, Paul VK, Aggarwal R, Agarwal N,

Mehta U, Sopory S, Natchu UCM, Bhatnagar S, Bal V, Rath S, Wadhwa N (2015)

Underweight full-term Indian neonates show differences in umbilical cord blood

leukocyte phenotype: a cross-sectional study. PLoS One (in press)

59. Rawal L, Pathak D, Sehgal N, Ali S (2015) Transcriptional dynamics of Homeobox C11

gene in water buffalo Bubalus bubalis. DNA Cell Biol ( in press).

60. Saida B, Dani P, Patnaik N, Agrawal B, Rajaratna T, Jaiswal A, Singh AK, Rani R

(2014) Haplotypes of polymorphic antigen processing genes for low molecular mass

polypeptides (LMP2 and LMP7) are strongly associated with Type 1 diabetes in North

India. J Diabetes Metab 5: 451.

61. *Santhanam SK, Dutta D, Parween F, Qadri A (2014) The Virulence polysaccharide Vi

released by Salmonella Typhi targets membrane prohibitin to inhibit T cell activation. J

Infect Dis 210: 79-88.

62. Saroj DC, Singh KH, Anant A, Biswal BK (2014) Overexpression, purification,

crystallization and structure determination of AspB, a putative aspartate

aminotransferase from Mycobacterium tuberculosis. Acta Cryst F70: 928-932.

63. Satija YK, Das S (2015) Tyr99 phosphorylation determines the regulatory milieu of

tumor suppressor p73. Oncogene (in press).

64. Saxena S, Khan N, Dehinwal R, Kumar A, Sehgal D (2015) Conserved surface

accessible nucleoside ABC transporter component SP0845 is essential for pneumococcal

virulence and confers protection in vivo. PLoS One 10: e0118154.

12

65. Shah U, Haquea MA, Zaidia S, Hassan MI, Islam A, Batra JK, Singh TP, Ahmad F

(2014) Effect of sequential deletion of extra N-terminal residues on the structure and

stability of yeast iso-1-cytochrome-c. J Biomol Struct Dyn 32: 2005-2016.

66. *Shah U, Haquea MA, Zaidia S, Hassan MI, Islam A, Batra JK, Singh TP, Ahmad F

(2014) Effect of sequential deletion of extra N-terminal residues on the structure and

stability of yeast iso-1-cytochrome-c. J Biomol Struct Dyn 32: 2005-2016.

67. Sharma M, Rawal L, Panwar D, Sehgal N, Ali S (2014) Differential expression of

Homeobox C11 protein in water buffalo Bubalus bubalis and its putative 3D structure.

BMC Genomics 15: 638-349.

68. Shembekar N, Mallajosyula VVA, Chaudhury P, Upadhyay V, Varadarajan R, Gupta SK

(2014) Design and characterization of a humanized monoclonal antibody that potently

neutralize 2009 pandemic H1N1 virus. Biotechnol J 9:1594-1603.

69. Sheoran S, Panda BP, Admane P, Panda AK, Wajid S (2015) Ultrasound-assisted

extraction of gymnemic acids from Gymnema sylvestre leaves and its effect on insulin

producing RINm-5F ß cell Lines. Phytochem Anal 26: 97-104.

70. Shrestha A, Srichandan S, Minhas V, Panda AK, Gupta SK (2015) Canine zona

pellucida glycoprotein-3: up-scaled production, immunization strategy and its outcome

on fertility. Vaccine 33: 133-140.

71. Shukla J, Gupta R, Thakur KG, Gokhale R, Gopal B (2014) Structural basis for the

redox sensitivity of the Mycobacterium tuberculosis SigK-RskA σ-anti-σ complex. Acta

Cryst D Biol Cryst 70: 1026-36.

72. Singh AK, Pandey RK, Siqueira-Neto JL, Kwon YJ, Freitas-Junior LH, Shaha C,

Madhubala R (2015) A proteomic based approach to gain insight into reprogramming of

THP-1 cells exposed to Leishmania donovani over an early temporal window. Infect

Immun doi:10.1128/IAI.02833-14.

73. Singh M, Bansal S, Kundu S,

Bhargava P,

Singh A,

Motiani RK,

Shyam R,

Sreekanth V,

Sengupta S, Bajaj A (2015) Synthesis, structure-activity relationship, and mechanistic

investigation of lithocholic acid amphiphiles for colon cancer therapy. Med Chem

Commun 6: 192-201.

74. Singh M, Kundu S, Reddy MA, Sreekanth V,

Motiani RK, Sengupta S,

Srivastava A,

Bajaj A (2014) Injectable small molecule hydrogel as a potential nanocarrier for

localized and sustained in vivo delivery of doxorubicin. Nanoscale 6: 12849-55.

75. Singh S, Suri A (2014) Targeting the testis-specific heat-shock protein 70-2 (HSP70-2)

reduces cellular growth, migration, and invasion in renal cell carcinoma cells. Tumor

Biol 35:12695-706.

76. Tripathi R, Ash D, Shaha C (2014) Beclin-1 p53 interaction is crucial for cell fate

determination in embryonal carcinoma cells. J Cell Mol Med 18: 2275-2286.

77. Tufail S, Owais M, Kazmi S, Balyan R, Khalsa JK, Faisal SM, Sherwani MA, Gatoo

MA, Umar MS, Zubair S. (2015) Amyloid form of ovalbumin evokes native antigen-

13

specific immune response in the host: prospective immuno-prophylactic potential. J Biol

Chem 290: 4131-4148.

78. Tyagi P, Gupta N, Jain A, Upadhyay P, Puliyel J (2015) Intra-gastric pressures in

neonates receiving bubble CPAP. Indian J Pediatr 82:131-135.

79. Upadhyay AK, Singh A, Mukherjee KJ, Panda AK (2014) Refolding and purification of

recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric

protein. Front Microbiol 5: 486.

80. Upadhyay M, Srivastava B, Jain A, Kidwai M, Kumar S, Gomes J, Goswami DG, Panda

AK, Kuhad RC (2014) Production of ganedoric acid by Ganoderma lucidum RCKB-

2010 and its therapeutic potential. Ann Microbiol 64: 839-846.

81. Yadav SK, Batra JK (2015) Ribotoxin restrictocin manifests anti-HIV-1 activity through

its specific ribonuclease activity. Int J Biol Macromol doi: 10.1016/j.ijbiomac.

2015.01.062.

Reviews / Proceedings

1. *Chopra A, Batra JK (2014) Antimicrobial activity of human eosinophil granule

proteins. Meth Mol Biol 1178: 267-281.

2. Dalai SK, Yadav N, Patidar M, Patel H, Singh AP (2015) Liver-stage specific response

among endemic populations: diet and immunity. Front Immunol doi:

10.3389/fimmu.2015.00125

3. Gupta SK (2014) Unraveling the intricacies of mammalian fertilization. Asian J Androl

16:801-802.

4. Gupta SK, Shembekar N (2015). Monoclonal antibodies. In Textbook of Biochemistry

and Human Biology (Eds. Talwar GP, Sarin SK, Hasnain SE). Prentice-Hall of India Pvt

Ltd, New Delhi, India. pp1233-1239.

5. Gupta SK (2014) Role of zona pellucida glycoproteins during fertilization in humans. J

Reprod Immunol doi: 10.1016/j.jri.2014.08.006

6. Jaijyan, DK, Singh H, Singh, AP (2014) Malaria liver stage parasites strategies for

immune evasion and host modulation: Implication for vaccine design. Austin J

Vaccines Immunother 1: 5.

7. Jain Y, Upadhyay P (2014) An inexpensive and 'do it yourself' infant warmer without

electricity. Natl Med J India 27: 55.

8. Kumari R, Kohli S, Das S (2014) p53 regulation upon genotoxic stress: Intricacies and

Complexities. Mol Cell Oncol doi: 10.4161/23723548.2014.969653.

9. Kumari R, Sen N, Das S (2014) Tumour suppressor p53: understanding the molecular

mechanisms inherent to cancer. Curr Sci 107: 786-794.

14

10. Mathur R, Shaha C (2014) Cell death in a kinetoplastid parasite, the Leishmania spp. In

Leishmania: Genomics, Molecular Biology and Control. (Eds. Adak, S and Dutta R,

Horizon Scientific Press, UK) pp 79-92.

11. Natarajan VT, Ganju P, Ramakumar A, Grover R, Gokhale RS (2014) Multifaceted

pathways protect human skin from UV radiation. Nat Chem Biol 10: 542-51.

12. *Rani R, Singh A (2014) Functional implications of MHC associations in autoimmune

diseases with special reference to Type1 diabetes, Vitiligo and Hypoparathyroidism. In

HLA & Associated Human Diseases, (Ed. Yongzhi Xi, inTech publisher, Rijeka,

Croatia) pp 201-221.

13. Sehgal L, Usmani A, Dalal SN, Majumdar SS (2014) Generation of transgenic mice by

exploiting spermatogonial stem cells in vivo. Methods Mol Biol 1194: 327-337.

14. Singh A, Upadhyay, V, Panda AK (2015) Solubilization and refolding of inclusion body

proteins. Methods Mol Biol 1258: 283-291.

15. Singh MS, Bhaskar S (2014) Nanocarrier-based immunotherapy in cancer management

and research. Immuno Targets Ther 3:121-134.

*in press last year, since published

Patents

Granted

1. Garg LC, Dixit A, Keshav G (2015) A recombinant vaccine against Clostridium

Perfringens infection and Epsilon (έ) toxin intoxication (European patent no. 2591108

granted on 11/03/2015).

2. Surolia A, Gupta S, Singh MP, Chattopadhyay T (2015) A method for treating metabolic

disorders including type 1 and type 2 diabetes mellitus (US patent no. 8940691 granted

on 27/01/2015).

3. Sengupta S (2015) RECQL4, the DNA helicase mutated in rothmund-thomson

syndrome, regulated mithochondrial function (European application no. 10793297.2

accepted on 10/11/2014).

4. Panda AK, Gopimohan R, Anish CK (2014) Biodegradable polymer scaffold and process

for preparation thereof (European patent no.08842301.7 granted on 19/12/2014 and

further validated in UK).

5. Panda AK, Gopimohan R, Anish CK (2014) Biodegradable polymer scaffold and process

for preparation thereof (Australia patent no. 2008315318 granted on 24/05/2014).

6. Panda AK, Singh MS, Updhayay AK (2014) Process for obtaining bioactive recombinant

protein from inclusion bodies (Australia patent no. 2008249543 granted on 05/06/2014).

15

7. Surolia A, Gupta S, Singh MP, Chattopadhyay T (2014) Process for preparing

supramolecular calcitonin assemblies (SCA) (European patent no.EP2282763 granted on

24/10/2014).

8. Surolia A, Gupta S, Singh MP, Chattopadhyay T (2014) Composition useful for treatment

of diabetes & disorder (Australia patent no. 2008354530 granted on 12/06/2014).

9. Surolia A, Gupta S, Singh MP, Chattopadhyay T (2014) Composition useful for treatment

of diabetes & disorder (Australia patent no. 2009200906 granted on 7/07/2014).

Filed

1. Bhattacharjee J, Das B, Nagarajan P, Upadhyay P (2015) A simple device for injecting

small volume (5-100µl) of liquid in small laboratory animals (Indian patent application

415/DEL/2015 filed on 13/02/2015).

2. Panda AK, Prasad A (2015) A method for efficient entrapment of water soluble

antibiotics in polymer scaffold to be used for wound dressing (Indian patent application

144/DEL/2015 filed on 16/01/2015).

3. Upadhyay V, Singh A, Panda AK (2015) Solubilization of inclusion body protein using

Trifluoroethanol (Indian patent application 3762/DEL/2014 filed on 18/12/2014).

16

Study of mucosal immune response

Principal Investigator Anna George

Ph. D. Students Lucas D‟Souza

Srijani Basu

Snehlata Gupta

Suman Gupta (since Jan 2015)

Collaborators V Bal, NII

S Rath, NII

UCM Natchu, THSTI, Faridabad

T Vaidya, CCMB, Hyderabad

Theme of research

The laboratory works in the broad area of activation, differentiation, survival and function of

immune cells in systemic organs and at mucosal sites. We attempt to identify molecules that

are involved in modulation of cellular differentiation pathways and promotion of cell suvival,

to dissect mechanisms underlying the induction of T cell tolerance, and to understand how

the microbial flora of the gut and the immune system interact with and influence each other.

Objectives

Ongoing research efforts in the laboratory revolve around the following broad areas:

a) Control of B cell differentiation: We are assessing the role of CD40 ligation on B cell

differentiation. Approaches include stimulating B cells with LPS in the presence or absence

of anti-CD40 to dissect pathways in vitro and the use of CD40-null and TCR-null mice to

assess the role of CD40 ligation under homeostatic conditions in tuning differentiation

thresholds.

b) Plasma cell survival: We are attempting to characterize factors that are involved in

promoting plasma cell survival under normal and pathological conditions.

c) Microbial homeostasis in the gut: We are attempting to estimate microbial diversity in the

intestine of various inbred and immunodeficient mouse strains.

d) Oral tolerance: We have initiated experiments to probe the possible role of innate cells in

the induction of oral tolerance.

Work reported in 2013-2014

In our analysis of B cell antigen presentation, we reported that movement of endocytosed

BCR to LAMP1+ compartments is poor in beige mice which carry the Lyst mutation, with a

substantial fraction remaining outside such compartments even at 120 min as compared to its

rapid delivery to lysosomes in wild-type B cells. Further, E-I-Ab complexes that are formed

following pulsing of activated B cells with E-GST were located largely in LAMP-1+

compartments in wild-type cells and mostly outside such compartments in beige B cells,

indicating that peptide loading occurs at distinct locations in B cells from the two strains. We

also reported prolonged co-localization of the internalized BCR with pErk and the early

endosomal marker Rab5 in beige B cells. Together, the data indicate that BCR-antigen

17

complexes can continue to signal in early endosomes and that peptide loading on MHC-II

molecules can also occur here, although the process is less efficient than in lysosomes.

In our study on plasma cell (PC) survival, we extended our earlier findings that Nos2

deficiency compromises PC viability in vitro to PC function by scoring for decline in

antibody secreting cells (ASCs). We reported that early activation (CD69 and MHC-II

upregulation), proliferation (CFSE dilution) and terminal differentiation (Pax5, Bach2, IRF4

and Blimp1 transcripts and PC generatioin) were unaffected in Nos2-/- B cells, thus limiting

the effect of iNOS-deficiency specifically to formed PCs. We also reported that iNOS-

deficiency led to lower PC survival in vivo, when scored as decline in antigen-specific ASCs

in spleen and bone marrow over time post immunization with NP-Ficoll, or immunization of

bone marrow chimeras with NP-OVA, with ASCs tracked in cells sorted from the two

donors. Additionally, we tested whether iNOS inhibition could dampen an established

antibody response by treating mice daily with aminoguanidine from day 28 post

immunization (after allowing for a primary response, germinal center formation and isotype

switching) and reported that aminoguanidine treatment led to a dramatic fall in titers

specifically in wild-type mice. These experiments were also confirmed by tracking ASCs

over time in immunized bone marrow chimeras. Thus, together with earlier data, we have

shown that iNOS promotes PC survival via a pathway that involves activation of PKG

leading to modulation of ER stress and caspase activation, and pro-survival mediators such as

IL-6 feed into this pathway by inducing iNOS. Also, iNOS inhibition can lead to down-

modulation of an established antibody response by adversely affecting PC survival.

Progress of work during the current reporting year (2014-2015)

Over the current reporting year, we have been following up our previously reported findings

that CD40 ligation can inhibit terminal differentiation of B cells (and promote memory

generation) by a pathway that involves activation of JNK and inhibition of Blimp-1. We first

tracked Blimp-1 transcript levels by qPCR over a 24 h period following stimulation of naïve

IgD+ B cells with LPS in the presence or absence of anti-CD40 and we report that while

Blimp-1 is induced in LPS-cultures, addition of anti-CD40 prevents this induction and

actively represses Blimp-1 transcripts within 12 h to levels that are lower than in the starting

population. Blimp-1 is also repressed when IgD+ B cells are cultured with anti-CD40 alone,

with levels similar to those in sort-purified memory B cell subsets (IgG+ cells from the

spleen and IgA+PNA- cells from Peyer‟s patches). Blimp-1 expression in germinal center B

cells, purified as B220+IgA+PNA+ cells from Peyer‟s patches was lower than in IgD+ cells

but higher than in memory cells, as expected from an ensemble average of dividing cells and

cells preparing to differentiate into memory cells and plasmablasts.

The finding that anti-CD40 alone can repress Blimp-1 transcripts suggests that the

transcription factor may be under active repression in B cells in vivo via B-T interaction. In

support of this, we found that Blimp-1 transcripts were higher in IgD+ B cells from CD40-/-

mice as compared to cells from their wild-type (Balb/c) counterparts. To confirm lineage

specificity of the phenotype, we generated wild-type/CD40-/- bone marrow chimeras, sorted

the two donor populations (as B220+IgD+CD40+ and B220+IgD+CD40-) from the spleens

of the reconstituted mice 2 months later and estimated Blimp-1 transcripts in the sorted cells;-

they were elevated in the CD40-/- donor cells. Further, Blimp-1 transcripts were higher in

TCR mice (that we generated in-house from TCR TCR crosses) than in their

wild-type (B6) counterparts, and treatment of B6 mice with anti-CD40L antibody lead to an

increase in Blimp-1 transcripts at 72 h as compared to cells from mice treated with an isotype

18

control. Finally, treatment of TCR-- B cells with anti-CD40 (to provide the ligand that is

absent in these mice) led to Blimp-1 repression but, as expected, had no effect on CD40-/- B

cells. Together, the data indicate that ligation of CD40 on B cells with CD40L on T cells

keeps Blimp-1 repressed in vivo.

The rapid downmodulation of Blimp-1 by CD40 ligation, before B cell proliferation has been

initiated, indicates that cell fate determination events may occur very early in responding B

cells and suggests that CD40 signaling may have a dominant role in suppressing PC

generation. In support of this, we found that anti-CD40 could inhibit PC generation if added

as late as 48 h after LPS stimulation. We also found significant inhibition if the antibody was

washed out 24 h after culture initiation. Together, the data indicate that CD40 ligation around

the time of B cell activation, such as provided by interaction of antigen-specific B cells with

activated T cells during immunization, can modulate B cell differentiation decisions and the

finding that CD40 ligation can downmodulate Blimp-1 in naïve B cells suggests that CD40

may provide a 'constitutive' signal in vivo of relevance for such decisions by altering the

differentiation-threshold for B cells. A prediction that follows is that CD40-null B cells are

likely to be more poised towards terminal differentiation and hence may show enhanced

generation of PCs upon stimulation in vitro and that CD40-null mice may generate enhanced

Ab responses following immunization with a T-independent Ag. We tested this by

stimulating sort-purified IgD+ B cells, follicular B cells and marginal zone B cells from wild-

type and CD40-/- mice with titrating doses of LPS and found that all three populations from

CD40-/- mice undergo terminal differentiation earlier and to lower doses of LPS than wild-

type B cells. Similar results were seen with other Tlr ligands. Further, While CD40-/- mice

predictably have lower levels of serum IgG, they show significantly higher levels of serum

IgM, have higher frequencies of antibody secreting cells in the spleen and also make a higher

IgM response following immunization with NP-Ficoll. Tlr- and IgM- expression are similar

on B cells from the two strains as are B cell activation and proliferation in response to LPS.

Thus, in the absence of CD40-CD40L interactions in vivo, B cells express higher levels of

Blimp-1 and are more poised towards terminal differentiation when activated.

Attempts are currently being made to understand the mechanism leading to the rapid loss of

Blimp-1 transcripts following CD40-triggering. In keeping with our previously reported data,

we find that JNK is involved, as addition of a JNK inhibitor along with anti-CD40 to wild-

type B cells leads to an increase in Blimp-1 and conversely, surrogate activation of JNK in

CD40 B cells with sorbitol (to bypass the absent CD40-mediated signaling) leads to a

decrease. To determine whether mRNA stability is altered by CD40 signaling, we added

actinomycin D to B cells stimulated with LPS in the presence or absence of anti-CD40 and

found that while Blimp-1 mRNA has poor stability (as reported by others), the rate of decline

in the presence of actinomycin D was identical in the two samples. Thus, preliminary

experiments indicate that issues of message stability are not involved. We are currently

looking for the involvement of Hrd1 (which has been reported to mediate Blimp-1

ubiquitination) and miRNAs in the repression of Blimp-1 mRNA.

Since the generation of plasma cells and memory cells are mutually exclusive events, and

since IgM+ cells form a significant fraction of the memory B cell pool, we also tested

whether the enhanced tendency to terminal differentiation in CD40-null mice was

accompanied by lower frequencies of memory B cells to environmental antigens. B cell

memory been classified into distinct subsets based on the expression of CD73, CD80 and

CD273 and hence we also assessed their relative proportions in unmanipulated WT and

CD40-null mice to see whether CD40 signaling through T-B interactions affected the

19

generation of any or all of these subsets. IgM memory B cells were identified in splenocytes

as B220+IgM

+ IgD

-CD93

-CD21/35

-CD23

+ cells and their frequency as well as the frequency

of each IgM memory subset was estimated. IgM memory cells were found to be lower in the

spleens of CD40-/- mice, with severe depletion of the CD73+ population.

Together, our data indicate that CD40 actively represses Blimp-1 in vivo and prevents early

differentiation of activated B cells into plasma cells. This presumably supports their

progression into germinal centers and the acquisition of somatic mutations before they

undergo terminal differentiation, and provide a mechanistic link between memory generation

and the requirement for germinal centers and T cells. They also demonstrate that a specific

fraction of IgM memory is T cell dependent.

Future plans

We would like to carry forward ongoing research efforts in the lab.

Last year, we reported that iNOS supports plasma cell survival by modulating ER stress and

facilitating the ability of plasma cells to respond to pro-survival molecules such as IL-6 and

APRIL. We also reported that inhibiting iNOS with aminoguanidine could dampen an

ongoing antibody response in vivo. We would like to take these results further and explore

whether the findings have clinical consequences. Preliminary experiments will focus on

whether aminoguanidine treatment can lower anti-DNA antibodies that develop with aging in

the autoimmune-prone mouse strain NZM, and on characterizing the viability of plasma cells

in this mouse (as compared to age-matched wild-type mice) as well as their response to pro-

survival factors.

We have initiated a series of exploratory experiments to determine whether mouse strains

with specific mutations that affect immune function show differential skewing towards

memory generation versus terminal differentiation in their response to environmental

antigens, and whether any of these mutations are involved in influencing plasma cell survival.

Thus, the frequency of B cell memory subsets (switched, unswitched, and subsets identified

by specific markers) will be determined in the spleen, serum Ig isotypes and plasma cell

frequencies in the spleen measured, and survival of isolated plasma cells determined if we

can get sufficient numbers sorted. We would like to follow these up with tracking the

response of various strains to immunization with a T-independent antigen over time (peak

response versus decay).

Our lab is interested in understanding microbial and immune cell homeostasis in the intestine

and we have embarked on a series of experiments aimed at estimating the microbial diversity

in the intestine of various mouse strains and possible links to IgA responses in the host.

Initially, taxa that are preferentially coated with IgA will be identified in wild-type mice by

sorting IgA-coated and IgA-uncoated bacteria from fecal pellets and amplifying specific

selected taxa by genomic qPCR. Comparisons will be made with pellets from RAG-/- mice.

We will also look at diversity in wild-type and RAG-/- mice following co-housing of adults,

and in F2 pups generated from wild-type X RAG-/- crosses. Next, we will determine the

diversity and distribution of strains into the IgA-coated and uncoated pool in various mutant

mouse strains. We are also exploring the utility of Vitamin D as an adjuvant for the

promotion of a mucosal immune response that is measured as IgA in serum, saliva and feces

following subcutaneous immunizations. This is aimed at circumventing the various

difficulties associated with mucosal priming.

20

Action taken on the RAP/SAC 2014 recommendations

RAPSAC members were satisfied with the progress of work that was presented formally. In

informal discussions it was suggested that we might want to look for in vivo correlates of our

in vitro findings linking CD40 stimulation to inhibition of terminal differentiation. This has

been done and progress is reported above.

Publications

Original peer-reviewed articles

1. Rane S, Das R, Ranganathan V, Prabhu S, Das A, Mattoo H, Durdik JM, George A, Rath

S, Bal V (2014) Peripheral residence of naïve CD4 T cells induces MHC class II-

dependent alterations in phenotype and function. BMC Biol 12: 106-125.

21

Understanding the regulation of intracellular transport: Role of GTPases

Principal Investigator Amitabha Mukhopadhyay

Project Associates Ruchir Rastogi

Manglesh K Singh

Amir Kumar Singh

Vijay Singh

Deepika Gupta

Ph. D. Students Pawan Kishor Singh

Jitendra Kumar Verma

Smriti Parashar

Kamal K Gupta

Anjali Kapoor

Chandni Sood

Themes of research

Major theme of the project is to understand the regulation of intracellular trafficking and its

modulation by intracellular pathogens as well as in different pathological conditions. One of

the main goals of this project is to understand the mechanism of survival of intracellular

pathogens in host cells by modulating the host trafficking pathway. We are also trying to

understand the regulation of intracellular trafficking pathway in a protozoan pathogen like

Leishmania using hemoglobin endocytic pathway as we have shown that parasite acquires

heme by the degradation of internalized hemoglobin. We have also initiated the studies on

cytokine mediated modulation of intracellular trafficking.

Objectives

Phagocytosis is an important process in host defense and is mediated by complex interactions

between defined intracellular compartments. The final fate of the nascent phagosomes usually

culminates with the fusion of lysosomes. But some invading microorganisms modulate this

central process for their survival in the phagocytic cells. The major objectives of the present

investigations are:

a. Modulation of intracellular trafficking in host cells by various intracellular pathogens.

b. Determination of the role of various cytokines in the modulation of phagosome

trafficking.

Evidences from a variety of sources, have established that transport of cargo along the

endocytic pathway requires a series of highly coordinated and specific vesicle fusion events

regulated by small GTP binding proteins of the Rab family. Not much is known about the

regulation of endocytosis and intracellular trafficking in protozoan parasites. The major

objective of the project is to understand how intracellular pathways are regulated in

Leishmania and role of these pathways to target the hemoglobin to the degradative

compartment to generate heme, require for parasite survival.

c. Mechanism of intracellular trafficking in Leishmania.

22

Work reported in 2013-2014

a. Modulation of intracellular trafficking in host cells by various intracellular pathogens

It is now well evident that several intracellular pathogens modulate host cellular machinery

for their benefit through some of their own proteins, collectively known as effectors.

Previously, we have identified and determined the role two such effectors namely SopE and

SipC from Salmonella which modulate host cell trafficking pathways by recruiting Rab5 and

Syntaxin6, respectively. In earlier studies, we have shown that live-Salmonella-containing

phagosomes specifically recruit significantly higher amounts of Syntaxin8. Last year, we

have reported, SipA, an effector protein from Salmonella specifically binds with host

Syntaxin8. Subsequently, using several truncated mutants of SipA, we have shown that N-

terminal end of SipA (SipA1-242

) binds with Syntaxin8 possibly mimicking as cognate

SNARE.We have also reported last year that Leishmania infection in macrophages triggers

the expression of Rab5 in the host cells.

b. Mechanism of intracellular trafficking in Leishmania

Previously, we have identified a specific receptor (HbR) for hemoglobin in Leishmania and

characterized the trafficking of hemoglobin from cell surface to lysosomes via Rab5 and

Rab7 regulated process. Last year, we have reported that hemoglobin endocytosis in

Leishmania is a clathrin-dependent process (BBA. Mole. Cell Res. 2013. 1833: 1065-1077).

Moreover, we have also reported that immunization with HbR-DNA induces complete

protection against virulent Leishmania donovani infection in both BALB/c mice and hamster

by inducing Th1 response. Thus, our studies have demonstrated that HbR is a potential

vaccine candidate against visceral leishmaniasis (Science Transl. Med. 2013. 5:202ra121).

We have also reported that cloning and expression of putative epsilon subunit of ATP-

synthase from Leishmania and our preliminary data have indicated that this protein binds

with HbR. In order to understand the mechanism of haemoglobin internalization via clathrin-

mediated endocytosis in Leishmania, we have also reported the cloning and expression of

dynamin homologue from Leishmania.

Progress of work during the current reporting year (2014-2015)

a. Modulation of intracellular trafficking in host cells by various intracellular pathogens

Previously, we have shown that N-terminal of SipA binds with Syntaxin8. In the reporting

period, we have tried to map the region of Syntaxin8 important for the interaction with SipA.

Accordingly, we have made two truncated mutants of Syntaxin8 namely Syntaxin81-105

(N

terminal Syn8), Syntaxin8106-236

(C terminal Syn8). Our results have shown that Syntaxin8106-

236 binds with SipA whereas no binding of Syntaxin8

1-105 with SipA is detected. Further

analysis of Syntaxin8 has revealed that amino acid residues span between 145-209 is the

SNARE motif of Syntaxin8. Therefore, we have included the SNARE motif of Syntaxin8

into the N-terminal region and made another truncated protein, Syntaxin81-209

(N terminal

with SNARE motif). We have found that Syntaxin81-209

binds with SipA indicating that

SNARE motif of Syntaxin 8 is important for binding with SipA.

Previous studies have shown that Salmonella infection activates host Caspase3 and cleaves

SipA into two fragments. It has been reported that C-terminal of SipA binds and localized

with host actin, however, role of N-terminal fragment of SipA in the trafficking of

23

Salmonella is not clearly understood. In order to understand the role N-terminal fragment of

SipA, we have expressed SipA:WT, SipA1-435

or SipA436-685

as N-terminal 3X-FLAG fusion

protein in HeLa cells. Our results have shown that SipA:WT and SipA436-685

colocalizes with

phalloidin labeled actin. Whereas, SipA1-435

is found to be localize on distinct vesicular

membrane structure. Subsequently, we have coexpressed SipA or its truncated protein along

with GFP-Syntaxin8 in HeLa cells. Interestingly, we have found that SipA1-435

significantly

colocalized with Syntaxin8. In contrast, SipA:WT and SipA436-685

are found to be colocalized

with actin. Currently, we are analyzing the functional significance of SipA-Syntaxin8

interaction in Salmonella trafficking in host cells.

Last year, we have also reported our preliminary observation that Leishmania infection in

macrophages triggers the expression of Rab5 in the host cells. In the reporting period, we

have tried to understand the mechanism of Rab5 overexpression in macrophages by

Leishmania infection. In the reporting period, we have shown that Leishmania infection

specifically overproduce Rab5a in macrophages whereas levels of Rab3, Rab4 and Rab11 are

not altered by Leishmania infection in macrophages. Moreover, real time PCR analyses have

shown that overexpression of Rab5a by Leishmania infection is due to enhance level of

Rab5a specific mRNA indicating transcriptional activation of Rab5a. Subsequently,

microRNA profiling reveals that expressions of large numbers of microRNA are modulated

in infected macrophages in comparison to uninfected cells. These results show that the

expression of mir-494 is downregulated in Leishmania infection. Interestingly, bioinformatics

analysis indicates that 3/-regulatory sequence of Rab5a has mir-494 binding site. Therefore,

we have cloned the 3/-regulatory sequence of Rab5a in pmirGLO (pmirGLO-3

/Rab5a)

luciferase containing vector. In order to determine the role of mir-494 in regulating Rab5a

expression, Hela cells were co-transfected with mir-494 and pmirGLO-3/Rab5a and

luciferase activity was measured after 48 hrs. Our results have shown that mir-494

significantly inhibits the transactivation of luciferase. Finally, we have shown that

transfection mir-494 specifically reduces the expression of Rab5a mRNA and protein in

HeLa and THP-1. Taken together, these results indicate that mir-494 regulates the expression

of Rab5a in HeLa cells and macrophages. Subsequently, attempts were made to understand

how Leishmania infection in macrophages upregulates the expression of Rab5. Our results

have shown that Leishmania infection inhibits the expression mir-494 in macrophages.

Finally, we have shown that Leishmania infection degrades AP1 complex and thereby blocks

the synthesis of mir-494. Studies are in progress to determine functional significance of

Rab5a overexpression in the trafficking of Leishmania in macrophages.

b. Mechanism of intracellular trafficking in Leishmania.

Initially, we have shown that early step of hemoglobin endocytosis in Leishmania is regulated

by Rab5 which has two isoforms namely, LdRab5a and LdRab5b. Subsequently, using

parasites overexpressing respective Rab5 isoforms, we have shown that Rab5a regulates the

fluid-phase endocytosis of HRP and dextran whereas receptor-mediated endocytosis of Hb is

regulated by Rab5b in Leishmania. To unequivocally prove the role of Rab5 isoforms in

various modes of endocytosis in Leishmania, we tried to generate Rab5a and Rab5b null

mutant Leishmania in the reporting period. We have found that both LdRab5a and LdRab5b

null mutant cells were lethal indicating their essential functions in parasites. However, we

could rescue LdRab5b-/-

cells by hemin addition as parasite acquires heme from the

degradation of internalized hemoglobin. These cells have shown severe growth defect and

morphological analysis reveals enlarged flagellar pocket. Finally, we have shown that

LdRab5b-/-

cells are unable to internalized Hb but HRP uptake is not affected. In contrast,

24

about 50% inhibition of HRP uptake is observed in LdRab5a+/-

Leishmania without affecting

Hb-endocytosis. Interestingly, we have observed that genistein differentially modulates the

expression of LdRab5 isoforms and accordingly regulates respective modes of endocytosis

indicating role of receptor tyrosine kinases in various modes of endocytosis in Leishmania.

This is the first demonstration that Rab5a and Rab5b differentially regulate fluid-phase and

receptor-mediated endocytosis.

We have also reported earlier that epsilon subunit of ATP-synthase (Ld-ATPSy) from

Leishmania binds with HbR. In the reporting period, we tried to characterize the role of this

protein in haemoglobin endocytosis in Leishmania. To be an adaptor molecule in clathrin-

mediated haemoglobin endocytosis, Ld-ATPSy should bind with both HbR and clathrin. Our

results have shown that GST-HbR binds with His-LdATPSy in protein-protein interaction.

Similarly, immobilized GST-LdATPSy can specifically bind with HbR from Leishmania

lysate. Moreover, we have found that 5 min internalization of Hb-Alexa Red colocalized with

GFP-ATPSy in Leishmania. Taken together, these results demonstrate that HbR specifically

binds with Ld-ATPSy and play a role in Hb endocytosis.

Subsequently, we have determined the binding of Ld-ATPSy with clathrin. Accordingly,

GST-Ld-ATPSy was immobilized on glutathione-Sepharose beads and incubated in the

presence of 3 mg of Leishmania lyate prepared from GFP-Clathrin overexpressed cell for 2 h

at 4 °C. Beads were washed six times with PBS to remove unbound proteins and finally,

Western blot analysis was carried out using anti-GFP antibody. Our results have

demonstrated that GST- Ld-ATPSy binds with Ld-clathrin from Leishmania lysate.

Previously we have shown that Clathrin (Ld-CHC) from Leishmania is about 180 kD protein

and composed of three major domains namely Treminal, Distal and Proximal domains. In

order determine which domain of LdCHC binds with epsilon subunit of ATP-synthase in

Leishmania, we have cloned and expressed each of the domains separately as GST fusion

protein. Subsequently, different domains were immobilized on glutathione-Sepharose beads

and incubated with equimolar concentration of His-ATPSy for 2 h at 4 °C. Beads were

washed six times with PBS to remove unbound proteins and Western blot analysis was

carried out using anti-ATP synthase antibody. Our results have shown that epsilon subunit

ATP synthase predominantly binds with Terminal Domain of Ld-Clathrin. Thus, we have

demonstrated that epsilon subunit ATP synthase is a novel adaptor in clathrin-mediated

hemoglobin endocytosis in Leishmania.

Earlier, we have reported cloning and expression of dynamin homologue from Leishmania. In

the reporting period, we have made Ld-Dynamin:K39A and Ld-dynamin:T60F mutants. We

have expressed Ld-Dynamin:WT, Ld-Dynamin:K39A and Ld-Dynamin:T60F mutants either

as N-terminal GST or GFP tag. Subsequently, we have determineted the GTP binding and

GTPase activities of these proteins. Our results have shown that Ld-Dynamin:WT and Ld-

Dynamin:K39A bind comparable amount of GTP, whereas significantly less binding of GTP

is detected with Ld-Dynamin:T60F mutant. However, Ld-Dynamin:K39A and Ld-

Dynamin:T60F mutants have shown significantly reduced GTPase activity in comparison to

Ld-Dynamin:WT. In order to determine the role of Ld-Dynamin in hemoglobin trafficking,

we have overexpressed these proteins individually in Leishmania as GFP fusion protein.

Interestingly, we have found that Ld-Dynamin:WT and Ld-Dynamin:K39A predominantly

localize in the flagellar pocket hemoglobin binds with its receptor in clathrin-coated pits. In

contrast, Ld-Dynamin:T60F mutant failed to localize in the target compartment. Detail

studies to determine the role of dynamin in hemoglobin endocytosis are in progress using

these mutants.

25

Future plans

1. Studies are in progress to determine role of Syntaxin8 and SipA interaction in the

intracellular trafficking of Salmonella in host cells.

2. Studies are in progress to determine the functional significance of Rab5a overexpression

by Leishmania infection in macrophages.

3. We have characterized epsilon subunit of ATP-synthase as a novel adaptor which binds

with both HbR and Clathrin in Leishmania. Studies are in progress to make a ATP-

synthase null mutant Leishmania as well as to prepare a dominant negative mutant of

ATP-synthase which will be unable to bind either HbR or clathrin. These reagents will be

used to determine the role of epsilon subunit of ATP-synthase in hemoglobin endocytosis

in Leishmania.

4. Studies are in progress to determine the role of dynamin in the internalization of

hemoglobin in Leishmania.

5. Studies are in progress to determine the role of Rab4 and Rab11 in receptor recycling in

Leishmania.

Action taken on RAP/SAC 2014 recommendations

Clarifications sought were provided to the satisfaction of the RAP/SAC members. No

specific follow up actions was suggested.

26

Study on expansion and plasticity of bone marrow stem cells

Principal Investigator Asok Mukhopadhyay

Research Associates Prakash Baligar

Surender K. Sharawat (till July 2014)

Zahid Akhtar (since Sep 2014)

Ph. D. Students Abinaya Sundari T

Veena Kochat

Zaffar Iqubal

Research Fellows Snehashis Mukherjee

Shyam Krishna M

Collaborators SK Sarin, ILBS, Delhi

N Threhan Pati, ILBS, Delhi

L Kumar, AIIMS, Delhi

S Kumar, AIIMS, Delhi

S Mathur, AIIMS, Delhi

M Srivastava, NII

P Nagarajan, NII

J Teckman, SLU, St. Louis, USA.

Theme of research

Bone marrow (BM) niche controls self-renewal and differentiation of HSCs. To understand

the regulation of hematopoiesis and the effect of aging on stem cells, it is necessary to

decipher stem cell niche in different age groups of mice. A comprehensive knowledge on

hematopoietic niche will help to mimic an ex vivo expansion culture in which both stem-ness

and engraftability of cells can be maintained. It is now known that hematopoietic cells are

involved in regeneration of many non-hematopoietic organs. Ex vivo cultured cells seem to

facilitate transplantation for hematological reconstitution as well as treating other diseased

organs. The overall themes of our research involve the study of stem cell niches, plasticity of

BM-derived stem cells, and the role of BM-derived cells in progression of cancer.

Objectives

We intend to dissect HSCs niche to elucidate its function in marrow regeneration, as well as

their role in various pathological conditions of solid organs, and plasticity of adult stem cells.

The objectives are as follows:

1. Molecular control of self-renewal and engraftability of HSCs in mice.

2. To understand liver regeneration by BM-derived cells and to elucidate the mechanism of

hepatic differentiation.

3. Role of BM cells in stem-ness and cancer progression.

4. Mechanistic insight in the regeneration of neurons by MSC-derived precursor cells.

5. Study of molecular interplay during fibrosis and normal regeneration of tissue.

27

Work reported in 2013 -2014

A. Hematopoietic stem cells niche and marrow regeneration

Using shRNA approach and specific inhibitor of ErbB pathway, we showed that in absence

of Amphiregulin, Stat5 could not be activated in LSK cells leading to apoptosis.

B. Plasticity of BM-derived cells

We showed that fetal liver MSCs-derived domapinergic neurons rescue parkinsonian mice

from motor neuron defect. This functional evaluation of the cells was further supported by

immunohistochemical analyses of brain tissue and synthesis of dopamine.

In hemophilia A mouse model, allo-antigen sensitized Treg cells (sTregs) were found to

prevent rejection of allogeneic uncommitted bone marrow cells, which lead to engraftment

and differentiation of donor cells into hepatocyte-, endothelial- and Kupffer-like cells. By

inter-phase FISH analyses, we have confirmed that cell fusion in this model was low. The

donor derived hepatocytes gained expression of liver enriched transcription factors (GATA4,

GATA6, HNF3α, HNF3β, HNF1α, HNF1β, HNF4α, HNF6, OC-2, CEBPα and CEBPβ),

liver specific proteins (TDO, Albumin, FVIII, CK18, E-cadherin and CYP1A2).

In another liver disease model of human alpha1-antitrypsin (AAT) deficiency, we have

demonstrated that the hallmark of the disease, the formation of diastase resistant PAS-stained

globules in hepatocytes, was significantly reduced. Not only that, abnormal glycogen

metabolism in the disease mice was also partially rectified.

C. Ovarian cancer

We have demonstrated that in ascetic fluid the tumor cells were associated with

hematopoietic phenotype. To understand genetic changes in these hemato-epithelial cells

with respect to EpCAM-expressing tumor cells three sets of samples were subjected to

microarray analyses.

D. Muscle regeneration and fibrosis

In previous year, we have reported the role of NF-B pathway in aberrant generation of

skeletal muscle in mouse.

Progress of work during the current reporting year (2014-15)

A. Hematopoietic stem cells niche and marrow regeneration

The functional role of Areg gene was studied by silencing its expression in stromal cell line

using shRNA technology. Murine bone marrow HSCs was cultured in the presence

of Areg+ and Areg

- stromal cell lines to evaluate the functional role of this gene. Previously,

we showed that Areg- stromal cells does not support HSCs in culture rather cells underwent

apoptotic death and Areg-stat5 signaling helps in rescue and survival of HSCs. To assess the

downstream signaling of stat5 related to the regulation of apoptosis in HSCs, we evaluated

the pro-apoptotic and anti-apoptotic genes namely Bad, Bax, Bcl-XL, Socs and Myc. We

observed up-regulation of pro-apoptotic genes in Areg-

stroma-dependent culture of HSCs

and the result was inversed in wild-type. For the assessment of our hypothesis on extrinsic

28

pathway of apoptosis in HSCs, subsequent results confirmed that apoptosis of HSCs in Areg-

stroma-dependent culture was FasR mediated involving the activation of caspase 3/7. Further,

in order to prove that Areg is not required for the survival of stromal cells, unlike in HSCs,

we checked Stat5 phosphorylation in both cell types (Areg+

and Areg- stroma). Results

showed that no Stat5 was activated in both wild-type and Areg- culture. This concluded that

Areg signaling of stat5 phosphorylation is particular to HSCs and not to the stromal cells.

New studies will focus on essential role of Areg in the bone marrow stroma in vivo.

B. Plasticity in BM cells

The cellular fate of adult stem cells has been found to be altered in pathological conditions

where the lineage barrier is overwritten by cellular reprogramming, which can occur either by

heterotypic cell fusion or by direct differentiation. Earlier, in acute liver injury model, we

have shown that a fraction of uncommitted BM cells can change their phenotype into

hepatocytes. We isolated these BM-derived hepatocytes and performed a comprehensive

transcriptional profiling to analyze the changes in gene expression that accompanied during

this phenotype conversion. We found a large similarity in gene expression profile between

BM-derived hepatocytes and primary hepatocytes, about 15946 genes were commonly

expressed. However, we also identified 1113 and 605 genes that were exclusively expressed

in primary hepatocytes and BM-derived hepatocytes, respectively. By gene ontology analyses

we found that even though BM-derived hepatocytes exhibited some dissimilarity in gene

expression profile with respect to primary hepatocytes, there was induction of hepatic

transcriptional program with concomitant decline of hematopoiesis related program.

We further investigated the epigenetic mechanisms that co-ordinate these transcriptional

changes by ChIP-qPCR analysis for histone modifications. Enrichment of activating histone

marks (H3K4me3and H3K9Ac) were found at promoters of crucial hepatic transcription

factors like HNF4α, HNF1α, HNF3α, HNF3β, CEBPα, CEBPβ, HNF6 and GATA4, whereas

repressive marks (H3K27me3 and H3K9me3) were reduced in these loci after

reprogramming, in which H3K27me3 mark was prominent. In contrast to these findings we

found enrichment of repressive marks (H3K27me3 and H3K9me3) at the hematopoietic gene

promoters like CD45 and GATA2 in BM-derived hepatocytes. Further analysis of DNA

methylation patterns at the promoters of hepatic and hematopoietic genes will determine the

stability of this molecular reprogramming. We examined the role of EZH2 and JMJD3, both

of which are chromatin modifying enzymes that antagonistically regulate methylation status

of H3K27 in mediating the loss of H3K27me3 mark in BM-derived hepatocytes following

ChIP-qPCR. JMJD3 can actively demethylate H3K27me3 and remove this repressive mark.

Those promoters studied here possess both activating mark (H3K4me3) and repressive mark

(H3K27me3) in the Lin- BMCs, removal of the silencing mark can mediate transcriptional

initiation. Binding of JMJD3 to the promoters of hepatic transcription factors along with loss

of EZH2 was observed in BM-derived hepatocytes, whereas in Lin- BMCs these promoters

were enriched for EZH2.

To understand the role of JMJD3 and EZH2 in hepatic specification of Lin-

BMCs, we

investigated whether their expressions undergo any change in response to induction by

growth factors like HGF, EGF and oncostatin M in culture. In vitro differentiation of Lin-

BMCs in hepatic phenotype showed EZH2 expression to decline and JMJD3 expression to

increase similar to that were observed in studies in vivo. Gene expression analysis revealed

increase in expression of some hepatic genes like HNF4α, HNF3α, albumin, CK18 and E-

cadherin. Immunocytochemical analysis showed that the differentiated cells, expressing

29

HNF4α in the culture, also exhibited intense nuclear staining for JMJD3. However, the levels

of EZH2 and H3K27me3 in these cells were much lower than that in Lin- BMCs. Further

experiments using an inhibitor for JMJD3 (GSK-J1) will prove the definitive role of JMJD3

in hepatic differentiation of Lin-

BMCs under inductive conditions, comparable to the

prevailing regenerative environment in the liver with acute injury.

In another project, we have crossed NOD-SCID (female) with PiZ transgenic mice (male),

the F1 generation mice so formed were again intercrossed. In that way, we have generated F3

SCID-PiZ breeding pairs; the homozygous mice were screened by PCR with respect to

double mutant (hPiZ and Pkrdc genes). The homozygous mice were expanded and further

characterized with respect to double mutation of genes, serum hAAT level, and liver

pathology. Human MSCs were subjected to transplantation in the properly characterized

diseased mice. The mice are due for the analysis to assess the therapeutic potential of the

cells.

Earlier, we have shown that CD45-expressing BM cells have more anti-fibrogeneic potential

than MSCs in CCl4-induced liver fibrosis model. During past one year we have established

the molecular mechanisms that are responsible for diverse functional difference between two

cell types. In vitro experiments with activated hepatic stellate cells showed that MSCs

conditioned medium contains potent fibrogeneic TGF, which induced stellate cells for

myofibroblastic differentiation leading to the synthesis of more -SMA and collagen I. In

addition, these cells were also found to secrete IGF-1 and PDGF, potent anti-apoptotic and

mitogen for hepatic stellate cells. The combined effect of these factors resulted pro-fibrotic

environment. On the other hand, CD45-secreted FasL facilitated apoptotic death of activated

stellate cells. Again, as INF was secreted by these cells, the TGF pathway was also

inhibited, thus myofibroblastic differentiation did not occur. Overall, CD45 cells facilitated

death of activated hepatic stellate cells, whereas MSCs induced their myofibroblastic

differentiation.

C. Ovarian cancer

Microarray gene expression profiling of two human ovarian cancer samples revealed that

about 1728 genes were upregulated in EpCAM+CD45

+ cells with respect to EpCAM

+ tumor

cells. The Upregulated genes were clustered into 10 different functions (apoptosis, drug

resistance, stem cell markers, immune surveillance, metabolism, migration, cell signaling,

proliferation, cell cycle, angiogenesis) relating to the cancer. Subsequently, we validated

microarray data in 3 genes of each panel of 4 important functions (drug resistance, stem cell

marker, apoptosis and immune surveillance). Taking into account of microarray data and

qPCR validation, we propose that EpCAM+CD45

+ cells have acquired gene expressions in

terms of aforementioned functional properties, which will be further authenticated by

functional assay.

In view of the current findings, without ruling out the possibility of fusion with hematopoietic

cells (our previous study in mouse model), we further propose that exosomes secreted by

stromal cells could influence the tumor cells leading to the change of phenotype and

subsequent functional gain by tumor cells. In this direction, we are pursuing investigation on

the molecular cargo, secreted by tumour stromal cells. The exosomes from the ascetic fluid of

human ovarian carcinoma have been isolated for further characterization.

30

Future plans

1. Pathway analysis (wet and dry lab based experiments) of new hematopoietic genes (e.g.

amphiregulin, etc.) and their functional studies.

2. DNA methylation and gene expression studies in BM-derived hepatocytes.

3. In vitro/ in vivo studies to substantiate functional gain in EpCAM+CD45

+ cells, and

elucidating the role of exosomes in reprogramming of hemato-epithelial cells.

4. Stage specific differentiation of human MSCs into dopaminergic neurons and

investigation on epigenetic mechanisms involved.

5. Evaluation of the therapeutic potential of human MSCs in chimeric SCID-PiZ diseased

model.

Action taken on the RAP/SAC 2014 recommendations

Scientific and technical queries raised by the members were answered.

31

Fine Tuning of NF-kappaB Signaling

Principal Investigator Soumen Basak

Ph. D. Students Balaji Banoth

Payel Roy

Tapas Mukherjee

Sachendra Singh Bais

Meenakshi Chawla

Budhaditya Chatterjee

Collaborators V Bal, NII

S Rath, NII

Theme of research

Biochemical and genetic studies have orchestrated molecular connectivities between

apparently “insulated” signal transduction pathways. Yet, a role of such interconnectedness

in regulating stimulus responsive behavior of the cell system remains unclear. In an

integrative approach, which combines experimental and mathematical studies, we have been

exploring physiological and patho-physiological consequences of interdependent regulations

of cell signaling pathways. In an ongoing program, we have shown that network interactions

via the NF-B system allows for fine-tuning of inflammatory response by

microenvironmental cues. In a newly proposed program, we will further attempt to delineate

the pathophysiological consequence of altered network circuitry in multiple myeloma.

Objectives

A. On going program – Exploring the signalling crosstalk between developmental

LTR and inflammatory TLRs

We will further extend our study that addresses the regulatory role of lymph node inducing

LTR in fine-tuning inflammatory response to TLR. First, we will examine the molecular

basis underlying the duration code that imparts stimulus selectivity in crosstalk control of

innate inflammatory response. Second, we will investigate the physiological role of crosstalk

control in murine infection model.

B. New program – Investigating signal transduction via perturbed cellular network in

multiple myeloma

In this newly proposed research program, we will investigate how myeloma associated

activating mutations in the non-canonical pathway alter TNF induced inflammatory signal

32

transductions. A plausible role of the convergence of tumor promoting TNF signalling and

cell-intrinsic mutations in generating resilience to apoptosis in multiple myeloma cells will

also be addressed.

Work reported in 2013-2014

A. On going program – Exploring the signalling crosstalk between developmental

LTR and inflammatory TLRs

We have earlier reported that LTR modulates TLR4 induced late expressions of NF-B

target genes through crosstalk. We have presented biochemical and genetic evidence

suggesting that Nfkb2 functions as a mediator of crosstalk. Although, we have noted that a

duration code insulates transient inflammatory signals from crosstalk amplifications, the

mechanisms were elusive at the time of submission of the last year‟s report.

B. New program – Investigating signal transduction via perturbed cellular networks in

multiple myeloma

We are submitting this as a new research proposal for consideration to RAP-SAC.

Progress of work during the current reporting year (2014-2015)

A. On going program – Exploring the signalling crosstalk between developmental LTR

and inflammatory TLRs

Previously, we have shown that Nfkb2 mediates crosstalk synergy between LTR and TLR4,

but IL-1R pathway was insulated from crosstalk amplifications. Here, we have analyses the

underlying molecular mechanism that discriminates between inflammatory signals. Also, we

have established a physiological role of cross-regulation in gut immunity.

33

i) Molecular mechanism underlying the duration code

First, we compared inducible expression of the crosstalk-mediator Nfkb2 in response to LPS

or IL-1. As opposed to rapid Ib expression, LPS induced Nfkb2 mRNA with a delay (top

panel, Figure 1A). Similarly, chronic TNF treatment induced Nfkb2 mRNA in WT MEFs

with an explicit 1h delay (Figure 1B) that was also incorporated in the current mathematical

model. When stably expressed from an exogenous kappaB site driven promoter in Nfkb2-/-

MEFs, Nfkb2 mRNA was readily induced by TNF in these cells, termed delay null mutants

(Figure 1B). Remarkably, IL-1 treatment was ineffective in activating the expression of

Nfkb2 mRNA in WT MEFs, despite the early induction of Ikb (bottom, Figure 1A). We

could rescue this defect in Nfkb2 mRNA induction by IL-1 in the delay null cells, both

computationally (Figure 1C) and experimentally (Figure 1D). Our results suggested that a

promoter intrinsic delay necessitates persistent canonical signal for RelA mediated induction

of pro-synergistic Nfkb2. Such delay encoding insulated IL-1R signaling, which transiently

activates IKK2 and RelA by restricting Nfkb2 mRNA expressions. Our studies also explained

Figure 1. Induction of Nfkb2 expressions by canonical signal is required for

crosstalk. (A) Relative levels of Nfkb2 and Ikba mRNAs in WT MEFs during LPS or

IL-1 signaling. (B) TNF induced delayed expression of Nfkb2 mRNA in WT MEFs

and rapid production in the delay null cell, an engineered Nfkb2-/-

cell-line with

Nfkb2 mRNA being expressed from an exogenous NF-kB dependent promoter. (C)

Simulations comparing IL-1 induced Nkfb2 mRNA expressions in WT or delay null

mutant, which lacks the transcriptional delay in the Nfkb2 mRNA expression. (D)

Quantitative RT-PCR revealing IL-1 induced expression of Nfkb2 and Ikba mRNAs

in the delay null cells.

34

the requirement for the long-duration NIK-IKK1 signals in targeting this late acting p100

Nfkb2 feedback for RelA:p52 dimer generation.

In sum, we elucidated a crosstalk mechanism that discriminates between TLR4 engagement

and concomitant cell activation through TLR4 and LTβR. Negative feedbacks by IκBα and

p100/IκBδ coordinately terminate canonical TLR4 response. But, Nfkb2 functions pro-

synergistically upon costimulation; in a positive feedback loop, non-canonical LTβR signal

targets the newly synthesized p100, abundantly produced by TLR4, to potently generate

RelA:p52 dimers in sustaining inflammatory RelA NF-κB responses. Although, emergent

crosstalk is controlled by several biochemical constrains, the transcriptional delay intrinsic to

the Nfkb2 promoter appears to be critical for the duration code.

ii) A mouse gut-infection model depicts the physiological significance of crosstalk

regulations:

In addition to its role in lymph node development during embryogenesis, recent studies have

illustrated a requirement for LTR in innate immune responses in adult mice. Given our

identification of a costimulatory function of LTR in inflammatory RelA activation, we

asked if signal integration via the NF-B system could explain the epithelial requirement of

LTR in innate immunity.

First, we biochemically analyzed NF-B activation in intestinal epithelial cells (IECs)

derived from WT mice intraperitoneally injected with antagonistic LTβR-Ig or a control-Ig

one day prior to oral infection with enteric pathogen Citrobacter rodentium. Upon

colonization, Citrobacter initially triggered epithelial accumulation of p100 that was fully

processed into p52 by day5 generating RelA:p52 dimer in control-Ig, but not LTβR-Ig,

treated mice. Perturbing LTβR signal attenuated NF-B activation with more obvious defects

at day5. Likewise, pathogen-responsive RelA activation in IECs derived from Nfkb2-/-

mice

was severely weakened at day5 that led to significantly reduced expressions of the RelA

target chemokines encoding KC and MIP-2α as compared to WT mice. Indeed, infected

Nfkb2-/-

mice exhibited diminished neutrophil recruitment in the lamina propria, as revealed

by anti-myeloperoxidase immunostaining of the colon sections. Sustained epithelial RelA

activity that relies on LTR mediated processing of pathogen-induced p100 into p52,

therefore, mirrored our MEF based analyses depicting crosstalk between canonical and non-

canonical signaling. Collectively, our results connected the previously reported epithelial

requirement of LTβR and NIK in innate immune response to the NF-B system in reinforcing

RelA activity through Nfkb2 mediated crosstalk control. Subdued epithelial NF-κB activation,

and not hyper-induction, in IECs from infected Nfkb2-/-

mice also suggested that a dominant

precursor function of p100 supplying RelA:p52 dimer prolongs RelA response within the

intestinal niche.

35

B. New program – Investigating signal transduction via perturbed cellular networks in

multiple myeloma

It is thought that tumor microenvironment derived chronic cytokine signals lead to aberrant

dynamic control in the canonical pathway with persistently elevated RelA activity, which

offers survival advantage to transformed cells in carcinomas. Curiously, a significant

enrichment of gain-of-function mutations in the non-canonical NIK-RelB/NF-B pathway

has been described in multiple myeloma. Although, small numbers of multiple myeloma

cells could be detected in the peripheral circulation, they largely reside in the bone marrow

niche, where stromal cells provide paracrine signals through pro-inflammatory cytokines,

such as TNF and IL6, to promote cancerous growth and cell-survival. Yet, a plausible

collaboration between pro-inflammatory cytokine induced canonical signaling and cell-

intrinsic activating mutations in the non-canonical pathway in multiple myeloma has not been

investigated.

i) TNF priming protects myeloma cells with activating mutations in the non-canonical

pathway from apoptotic death

To this end, we examined KMS28PE human myeloma cell lines (HMCLs) that expresses

cIAP1/cIAP2 only at low levels owing to genetic aberration, and JK6L cell-line, which

harbors an inactivating mutation in Nfkb2, for their resilience to apoptotic stimuli. As a

control, we utilized OciMy5 cell-line, which lacks non-canonical mutations, but possess an

amplification of Nfkb1. Our cell death assay revealed that priming with TNF at 8h prior to

TRAIL exposure resulted in ~50% protection from TRAIL mediated death in KMS28PE and

JK6L, but not in OciMy5, cell-lines (Figure 2A).

Figure 2: TNF priming imparts resilience in

Multiple Myeloma cells to apoptotic TRAIL

(A) TRAIL mediated cell death was assessed at

16h post-stimulation using trypan blue dye

exclusion assay in the indicated HMCLs

primed with TNF for 6-8h and presented

relative to the cell death induced in the

respective naïve cells treated with TRAIL

alone. (B) Nuclear NF-kB activity induced in

indicated HMCLs by TNF in a time course was

resolved in EMSA. The faster migrating

complex, indicated with a green arrowhead,

consists of RelB and the slower migrating

complex, denoted with a magenta arrow,

composed of RelA dimers.

36

ii) TNF activates a perpetuating RelB:p50 response in a subset of myeloma cells:

TNF is known to mediate its pro-survival functions by activating the canonical RelA/NF-κB

pathway. Our EMSA analyses demonstrated that TNF induces nuclear RelA:p50 dimer in

both OciMy1 and JK6L cell-lines, albeit transiently with only residual RelA DNA binding

activity at 8h post-stimulation (Figure 2B). Although subjected to similar post-induction

attenuation, TNF weakly induced RelA/NF-κB activity with a delayed onset in KMS28PE

cells, likely due to depleted cIAP1/cIAP2 levels. Strikingly, TNF stimulated an additional

NF-κB DNA binding complex composed of RelB:p50 dimer in both KMS20PE and JK6L,

but not in OciMy5 cell line (Figure 2B). Unlike deteriorating RelA:p50 activity, however,

RelB:p50 dimers perpetuated in the nucleus during TNF signaling attaining a peak at 8h post-

stimulation. We have also observed a rather low level of basal RelB activity in KMS28PE

and JK6L cell-lines.

Future plans

A. On going program – Exploring the signalling crosstalk between developmental

LTR and inflammatory TLRs

Our preliminary studies revealed remarkable differences in crosstalk control between

myelomonocytic cells and those of the epithelial origin. Yet, signal induced synthesis of the

crosstalk mediator Nfkb2 were comparable in these two cell types. In the coming year, we

will characterize the molecular basis underlying cell-type specificity of crosstalk between

LTR and TLR4 signalling. Using advanced multi-parametric analyses, we will generate

experimentally testable hypothesis. Those will then be validated using biochemical and

genetic tools. As a proof of concept, we will finally attempt to generate engineered cells with

swapped cell type specificities. In sum, we propose that quantitative analyses of cell

signalling pathways in a computation model may offer insights on physiological significance

of emergent system properties.

B. New program – Investigating signal transduction via perturbed cellular networks in

multiple myeloma

In an interdisciplinary study, which combines biochemistry, genetics and mathematical

modelling, we will further examine the molecular mechanism underlying altered utilization of

NF-B dimers by TNF in multiple myeloma. Second, we will address potential overlap

between RelA and RelB containing NF-B dimers in mediating kappaB driven gene-

expressions during inflammatory signalling or their functional redundancy in anti-apoptotic

pathway. Finally, we will study if alter dimer utilization indeed propagates prosurvival

response to TNF in myeloma cells. We hope that our analyses will shed light into the

pathophysiological consequences of disease-associated perturbations in the cell-signalling

network.

37

Action taken on the RAP/SAC 2014 recommendations

The project described under (A) was extensively discussed with the RAP-SAC members in

April 2014. Currently, the manuscript originated from the work is under revision in an

international peer-reviewed journal. We are preparing a second manuscript that focuses on

cell type specificity of crosstalk control. Subscribing to the recommendation of RAP-SAC

panel, the laboratory has engaged into collaborative studies with other research groups to

further our understanding on cell-signaling mechanisms underlying immune processes. We

have already published one collaborative paper, while a second manuscript is under revision.

Of note, project described in (B) is a new project and was not discussed earlier.

Publications

Original peer-reviewed articles

1. Almaden JV, Tsui R, Liu Y-C, Birnbaum H, Shokhirev MN, Ngo K, Davis-Turak J, Otero

D, Basak S, Rickert RC, Hoffmann A (2014) A Pathway switch directs BAFF signaling to

distinct NFB transcription factors in maturing and proliferating B cells. Cell Rep 9:

2098–2111.

38

Ribonucleases and heat shock proteins: involvement in host defense

Principal Investigator Janendra K Batra

Project Associates Manish Gupta

Ubaid Ullah Shah

Ph. D. Students Ayush Attrey

Owais Rashid Hakiem

Priyanka Parijat

Virendra K Patel

Alla Singh

Prajna Tripathi

Collaborator F Ahmad, Jamia Millia Islamia, Delhi

Theme of research

The work is focused on the following two major themes.

1. Investigation of the role of human ribonucleases, particularly eosinophil ribonucleases,

eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) in host defense.

Human ribonucleases, and naturally occurring protein toxins are being explored to design

knowledge-based recombinant toxins.

2. Investigation of crucial housekeeping proteins of M. tuberculosis for their role in survival

and virulence of the pathogen. Caseinolytic protease (Clp) regulates the expression of

virulence genes, and also help bacterial pathogens in countering stress in the host. RNase P is

structurally completely different in bacteria and human. The functioning of Clp machinery,

and RNase P mediated tRNA maturation is being investigated in M. tuberculosis.

Objectives

- Investigation of molecular mechanism of biological actions of human ribonucleases

and their role in host defense. Our study is focused on understanding the mechanism of

action of ECP and EDN, identification of their intracellular targets, and their modulation

during eosinophil differentiation and maturation.

- Construction and evaluation of recombinant toxins as potential therapeutics. The aim

is to rationally design, engineer and characterize specific and potent recombinant toxins

for targeted therapy employing human RNases, cell death inducing proteins of human

origin, ribosome-inactivating protein, saporin, and ribonucleolytic toxin, restrictocin.

- Investigation of the involvement of Clp proteases in pathogenic mechanism of M.

tuberculosis. We are investigating the molecular basis of interplay between substrate,

adaptor and various members of the Clp family to understand their involvement in protein

39

homeostasis and in turn in the survival and pathogenesis of M. tuberculosis. Role of these

proteins is being investigated in the persistence of the pathogen in host.

- Structure-function analysis of ribonuclease P of M. tuberculosis. The objective of our

study is to understand the mechanism of function of the mycobacterial RNase P. The role

of unique determinants of the mycobacterial enzyme is being investigated in its functional

mechanism.

Work reported in 2013-2014

Construction and evaluation of recombinant toxins as potential therapeutics

Saporin and restrictocin both were shown to inhibit HIV propagation in two model cell lines.

Further, it was demonstrated that for the anti-HIV activity of saporin its DNA fragmentation

activity is important. Restrictocin was shown to manifest its anti-HIV activity through its

specific RNase activity.

Earlier, we had shown that saporin induces apoptosis in mammalian cells through the

intrinsic pathway. Our further studies established that saporin induces loss of mitochondrial

membrane potential accompanied by reactive oxygen species generation. SAP/JNK, ERK and

p38 were phosphorylated in cells treated with saporin. The levels of p21WAF/CIP1, a cell

cycle protein involved in cell growth inhibition, and ER stress regulating protein, Bip

decreased with time in saporin treated cells.

Investigation of the involvement of Clp proteases in pathogenic mechanism of M.

tuberculosis

In M. tuberculosis genome, the upstream region of groES and hrcA genes contains the

probable CIRCE DNA binding site for the transcriptional repressor, HrcA. The recombinant

HrcA of M. smegmatis and M. tuberculosis, purified by denaturation and renaturation from E.

coli inclusion bodies, was unable to bind to this DNA. However, the recombinant HrcA

bound to the target DNA in the presence of mycobacterial cell extracts devoid of HrcA

protein suggesting that other protein(s) are required for HrcA binding to DNA. Similarly, the

DNA binding properties of another transcriptional repressor, HspR of M. smegmatis and M.

tuberculosis were analysed using a DNA fragment from the HAIR motif, upstream of dnaK

gene. Both HspRs bound to the HAIR DNA, however they showed some non-specific

binding to other DNAs as well.

We have phenotypically characterized the effect of heat stress on knockout strains of hrcA

and clpB genes of M. tuberculosis CDC1551. The growth of the clpB knockout was

significantly reduced. Our preliminary observations indicate that ClpB is required by the

pathogen for its survival under heat stress.

Structure-function analysis of ribonuclease P of M. tuberculosis

Eight variants of the M. tuberculosis RNase P protein component were generated that

contained the unique residues substituted to those present in E. coli and B. subtilis. The

holoenzymes reconstituted with F23A, A70K and R72A variants showed significantly

reduced activity; those with A77F and D124S had partial activity; whereas V27F, R72L and

R93A mutants showed non-specific activity on pre-tRNAAla.

40

Progress of work during the current reporting year (2014-2015)

Investigation of molecular mechanism of biological actions of human ribonucleases and

their role in host defense

Mechanism of ECP mediated cytotoxicity

Recombinant ECP showed cytotoxic activity in Jurkat cells with an ID50 of 1.0-1.5 µM. ECP

treatment of Jurkat cells was found to stimulate the phosphorylation of p38-MAPK and

ERK1/2. Further, ECP and ECP-H15D/H128D, a mutant of ECP lacking ribonuclease

activity, induced the activation of caspase 3, caspase 8 and PARP but not caspase 9,

indicating that for the apoptotic activity of ECP its RNase activity is not required. The

involvement and identification of cellular signaling pathways in ECP mediated cytotoxicity is

being confirmed using pathway specific inhibitor.

Investigation of the involvement of Clp proteases in pathogenic mechanism of M.

tuberculosis

Stress regulation and persistence mechanisms in Mycobacteria

We have further characterized the M. tuberculosis transcriptional repressor, HspR (MtHspR)

for its biochemical properties and also investigated the mechanism by which it may function

under heat stress. MtHspR was found to exist as a mixture of dimeric and monomeric protein.

We have demonstrated that HspR of M. tuberculosis independently binds to the HAIR DNA

element, present upstream of dnaK and clpB genes. The Tm of MtHspR was calculated to be

66°C. The HAIR binding activity of HspR was intact upto 60°C indicating that MtHspR on

its own is not the sensor of heat stress. We studied the heat sensitivity of MtHspR-HAIR

binding in the presence of M. tuberculosis and E. coli cell extracts and found that proteins

from the cell extract make the MtHspR-DNA interaction heat responsive. This suggests that

negative regulation of heat shock protein expression by HspR requires some other protein(s)

from the cytosol. We found DnaK to bind the M. tuberculosis HspR-HAIR complex. Further

detailed analysis of M. tuberculosis DnaK-HspR-HAIR interaction suggested that the DnaK

specifically binds to the HspR-HAIR complex and that the monomeric form of DnaK is most

favored state. The study also indicates that M. tuberculosis DnaK-HspR-HAIR binding is

affected by heat stress, especially so in the presence of the substrate protein, α-casein, which

supports the feedback loop mechanism, however, this does not lead to the destabilization of

MtHspR-HAIR complex. The HspR-HAIR binding in the presence of increasing amounts of

cell extracts led to appearance of two heat sensitive complexes of different mobilities. Mass

spectrometric analysis of these two complexes indicated the presence of tryptophanase and

phosphoglycerate kinase (PGK) in these two complexes respectively.

The biochemical properties of HspR of M. tuberculosis and M. smegmatis were also to

investigate if a unique 10 residue insertion at the C-terminus of MtHspR is involved in DNA

binding. However, ther was no difference in the DNA binding and heat sensing properties of

the two proteins indicating that this stretch of 10 amino acids is not involved in the binding

activity of HspR to HAIR element.

41

Structure-function analysis of Clp proteins of M. tuberculosis

We cloned and expressed M. smegmatis Clp proteases, ClpP1 and ClpP2 in M. smegmatis.

The proteins were purified to homogeneity. These proteins were functionally characterized

for their peptidase activity using Z-GGL-AMC as the substrate with two different activators

namely, Z-Leu-leu and Z-leu-leucinal. Though we found the proteins to be functionally

active, the activator didn‟t seem to play a significant role in the reaction, contrary to what has

been reported with M. tb ClpP1 and P2 proteins. We are currently reconstituting the M. tb

protease for a comparative analysis.

To understand the mechanism of ClpB chaperone mediated protein disassembly in M.

tuberculosis, we cloned and expressed, in E. coli, the proteins of M. tb ClpB and DnaKJE

chaperone complex namely, ClpB, ClpBΔN, DnaK, DnaJ1 and GrpE. These proteins were

purified to homogeneity. Tms of both ClpB and ClpBΔN proteins were found to be around

60°C, suggesting that both the proteins are stable under heat stress. Both, ClpB and ClpBΔN

showed a mixed population of hexameric and monomeric proteins. ClpB and ClpBΔN were

found to have inherent ATPase activity. The ATPase activity of ClpB and ClpBΔN proteins

was assayed in the presence of substrates α- and κ- casein and poly-L lysine. We observed

that while in the case of ClpB there was around 2-fold increase in ATPase activity in the

presence of casein, there was not a very significant increase with polylysine. In case of

ClpBΔN, there was not much increase in its ATPase activity in the presence of κ- casein but

significant increase in the presence of α- casein and polylysine. ClpB and ClpBΔN are,

therefore, differently modulated by their different substrates.

Molecular chaperones hold the protein substrates under stress conditions and prevent them

from unfolding or aggregating. We tested the prevention of aggregation ability of ClpB and

ClpBΔN. ClpB showed a high prevention of aggregation activity and 0.5 µM was able to

prevent upto 80% aggregation of luciferase as a result of heat treatment. ClpB had

comparable activity even in the absence of ATP. ATP analogues showed even better activity,

suggesting that while the ATPase activity of the protein does not play a part in prevention of

aggregation, the oligomeric state of the protein is crucial. ClpBΔN showed significantly less

prevention of aggregation activity as compared to that of ClpB. Also, unlike ClpB full length

protein, ClpBΔN showed no prevention of aggregation in the absence of ATP.

Structure-function analysis of ribonuclease P of M. tuberculosis

Bacterial RNase P, including that of M. tuberculosis, is a ribonucleoprotein containing a

catalytically active RNA subunit and a protein subunit. The protein subunits of bacterial

RNase P are catalytically inactive. In an attempt to reconstitute the M. tuberculosis RNase P

in vitro, we expressed the protein subunit in E. coli and purified it from the inclusion bodies.

The purification was achieved using two different strategies. In the first strategy, the RNase P

protein was denatured, renatured in vitro and further purified by cation exchange

chromatography after renaturation. Whereas, in the second strategy, the protein was purified

by cation exchange chromatography under denaturing conditions in 8M urea and

subsequently renatured in vitro. The two proteins differed remarkably in their properties. The

protein that was purified after renaturation showed a significant and specific inherent RNase

P activity in low salt, which was inhibited in the presence of high salt. Whereas, the protein

purified under denaturing conditions and renatured subsequently did not contain any inherent

RNase P activity. The presence of catalytically active RNA subunit, as a contaminant, in both

the preparations was ruled by various assays. It appears, that the M. tuberculosis RNase P

42

protein subunit is capable of attaining a conformation in vitro that is catalytically competent.

Further, validation of these observations is underway.

The various mutants of RNase P, generated earlier were purified under denaturing conditions

were further characterized for catalysis. The study shows that in M. tuberculosis RNase P

protein subunit residues V27, A70 and R72 are involved in substrate recognition; H67, F23,

A77 and R93 are involved in providing the RNA subunit an active conformation in the

holozyme. Attempts are on to generate a conditional RNase P protein knockout strain of M.

tuberculsos to investigate the role of protein subunit in RNase P catalysis in vivo.

Future plans

The mechanism of transcription repression of heat shock genes in M. tuberculosis will be

further delineated. Interaction of the HspR and HrcA repressors with the respective DNA

targets will be investigated by generating mutants of the two repressor proteins. Our

preliminary studies have indicated involvement of other proteins in the repression

mechanisms of Hspr and hrcA. These interacting partners will be identified and the overall

mechanism of transcription repression will be investigated using in vitro model assays.

Hsp60 and Hsp70 operons are respectively regulated by hrcA and hspR. The regulation

mechanism and interaction proteins of Hsp60 and Hsp70 complex with hrcA and hspR

respectively will be investigated. Using the wild type, and hspR and hrcA knockout strains of

M. tuberculosis, the role of these repressors in various stress conditions will be investigated

on the growth and infectivity of the pathogen. The observations will be validated by

complementation, by over expressing the knocked out targets in respective mutant strains.

The complementation studies will also be conducted with the clones of HspR and HrcA

protein mutants.

By structure function analysis interaction between ClpC, ClpX and ClpP and their substrates

will be investigated to understand the functioning of these proteins in M. tuberculosis.

Mechanism of protein aggregation, disaggregation and cleavage involving caseinolytic

proteases in M. tuberculosis will be investigated. Using a ClpB knockout strain of M.

tuberculosis, role of ClpB in stress tolerance and infectivity will be investigated.

The known inhibitors of ClpP proteolytic activity will be investigated for their mechanism of

action. Using studies with the known inhibitors as the basis, novel inhibitors specific for M.

tuberculosis Clp family will be identified. It is proposed to identify novel inhibitor scaffolds

by in silico docking studies. Further, a library of small molecules will be screened to identify

inhibitors specifically interfering with the functioning of caseinolytic proteases.

The recombinant anti-HIV and anti-cancer proteins developed will be further analysed in

different model systems for their efficacy. The anti-HIV protein molecules developed during

the study have an issue of toxicity at higher dosage. These molecules will be further

engineered by rational mutagenesis to reduce toxicity without compromising on the anti-viral

activity. The anti-HIV activity of human RNase, eosinophil derived neurotoxin will be

investigated in model cell systems.

We have identified several toxic proteins of human origin namely, cytochrome C, DP5 and

SMAC which are involved in induction of apoptosis and necrosis. It is proposed to explore

some these apoptosis inducing human proteins in the construction of chimeric toxins targeted

43

against cancer. The developed molecules will be characterized for their efficacy in vitro in

cell line models and in vivo in mouse models.

The role of mycobacterial RNase P in pathogenesis of M. tuberculosis will be investigated by

inhibiting its expression in vivo. Structure-function analysis of RNase P will be continued.

Intracellular targets of eosinophil proteins will be validated. The role of identified targets will

be investigated in the activity of eosinophil proteins.

Action taken on the RAP/SAC 2014 recommendations

Clarifications sought, during the laboratory visit were provided to the satisfaction of the

RAP/SAC members.

Publications

Original peer-reviewed articles

1. Yadav SK, Batra JK (2015) Ribotoxin restrictocin manifests anti-HIV-1 activity through

its specific ribonuclease activity. Int J Biol Macromol doi: 10.1016/j.ijbiomac.

2015.01.062.

2. Haque MA, Shah U, Zaidi S, Hassan MI, Islam A, Batra JK, Ahmad F (2015)

Characterization of pre-molten globule state of yeast iso-1-cytochrome c and its deletants

at pH 6.0 and 25°C. Int J Biol Macromol 72: 1406-1418.

3. Haque MA, Zaidi S, Shah U, Prakash A, Hassan MI, Islam A, Batra JK, Ahmad F (2014)

In vitro and in silico studies of urea-induced denaturation of yeast iso-1-cytochrome c and

its deletants at pH 6.0 and 25°C. J Biomol Struct Dyn 23: 1-10.

4. *Shah U, Haque MA, Zaidia S, Hassan MI, Islam A, Batra JK, Singh TP, Ahmad F

(2014) Effect of sequential deletion of extra N-terminal residues on the structure and

stability of yeast iso-1-cytochrome-c. J Biomol Struct Dyn 32: 2005-2016.

Review/Proceedings

1. *Chopra A, Batra JK (2014) Antimicrobial activity of human eosinophil granule proteins.

Meth Mol Biol 1178: 267-281.

*in press last year, since published.

44

Molecular modelling of proteins and protein-ligand complexes using

knowledge-based approaches and all atom simulations

Principal Investigator Debasisa Mohanty

Project Associate A Madhumalar (DBT Woman Scientist)

Ph. D. Students Deepak

Chhaya Dhiman

Neetu Sain

Mansi Grover

Priyesh Agrawal

Collaborators RS Gokhale, NII/IGIB

S Sengupta, NII

P Sharma, NII

V Nandicoori, NII

Theme of research

The main theme of the research projects is to understand the structural principles that govern

folding of peptides/proteins to stable conformations and binding of various ligands to

proteins, and use these principles for developing novel computational approaches for

prediction of the structures of peptides/proteins and specificities of protein-ligand complexes.

Theses prediction approaches for structure and substrate specificity are being used to analyze

various genomes for identifying novel biosynthetic pathways, protein-protein interaction

networks and regulatory networks.

Objectives

The specific objective of the various projects are to investigate, whether the combination of

knowledge-based and ab initio approaches can be used for predicting the (1) substrate

specificity of enzymes involved in biosynthesis of secondary metabolites and novel post-

translational modifications (2) substrate specificity of various peptide recognition modules

(PRMs) like MHCs, kinases, PTB, PDZ and WW domains etc, (3) analysis of microRNA-

protein interaction networks.

Work reported in 2013-2014

A. Analysis of enzymes associated with biosynthesis of secondary metabolites and

lanthipeptides

Retro-biosynthetic enumeration of enzymatic reactions

The retro-biosynthetic approach of identifying genes associated with known metabolites

involves enumeration of various chemical transformations or enzymatic reactions which

would generate a given chemical moiety and identifying the enzymes that can catalyze the

given biochemical transformations. Corresponding to each generic reaction functional group

45

information of product were stored in a database as SMARTS. Then using Reactor module of

ChemAxon corresponding generic reaction is used to transform a given chemical moiety into

its precursor molecule and this process is continued till no other functional group is detected

in the compound.

Threading analysis for class II lanthioninesynthetase

Threading analysis of LanM indicated the possible structural similarity with PI3-kinase.

However, residues known to be important for elimination of phosphate were also found in the

sequence stretch aligning with kinase domain. It is possible that the kinase like domain is

catalyzing both phosphorylation and eliminylation by conformational rearrangements in the

loop regions.

B. Analysis of interaction networks involving kinases and other PRMs

Structure based analysis of disease associated nsSNPs on kinases

We have explored the role of disease associated nsSNPs in inducing conformational

transitions from inactive to active state by analyzing trajectories obtained from MD

simulations on CDK2. In fact, we have proposed structural roles for various disease

associated mutations based on the MD studies and classified them based on their roles in

maintaining structural integrity, catalytic function, interaction with other downstream

macromolecules and regulation of catalysis.

Analysis of human PDZ domains

The structure-based multi-scale approach developed earlier for predicting binding partners of

PDZ domains has been benchmarked on human PDZ using phage display data. Apart from

pair potential based prediction of PDZ binding peptides, we have also performed MD

simulation on complexes involving internal and C-terminus peptides for Par6 PDZ domain to

investigate the subtle conformational changes which are required for recognition of internal

peptides by PDZ domains.

C. Structure and dynamics of microRNA-protein complexes

MD simulations on AGO-miRNA-mRNA ternary complex revealed readjustments in the

miRNA-mRNA interactions, involving bulging out of one nucleotide (U5) at the miRNA side

and formation of a non watson-crick G:A base-pairing. Interestingly, in simulations on the

miRNA-mRNA duplex in absence of the AGO protein, no such readjustments involving non-

canonical interactions were observed. Therefore, our simulations highlighted the role of AGO

in determining specificity of miRNA target recognition involving non-canonical interactions.

46

Progress of work during the current reporting year (2014-2015)

A. Analysis of enzymes associated with novel post-translational modifications and

biosynthesis of lanthipeptides

Deciphering molecular basis of functional divergence in AMPylating enzymes

Fic domain was recently shown to catalyze AMPylation - transfer of AMP from ATP to

hydroxyl side chain of diverse eukaryotic proteins, ranging from RhoGTPases to chaperon

BiP. We have carried out a series of explicit solvent molecular dynamics (MD) simulations

upto 1 μs duration on apo, holo and substrate/product bound IbpA Fic domain (IbpAFic2).

Simulations on holo-IbpAFic2 revealed that binding of Mg+2

to α and β phosphates is crucial

for preserving catalytically important contacts involving ATP. Comparative analysis of the

MD trajectories revealed how binding of ATP allosterically induces conformational changes

in the distal switch II binding region of Fic domains thereby aiding in substrate recognition.

Our simulations have also identified crucial aromatic-aromatic interactions which stabilize

the orientation of the catalytic histidine for inline nucleophilic attack during AMPylation,

thus providing structural basis for evolutionary conservation of these aromatic residue pairs

in Fic domains. Based on analysis of interacting interface residue pairs which persist over the

microsecond trajectory, we identified a tetrapeptide stretch involved in substrate recognition.

Structure based genome-wide search revealed distinct conservation pattern for this stretch in

different Fic subfamilies, further supporting its proposed role in substrate recognition. In

additions, combined use of simulations and phylogenetic analysis has helped in discovery of

a new subfamily of Fic proteins which harbor a conserved Lys/Arg in place of inhibitory Glu

of the regulatory helix. We propose the novel possibility of autoenhancement of AMPylation

activity in this new subfamily via movement of regulatory helix, in contrast to autoinhibition

seen in most Fic proteins.

In silico analysis of lanthipeptides and prediction of their cyclization patterns

The precursor polypeptides of lanthipeptides are ribosomally synthesized as a polypeptide

chain having a C-terminus rich in Ser, Thr and Cys residues. The lanthionine synthase

enzymes encoded in neighboring genes post-translationally modify the Ser/Thr residues to

form Dha/Dhb. Lantibiotic Cyclases catalyze cyclization of lanthipeptides by formation of

thioether bond between modified amino acids Dha/Dhb and Cys. Since, a large number of

lanthipeptide synthase gene clusters have been identified in various bacterial genomes,

predicting the cyclization pattern of lanthipeptides is a challenging task in genome mining for

novel lanthipeptides. In order to develop a sequence based method for prediction of

cyclization pattern, a dataset of class I and class II lanthipeptides with known cyclization

pattern were compiled. Out of the 24 lanthipeptides in class I and 36 lanthipeptides in class

II, 15 and 16 lanthipeptides from class I and class II respectively were selected as training set

and remaining lanthipeptides constituted the test set. For each lanthipeptide in the training

set, the corresponding leader peptide sequence was scanned for sequence strings or sub-

sequences of the type Ser/Thr-(X)n-Cys or Cys-(X)n-Ser/Thr to enumerate all theoretically

possible cyclization patterns. Out of these sequence strings, the strings corresponding to

thiother linked Ser/Thr-Cys or Cys-Ser/Thr pairs in the lanthipeptides were included in the

positive set, while all other strings were included in the negative set. After compiling the

positive and negative set sub-sequences for class I and class II separately, SVM (SVMlight

)

was trained for each class. Given the sequence of a leader peptide, the trained SVM model

could distinguish the thioether forming Ser/Thr-(X)n-Cys or Cys-(X)n-Ser/Thr sequence

47

strings from strings which do not cyclize. Benchmarking on the test set indicated that, for

class I the prediction accuracy was 90.2 % with precision and recall of 90.5% and 70.4%

respectively. Class II had accuracy of 80.7% with precision/recall of 66.0%/67.4%

respectively. However, when datasets of both the classes were merged, the combined SVM

had an accuracy of 77.6% with precision and recall values of 57.0% and 78.1% respectively.

These results indicate that the sequence based machine learning approach can predict the

cyclization patterns of lanthipeptides with reasonable accuracy.

B. Analysis of interaction networks involving kinases and other PRMs

Analysis of internal peptide recognition by PDZ domains

PDZ domains are peptide recognition modules (PRMs) that interact with the short peptides of

length 5-7 residues, which are often present at the C-terminus of interaction partners.

However, internal peptides can also be recognized by many PDZ domains. Deciphering

interaction partners for PDZ domains is a topic of considerable current interest, because of

involvement of these modular recognition domains in many human diseases like cystic

fibrosis and schizophrenia. In our earlier work, we have developed structure based methods

for predicting interaction partners of PDZ domains which recognize C-terminal peptides.

Crystallographic studies have revealed that, certain PDZ domains utilize two different

conformations to interact with C-terminal or internal peptides. We have attempted to develop

computational methods for predicting non-canonical binding partners of PDZ domains by

analyzing the mechanistic details of internal peptide recognition which is facilitated by the

plasticity of the PDZ domain. A series of 1 µs explicit solvent MD simulations have been

performed on cognate and non-cognate complexes of the Par6 PDZ domain for which crystal

structures were available in complex with internal as well as C-terminal peptide. Analysis of

these MD trajectories revealed that the non-cognate complexes generated by interchanging

the bound peptides in the crystal structures converged to the conformations in cognate

complexes during the 1 µs simulation. Thus our MD simulations were able to reproduce the

conformational transitions involving movement of the carboxylate binding loop which is

required for recognizing internal peptides. It was found that in absence of bound peptide

carboxylate binding loop of Par6 PDZ domain adopts a closed conformation and internal

peptide recognition follows induced-fit mechanism. Interestingly MM-PBSA analysis

performed on the MD trajectories is also in qualitative agreement with the experimentally

determined dissociation constant values of cognate and non-cognate complexes. These results

suggest that it would be possible to predict non-canonical binding partners of PDZ domains

by MM-PB/SA analysis.

C. Analysis of microRNA-protein interaction network

Analysis of the role of TF-miRNA network in miRNA mediated gene regulation

Apart from identifying targets of miRNAs, predicting the level of repression of a target

mRNA by a given miRNA has been a challenging question Since gene regulation involving

miRNA, mRNA and TF functions as a highly coupled network, prediction of regulatory

activity of miRNA requires a network based analysis involving miRNA, mRNA and TFs.

Such network based analysis can also help in understanding how loss of key regulatory links

in these networks is associated with diseases like cancer. In order to demonstrate the utility

of this network based approach, we have selected the HIPPO signaling pathway as an

example. The list of genes (mRNA) and miRNAs having direct involvement in the hippo

48

pathway were compiled based on literature survey. Using these mRNAs and miRNAs as

query searches were carried out in miRTarbase and TRANSFAC database to identify

miRNA:mRNA, TF:mRNA, TF:miRNA and miRNA:TF interactions. From these, 282

TF:miRNA co-regulatory pairs (i.e. a TF regulating a miRNA or vice-versa and both

regulating a common mRNA) comprising 196 TF:mRNA and 89 miRNALmRNA

interactions were identified. This information was provided as input to the cytoscape

software to generate a miRNA:TF:mRNA regulatory network containing 102 nodes and 563

edges. Based on the network analysis, out of the total of 60 core component and regulator

genes, we were able to highlight 7 genes that might be playing pivotal roles in signaling of

hippo pathway. In addition, ING4 and hsa-miR-193b-3p came across as important

regulators. Our analysis also suggested that, FPM315 is involved in regulating the all

important genes involved in inhibition of YAP/TAZ, thus highlighting its importance in

growth control/tumor suppression.

Future plans

Co-occurring domains and cellular context involving temporal and spatial expression of

interacting domain pairs play a significant role in protein-protein interaction. Hence, it is

necessary to develop network based methods which incorporate context dependent

information by utilizing interaction preference of modular domains obtained from high

throughput experimental data. Therefore, we plan to develop multi-scale methods for

identification of interaction partners of PRMs (PDZ, Kinase and SH2 domains) by combining

context dependent information with structural details. We also plan to initiate a project on

analysis of signaling and protein-protein interaction networks in P. falciparum and

identification of novel modulators/inhibitors of signaling and PPI networks, in collaboration

with experimental groups from NII and other institutes. We also propose to analyze disease-

associated nsSNPs/SNVs present on peptide recognition modules and their interaction

partners.

We propose to expand the scope of our structural bioinformatics methods for fold to function

correlation and algorithms for retro-biosynthetic enumeration of biochemical transformations

to develop computational methods which can help in identifying genes involved in novel

PTMs and biosynthesis of secondary metabolites. These computational methods will

specifically help in deciphering biosynthetic pathways of ribosomally synthesized and post-

translationally modified peptides (RiPPs) which constitute a major class of natural products.

Major objectives of the in silico analysis will be understanding the evolution of PTM

catalyzing enzymes, evolution of secondary metabolite biosynthetic networks and

identification of global and pathway specific regulators of secondary metabolites.

We propose to carry out sequence and structure based analysis of RNA-protein interactions

associated with biogenesis and target recognition of miRNA. Since miRNA biogenesis and

target recognition involves interactions of RNA with several important protein domains, a

systematic structure based analysis of proteins involved in miRNA biogenesis will help in

deciphering molecular details of biogenesis and target recognition. Structural modeling and in

silico analysis of miRNA-mRNA-Argonaute ternary complexes can facilitate better

understanding of the mechanism of target recognition by miRNA. Similarly structure based

analysis of Dicer inhibition by LIN28 would help in determining specificity of such

interactions that might assist in identifying other regulators of miRNA processing. Our

preliminary work on In silico analysis of miRNA-mRNA-TF gene regulatory networks using

HIPPO signaling pathway as a test case has helped in identifying key regulators. We plan to

49

develop softwares for automating various steps involved in such network based analysis by

integrating high throughput data.

Action taken on the RAP/SAC 2014 recommendations

Scientific and technical queries raised by the members during interaction with the group were

answered. No specific suggestions were given by RAP/SAC members.

Publications

Original peer-reviewed articles

1. Khater S,Mohanty D (2014) Genome-wide search for eliminylating domains reveals

novel function for BLES03-like proteins. Genome Biol Evol 6: 2017-33.

2. Damle NP, Mohanty D (2014) Mechanism of autophosphorylation of mycobacterial

PknB Explored by molecular dynamics simulations. Biochemistry 53: 4715-26.

3. Kumar N, Damle NP, Mohanty D (2015) Getting phosphorylated: Is it necessary to be

solvent accessible? Proc Ind Natl Sci Acad (in press).

50

Structure, interaction and design studies involving regulatory peptides and

proteins

Principal Investigator Dinakar M Salunke

Ph. D. Student Sharad Vashisht

Project Associate Ashish Kumar

Project Assistant Sonam Roy

Collaborator K Kaur

Theme of research

The structural aspects of molecular recognition and its applications in analyzing the

mechanisms associated with specific regulatory events and in rational molecular design.

Objectives

1. Understanding the protein architecture and the structural biology of various regulatory

events.

2. Analysis of the structural principles of immune recognition and molecular mimicry.

3. Rational molecular design studies based on the above.

Work reported in 2013-2014

Although immune system is shown to be highly specific, degenerate specificity in immune

recognition is often observed. We had worked on understanding degenerate specificity of

antibodies using a peptide (DVFYPYPYASGS) and a sugar (methyl α D mannopyranoside)

as model antigens. The structures of 2D10 in apo and the antigen-bound forms (with the

sugar as well as the peptide) showed that no conformational flexibility in CDRs of 2D10,

instead it was evident that the plasticity in the interaction had helped in the manifestation of

molecular mimicry. One interesting aspect of the study was that even if the potential for

flexibility existed, it was not utilized while recognizing both ligands. In order to address this

conundrum, we had begun looking for other sugars and peptides, which can binds to the

2D10 antibody with comparable affinities. Crystallographic analyses of 2D10 bound to five

different sugars were carried out at high resolution. Comparison of the structures has shown

that the antigen-combining site for sugars is constituted of CDR H3, L1 and L3 only. All the

five sugars have an overlapping primary binding site (equivalent to the methyl α D

mannopyranoside interacting region). This primary sugar-binding site has been shown to

accommodate same/similar as well as dissimilar sugars by utilizing plasticity in the

interacting residues available in the antigen-combining site. The second sugar of the similar

disaccharides (α1-3-Mannobiose, α1-6-Mannobiose) have been adjusted in the same direction

but with utilizing different sets of interacting residues of the antibody paratope. However, the

second sugar of dissimilar disaccharide (lactose in comparison to α1-3-Mannobiose, α1-6-

Mannobiose) exploits different paratope space altogether. The trisaccharide (α1-3, α1-6-

51

Mannotriose) was accommodated in the same site by differential positioning of the second

and third sugar rings in the antibody paratope (in comparison to all disaccharides) as well as

by utilizing conformational flexibility in the paratope region (mainly in CDR L1). This study

had demonstrated that an affinity-matured antibody could utilize at least three different

strategies in order to accommodate structurally similar/dissimilar sugars.

In another line of research, the structural proteomics of allergy seed proteome of eggplant

(Solenum melongena) was explored. A 45 kDa protein, SM80.1 showing weak homology

with other 7S vicilins, was further refined building almost entire model of the protein. The

overall crystal structure of SM80.1 indicates that it is a homotrimer consisting of 393 residues

in each monomer of which only residues 274-293 are structurally disordered. A monomer

subunit is composed of two similar domains further subdivided into a core and a loop sub

domain. Each domain consists of 2 elements, a compact eight-stranded beta barrel having the

“swiss roll” topology and an extended flexible fragment containing several short alpha

helices. Another protein, SM80.2, Solenum melongena was also purified from the defatted

seed powder by 80% ammonium sulphate fractionation. The purified protein corresponds to a

molecular weight of 11.7 kDa as analyzed by mass determination using mass spectrometry.

N-terminal sequencing was also done for the purified protein which identified 20 residues of

the polypeptide. The protein was crystallized and ab initio structure determination taken up.

Crystallographic studies involving another protein, SM80.2, from Solenum melongena has

been in progress.

Progress of the work during current reporting year (2014-15)

Extensive studies have been done in this laboratory to understand the mechanism(s)

underlying the recognition of chemically dissimilar ligands by a common immune receptor

clearly proves that breakdown in the antigenic discrimination potential and therefore

degeneracy is often encountered in the immune system. These degenerate antibodies might

have an edge over a fast mutating virus such as Influenza A virus.

To address this issue we started screening the Tomlinson I + J human single fold synthetic

naïve phage display single chain antibody fragment libraries against a highly mutated

continuous epitope of Influenza A virus i.e. WTGVTQN. In this library, DNA encoding

millions of variable heavy (VH) and variable light (VL) chains linked by flexible glycine-

serine linker products are cloned into a vector which is then engineered to express scFv fused

to pIII minor capsid protein of filamentous bacteriophage of E. coli. The peptide was

synthesized and conjugated to the proteins KLH and BSA by glutaraldehyde conjugation

method. Phages carrying target antibody were isolated by four cycles of selection against

KWTGVTQN-BSA and KWTGVTQN-KLH conjugates. Eluted phages were then allowed to

infect TG1 bacteria in order to isolate individual phagemid clones. These individual clones

were then screened for their ability to bind the peptide epitope by monoclonal phage ELISA

and competitive ELISA. Positive clones were further confirmed by PCR to check for the

presence of full length VH and VK. Degenerate binding specificities were analyzed against 8

different analogues of the peptide. Currently, few selected clones are being subjected to

expression in soluble scFv form.

Observed cross-reactivity as seen in our work on both germline and affinity-matured

antibodies is in defiance to the conventional rules of specificity. Crystallographic studies on

antibody diversity have given newer insights into the mechanism of repertoire amplification

52

at the germline as well as at the matured stage offering interesting physiological perspectives

bringing out intriguingly new aspects of antigen recognition in humoral antibody response.

These studies provide interesting insights into physiological processes with out contradicting

any rules of structural biology, the reason for the existence of dichotomy in specificity and

degeneracy in the humoral response need to be addressed. With every new study, completely

novel modes of binding and unexpected features continue to emerge from antigen-antibody

complexes. Consolidation of these data in a coherent manner would help draw critical

inferences that complete the picture and possibly provide answers for the observed shift in the

paradigm in antigen specificity.

Germline gene encoded antibodies are expected to bind to the cognate antigen with high

affinity and specificity after affinity maturation. Yet, no perfect apposition has been observed

in the established paradigm and the vast amount of emerging data. It is anticipated that a

coherent analysis of global data would further shed light on understanding the nature of

antigen recognition. In order to dissect the apparent behaviour of the antibodies sharing

common ancestor while exercising their recognition potential, we have compiled structural

data on antibody-antigen complexes. Nearly 3500 antibody structures have been determined

of which more than 650 structures delineate the full extent of antibody-antigen interactions.

The final unique dataset is a compilation of 224 structures of which 148 mature antibodies

are of mouse origin encoded by 38 different germline genes and 76 mature antibodies are of

human origin encoded by 14 different germline genes. These mature antibodies that have

arisen from a common germline gene, bind to antigens that are chemically distinct. Plasticity

of combining site is possibly designed to disregard antigenic variations. Such an observation

in mature antibodies is rather enigmatic and hence needs to be addressed holistically. It is

therefore envisaged that the corresponding germline antibody should apparently recognize all

of those epitopes bound by the mature antibodies. Understanding structural correlation of

antigen recognition among mature antibodies is anticipated to provide information on how

antigen modulates antibody evolution during affinity maturation.

Continuing the studies on structural biology of plant seed proteomics, structure of SM80.2,

preliminary crystallographic studies of which were reported last year, was completed.

SM80.2 shows homology with members of prolamins family that includes non-specific lipid

transfer proteins and 2S albumin family proteins. The structure was deterrmined ab initio and

refined at a resolution of 1.87Å. The final refinement cycle at present gave an overall Rcryst

25% and Rfree of 28%. Ramachandran plot shows 174 residues (98.31%) in allowed region

and 2 (1.13%) in preferred region. Only one residue is present as outlier.

The overall structure of SM80.2 consists of 177 residues with continuous density. It consists

of two monomers in the asymmetric unit. This high resolution data helped in extracting the

complete amino acid sequence of SM80.2. Sequence analysis of SM80.2 showed 80 %

sequence similarity with non-specific lipid-transfer protein AP10-like from Solanum

lycopersicum. A peculiar unexplained continuous density was also found while refinement.

As this protein is a non specific lipid transfer protein and thus is involved in transferring of

lipids, we expect this continuous unexplained density may correspond to lipid moiety bound

to protein. Also this lipid binding could be linked to allergenic activity shown by non specific

lipid transfer protein. Identification of the lipid in the observed electron density is being

undertaken.

Future plans

Bioinformatics and crystallographic analyses of antigen-antibody binding as well as broader

aspects of host-pathogen interactions will be continued towards addressing dichotomy of

53

specificity and degeneracy of antigen recognition. Structural proteomics of plant seed

allergens will also be continued towards crystallographic analysis and structure-function

correlation.

Publications

Original peer-reviewed articles

1. Jain A, Salunke DM (2015) Purification, identification and preliminary crystallographic

studies of an allergenic protein from Solanum melongena. Acta Cryst F Struct Biol

Commun 71:221-225.

2. Khan T, Salunke DM (2014) Adjustable locks and flexible keys: plasticity of epitope-

paratope interactions in germline antibodies. J Immunol 192:5398-5405.

3. *Gill J, Jayaswal P, Salunke DM (2014) Antigen exposure leads to rigidification of

germline antibody combining site. J Bioinform Comput Biol 12:1450006.

*in press last year, since published

54

Reconstructing the chemico-cellular trestle to decipher biology of

tuberculosis and vitiligo

Principal Investigator Rajesh S Gokhale

Ph. D. Student Parul Ganju

Collaborators C Gadgil, CSIR-NCL, Pune

R Rani, NII

K Natarajan, JNU, Delhi

Theme of research

Our group is interested to elucidate mechanistic and spatiotemporal coherence of cellular

processes that result in two distinct pathological diseases – Tuberculosis (TB) and Vitiligo.

Although unrelated, both disorders involve decisive role of unusual metabolites- complex

lipids with very-long branched acyl chains produced by the TB pathogen Mycobacterium

tuberculosis (Mtb) and heteropolymeric structurally uncharacterized melanins produced by

melanocytes. Both these diseases are also characterized by unpredictable disease progression

profiles. TB manifests in active, latent, reactivated and dissemination phases. Vitiligo is a

chronic unstable depigmenting disorder that often shows symmetry in its manifestation.

While 1/6th of the Mtb genome encodes genes involved in lipid metabolism; the only well-

characterized function of melanocytes is to produce melanins. Our endeavor is to understand

how small molecule metabolic networks are elaborately tuned in nature and how these

pathways provide distinct advantages to the specific biological system and finally their

underlying implications in disease outcome.

Objectives

To summarize, the objectives of the studies proposed are:

i) Delineating networks and pathways underlying biosynthesis or degradation/recycling of

lipidic metaboilites in mycobacteria

ii) Identify factors involved in melanogenesis and decode spatiotemporal coherence

associated with melanocyte-keratinocyte biology

Work reported in 2013-2014

We reported role of IFN-γ signaling in maintaining skin pigmentation homeostasis through

regulation of melanosome maturation. Based on cellular, molecular and pathophysiological

studies, we proposed that strength, durability and temporal response of the IFN-γ response

could be a crucial factor for maintaining epidermal pigmentation homeostasis.

55

Progress of work during the current reporting year (2014-2015)

TNF-α and hypoxia crosstalk dictate Mtb modulation of osteoclast biology

Bacterial pathogens can habituate diverse cell types by modulating the host machinery

response. Mycobacterium tuberculosis (Mtb), being an obligate intercellular pathogen, is

primarily known to survive in macrophages. While Mtb pathogenesis is classically related

with lungs, there are increasing number of reports of mycobacterial infections of other

tissues. Presently, it is not clear how Mtb adapts to these unconventional niches. Previously

we reported that Mtb could survive in the osteoclast cells. We have now delineated

mechanism by which Mtb infection of macrophage precursor cells potentiates formation of

giant functional osteoclasts. Our studies show that Mtb downregulates the canonical RANK

signaling after the early lineage commitment and instead TNF-α levels along with hypoxic

response are the dominant activators of osteoclastogenesis. Interestingly, the DosR mutant of

Mtb, which is known to show compromised phenotype in hypoxic conditions, display

delayed osteoclast maturation kinetics. Our study reveals another fascinating example by

which Mtb exploits host-derived factors in order to establish chronic infection through

subversion of the protective host responses.

Elucidation of Cis-Trans Isomerases from Mtb genome

Fatty acids and lipids are often considered to be the major carbon source for Mtb growth

during dormancy conditions. Fatty acids are classically degraded to acetate units through -

oxidation enzymes. We earlier reported that classical FadA and FadB enzymes of Mtb could

not utilize oleic acid as carbon source. We show that this inability is due to the absence of

cis-trans isomerase function in the FadB protein. Since crotonase fold is known to catalyze

this function, we investigated in to a family of such proteins, annotated as Ech proteins in

Mtb genome. Based on genetic complementation as well as biochemical enzymatic assays,

we have identified several isomerases with varying catalytic activity and substrate specificity.

Our study delineates a novel family of enzymes that are crucial for Mtb utilization of

unsaturated fatty acids.

Future Plans

Our studies reveal a novel mechanism by which Mtb modulates osteoclast biology. It is

tempting to speculate that Mtb infection of extra-pulmonary bone and spine niches may be

outcome of migration of infected macrophage precursors through neighboring lymph nodes.

Interestingly, lymph nodes that drain into spine have bidirectional flow as opposed to others

organs and thus may account for substantially higher incidences of spine TB. The

synchronous adaptations of both host and pathogen may in future provide insights into

dynamics associated with survival and virulence.

Action taken on the RAP/SAC 2014 recommendations

No specific recommendation was made.

56

Publications

Original peer-reviewed articles

1. Haque AS, Patel KD, Deshmukh MV, Chhabra A, Gokhale RS, Sankaranarayanan R

(2014) Delineating the reaction mechanism of reductase domains of Nonribosomal

Peptide Synthetases from mycobacteria. J Struct Biol 187:207-14.

2. Shukla J, Gupta R, Thakur KG, Gokhale R, Gopal B (2014) Structural basis for the redox

sensitivity of the Mycobacterium tuberculosis SigK-RskA σ-anti-σ complex. Acta Cryst

D Biol Cryst 70: 1026-36.

Reviews / Proceedings

1. Natarajan VT, Ganju P, Ramakumar A, Grover R, Gokhale RS (2014) Multifaceted

pathways protect human skin from UV radiation. Nat Chem Biol 10: 542-51.

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Studies on immune response from antigen loaded biodegradable polymer

particles and protein refolding from inclusion bodies

Principal Investigator Amulya K Panda

Ph. D. Students Vaibhav Upadhyay

Jairam Meena

Divya Jha

Robin Kumar

Akansha Singh

Research Assistants Sudeepa Srichandan

Anupam Singh

Collaborators LC Garg, NII

PK Upadhyay, NII

A Qadri, NII

D Sehgal, NII

SK Gupta, NII

SP Vyas, DHG Vishwavidyalaya, Sagar

MZ Iqbal, Jamia Hamdard, Delhi

BP Panda, Jamia Hamdard, Delhi

KC Gupta, IITR, Lucknow

RC Kuhad, University of Delhi

GP Talwar, TRF, Delhi

S Choudhary, I CARE Eye Hospital, Noida

Theme of research

The theme of the project is to evaluate polymeric particle based delivery system for improved

immunogenicity of different antigens such as Tetanus Toxoid (TT), Hepatitis B surface

antigen (HBsAg), viral and carbohydrate (Vi polysaccharide and S. pneumoniae

polysaccharides) based vaccines. Another major research activity of the laboratory is the

analysis of inclusion body formation and development of mild solubilization processes for

improved recovery of bioactive proteins.

Objectives

The main objective of the project is to improve the immunogenicity of antigens entrapped in

biodegradable polymer particles. High-throughput refolding of inclusion body proteins into

bioactive form is another objective of the research group. Researches in the following areas

are conducted in the laboratory to achieve the objectives:

1. Analysis of immune response from antigen loaded polymer particles and evaluation of

adjuvant properties associated with polymeric particle formulation. Evaluation of memory

antibody response from polymer particle based immunization.

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2. Development of polymeric membrane as a scaffold for three dimensional growths of

animal cells and its application as an artificial skin equivalent.

3. Solubilization and refolding of inclusion body proteins from Escherichia coli. This

involves analysis of inclusion body formation during protein expression and understanding of

protein aggregation with an aim to recover higher amount of bioactive protein.

Work reported in 2013-2014

Vi polysaccharide and ZP3 recombinant protein entrapped in polymer particle were

evaluated for the generation of both primary and memory antibody response from single

point immunization. It was reported that for carbohydrate antigens such as Vi

polysaccharides and Pneumococcal polysaccharides (PCP), entrapping them in nanoparticles

leads to antibody isotype switching and generation of memory antibody response from single

dose immunization. This indicated that delivering the carbohydrate intracellularly using

polymer nanoparticle could be an alternative to the conjugate vaccine generally used to elicit

memory antibody response from carbohydrate antigens. Extensive studies were carried out

using spray dried alum powder as an adjuvant. Alum adsorbed vaccines are known to be

unstable to dehydration and lose their immunogenicity when lyophilized. Antigens (DT, TT

and Lysozyme) mixed with alum were spray dried to make dry powder vaccine formulations.

It was observed that spray dried alum in powder form elicited similar primary and secondary

antibody response when used along with DT or TT. Spray drying process was optimized for

the formulation of polymer particle entrapping recombinant Pneumococcal surface protein

(PspA) as a candidate antigen. Spray dried microparticles entrapping PspA showed good

aerodynamic properties. Entrapping PspA in polylactide microparticles by spray drying

retained its immunogenicity and proved the potential for non-invasive vaccine delivery.

Spray drying process was optimized as an alternative to solvent evaporation method for

preparation of polymer particles for vaccine delivery.

Our focus on inclusion bodies (IBs) has been on two aspects: (1) to improve the recovery of

bioactive protein and (2) to understand the nature of aggregation during inclusion body

formation. Previously we have reported that different sized inclusion body aggregates are

formed during expression of recombinant protein in E. coli. A novel solubilization method

using n-propanol in presence of low concentration of urea was developed. N-propanol in

combination with 2 M urea was found to be sufficient for the efficient solubilization of hGH

inclusion bodies. There are no reports on the presence of enzyme activity in inclusion bodies

of oligomeric proteins. Inclusion bodies of Asparaginase are found to be of non-classical

types which display significant enzyme activity when solubilized. This observation strongly

suggests the presence of native-like quaternary structures of proteins in inclusion bodies.

Progress of work during the current reporting year (2014-2015)

A. Immune response from polymeric particles entrapping antigens

(i) Improve immunogenicity of carbohydrate antigens using polymer particles

Polymeric particles entrapping protein/carbohydrate antigens are being routinely used in the

laboratory to improve their immunogenicity. Different sized PLA particle entrapping Vi

polysaccharides were used for immunization to delineate the size dependency of antibody

59

response. It was observed that unlike protein antigens, Vi polysaccharides entrapped in

nanoparticles induces better antibody response than that observed with microparticles. As

nanoparticle entrapped Vi polysaccharide was eliciting memory antibody response and

promoting antibody isotype switching, it was of interest to compare the results with conjugate

vaccine. Vi polysaccharide was conjugated to flagellin or PspA and to both the protein

antigens using ADH linker. It was hypothesized that conjugation of polysaccharides with

homologous protein antigens (Vi polysaccharide and flagellin from same pathogenic bacteria)

could be more protective, as antibodies against both antigens can work synergistically as

compare to single antigen. The conjugates were purified and characterized. Immunogenicity

evaluations of the conjugates both in free and in nanoparticulate form are under way. The

results will provide information whether plain polysaccharide entrapped in nanoparticles are

comparable or better than that of conjugate vaccines in terms of generating antibody

response.

Nanoparticle and microparticle formulation are being evaluated using immunopotentiator

along with the antigen. Quil A was entrapped along with antigens (Ovalbumin or PCP1) in

polymer particles and used for immunization. It was observed used of imunopotentiator along

with antigen in particle formulations lead to enhance antibody response from single point

immunization. For ovalbumin, co-entrapment of Quil A along with ovalbumin in PLA

particle elicited antibody titers 5 times better than that achieved with plain PLA particles.

Detail immunization studies are going on with other antigens to evaluate the usefulness of co-

entrapping Quil A along with antigens in polymer particles.

(ii) Formulation and adjuvant effect of dry powder alum

Extensive studies are being carried out using spray dried alum powder as an adjuvant. Alum

particles were characterized in detail and compared with liquid alum preparation. In spite of

the fact that the antigen was released quickly from spray-dried alum particles, antibody

response in mice was found to be similar to that observed with traditional alum adsorbed

antigen. Cellular uptake studies and cytokine profile while using alum powder formulation

were studied. It was observed that spray dried alum is less inflammatory than alum gel.

Different alum particle formulations entrapping antigen are being compared to that of alum

gel for generation of antibody response. Alum particle along with TLR agonist such as MPL,

CpG are being formulated to see the synergistic adjuvant effect while delivering them using

polymer particles.

B. Formulation of antibiotics entrapping PLA particles and its fusion to form

membrane for wound healing.

Our study aims at developing a novel method of scaffold fabrication by using polymeric

particles. We have reported the fabrication of polymer membrane as a passive wound

dressing material. Attempts were made to develop antibiotic releasing polymer membrane so

that the dressing material can be used for infected wounds. Entrapment of water soluble

antibiotics such as gentamycin and neomycin in polymer particle were optimized and these

particles were fused to form membrane. The drug release profiles from the membrane were

evaluated and it was observed that active antibiotics could be released from these polymer

membranes for a period of 20-30 hours continuously. Preliminary investigations were carried

out both in vitro and in vivo to prove the suitability of the antibiotic releasing polymer

membrane for wound healing application.

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C. Solubilization and refolding of inclusion body proteins

(i) Mild solubilization of inclusion body protein

Mild solubilization of inclusion body protein without using high concentration of chaotropes

is being extensively used now a day for high throughput recovery of bio active proteins. This

is because native-like protein structure is preserved during mild solubilization and this helps

in improve recovery of bioactive protein. We have been continuously exploring the use of

organic solvent for solubilization and refolding of inclusion body proteins. It was observed

that inclusion body aggregates can be solubilized using 50 % trifluroethanol. Use of urea (3

M) helped in better solubilization of inclusion body aggregates. Human growth hormone

inclusion bodies were successfully solubilized using TFE along with 3 M urea. The proteins

were then refolded into native conformation. This provides a new way of inclusion body

solubilization and has also been tested for inclusion bodies of different proteins.

Inclusion bodies of Asparaginase were found to be of non-classical types which display

significant enzyme activity when solubilized. This observation strongly suggests the presence

of native-like quaternary structures in inclusion bodies. Currently we are exploring the

structural basis of enzyme activity in these IBs employing various biophysical and

biochemical techniques. We are also studying the effect of expression conditions on the

nature of inclusion body aggregates.

(ii) Amyloid aggregation of globular proteins in physiological conditions

Studies are being conducted in the laboratory for understanding amyloid aggregation of

globular protein. Ovalbumin was used as a model protein and it was observed that by heating

at 80 OC, the protein aggregates into amyloid fibrils. Further it was observed that in presence

of organic solvent such as 5% TFE or n-propanol, ovalbumin aggregates into amyloid fibrils.

The presence of organic solvents (TFE or Propanol) increased the rate of aggregate

formation. We are currently studying how amyloid aggregates are being formed at

physiological conditions.

Future plans

Conjugate vaccine comprising of Vi polysaccharides and flagellin, Vi conjugated to PsPa will

be entrapped in polymer particles and its immunogenicity will be evaluated. Antibody

response generated from conjugate will be compared with the market available conjugates.

Immunogenicity of conjugate vaccine entrapped in polymer particles will be evaluated. T

cell deficient mice will be used for immunization to understand the role of T cell on

generation of antibody response from nanoformulation. Molecular mechanism of improving

the immunogenicity of polymer particle entrapped antigen will be studied in detail. More

emphasis will be given to understand the structure of protein in inclusion bodies showing

enzymatic activities. Suitability of TFE as a inclusion body solubilization agent will be

explored in detail for other proteins. Mechanism of organic solvent based solubilization of

protein aggregates will be analyzed in details.

Action taken on RAP/SAC 2014 recommendations

No specific recommendation was received during RAP/SAC 2014 presentation.

61

Publications

Original peer-reviewed articles

1. Anish C, Upadhyay AK, Sehgal D, Panda AK (2014) Influences of process and

formulation parameters on powder flow properties and immunogenicity of spray dried

polymer particles entrapping recombinant pneumococcal surface protein A. Int J

Pharm 466: 198-210.

2. Rajmohan G, Admane P, Anish CK, Panda AK (2014) Fusion and self-assembly of

biodegradable polymer particles into scaffold like and membranelike structures at room

temperature for regenerative medicine. Mol Pharm 11: 2190-2202.

3. Kumar R, Majumdar DK, Panda AK and Pathak DP (2014) Eudragit coated

microparticulate delivery of bovine insulin for oral delivery. Int J Res Pharm Chem 4:

698-712.

4. Khuroo T, Verma D, Talegaonkar S, Padhi S, Panda AK, Iqbal Z (2014) Topotecan-

tamoxifen duple PLGA polymeric nanoparticles: Investigation of in vitro, in vivo and

cellular uptake potential. Int J Pharm 473: 384-394.

5. Upadhyay M, Srivastava B, Jain A, Kidwai M, Kumar S, Gomes J, Goswami DG, Panda

AK, Kuhad RC (2014) Production of ganedoric acid by Ganoderma lucidum RCKB-

2010 and its therapeutic potential. Ann Microbiol 64: 839-846.

6. Bhatnagar P, Patnaik S, Srivastava AK, Mudiam MKR, Shukla Y, Panda AK, Pant AB,

Kumar P, Gupta KC (2014) Anti-cancer acitivity of Bromelain nanoparticles by oral

administration. J Biomed Nanotech 10: 3558-3578.

7. Ahmad J, Mir SR, Hohli K, Chuttani K, Mishra AK, Panda, AK, Amin S (2014) Solid

nano emulsion preconcentrate for oral delivery of paclitaxel: Formulation design, bio

distribution and scintigraphy imaging. Biomed Res Int 2014: 984756.

8. Upadhyay AK, Singh A, Mukherjee KJ, Panda AK (2014) Refolding and purification of

recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric

protein. Front Microbiol 5: 486.

9. Nand Kripa N, Gupta JC, Panda AK, Jain SK (2015) Development of a recombinant

hCG-specific single chain immunotoxin cytotoxic to hCG expressing cancer cells.

Protein Expr and Purif 106: 10-17.

10. Shrestha A, Srichandan S, Minhas V, Panda AK, Gupta SK (2015) Canine zona

pellucida glycoprotein-3: up-scaled production, immunization strategy and its outcome

on fertility. Vaccine 33: 133-140.

11. Kapoor R, Harde H, Jain S, Panda AK, Panda BP (2015) Downstream processing,

formulation developments and antithromobotic evaluation of microbial nattokinases. J

Biomed Nanontech 11: 1213-1224.

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12. Sheoran S, Panda BP, Admane P, Panda AK, Wajid S (2015) Ultrasound-assisted

extraction of gymnemic acids from Gymnema sylvestre leaves and its effect on insulin

producing RINm-5F ß cell Lines. Phytochem Anal 26: 97-104.

Review/Proceedings

1. Singh A, Upadhyay, V, Panda AK (2015) Solubilization and refolding of inclusion body

proteins. Methods Mol Biol 1258: 283-291.

63

Analysis of antigen processing and presentation

Principal investigator Satyajit Rath

Project Associate Chaitali Banerjee

Project Fellows Shalini Tanwar

Bahadur Singh Gurjar

Monique Odette Kamtchueng

Ph. D. Students Jasneet Kaur Khalsa

Renu Balyan

Amanpreet Singh Chawla

Atika Dhar

Danish Umar

Collaborators V Bal, NII

A George, NII

SS Majumdar, NII

S Bhatnagar, THSTI, Faridabad

A Bagga, AIIMS, Delhi

B Ravindran, RMRC, Bhubaneswar

A Sahu, NCCS, Pune

JM Durdik, Univ Arkansas, Fayetteville, USA

Theme of research

The aim of the ongoing programmes in this group is to examine the generation and activation

of T, B and antigen-presenting myeloid cells using multiple interlinked experimental systems.

Objectives

A variety of experimental approaches are taken to address the theme issues. The approaches

in current use examine APCs and pathways involved in antigen presentation to MHC class I

and class II-restricted T cells, and analyse the consequences of intracellular signal

transduction modulation for both development and responses of B cells, T cells and

macrophages using genetic as well as pharmacological tools.

Work reported in 2013-2014

A. The role of ligand density in controlling outcome of CD8 T cell activation

CD8 T cells can be activated within 1-3 h of exposure to peptide-MHC class I (MHCI) ligand

on antigen-presenting cells (APCs). Even if these activated CD8 T cells are then separated

from the APCs, they undergo activation, proliferation and differentiation on their own. As

summarised in previous annual reports, we have been using CD8 T cells from mice

transgenic for T cell receptors (TCRs) specific for peptide-MHCI complexes to address the

regulation of the quantitative parameters of CD8 T cell responses. Our earlier data indicated

that heterogeneity in an apparently homogenous population is responsible for the dose-

64

response relationship in the CD8 T cell response as measured by transcriptional induction of

early activation genes.

Our subsequent data have now also indicated that more complex components of the

activation response by naïve CD8 T cells, namely, the proliferation programme of 4-6

successive cell divisions in ~60 h, can also be a binary decision made by individual cells.

However, when ligand density was titrated down exponentially, data indicated that individual

T cells were indeed able to identify and respond differentially to very low levels of ligand

density in the form of delayed entry into cell cycle, associated with slower degradation of the

cell cycle repressor protein p27kip1. We further found that, over this ligand density range,

some successfully activated CD8 T cells also showed loss of cell-surface TCR, while some

did not, a decision made downstream from that outcome of early activation. High ligand

density led to efficient TCR internalisation, while very low ligand density induced almost no

TCR internalization. Further, the decision to internalize TCRs determined the length of the

lag phase before cell cycle entry in responding CD8 T cells. A variant low-affinity ligand

induced CD8 T cell behaviour similar to that in response to low density of high-affinity

ligand. These results demonstrate that T cells make two serial decisions upon encounter with

cognate pMHC complexes, whether to be activated at all, and then whether to internalize

TCRs or not. These two relatively early decisions influence the number of precursor cells

recruited and their speed of cell cycle entry. These decisions appear insensitive to ligand

density and/or affinity in individual naïve CD8 T cells, but are instead determined by

heterogeneity in the response-capable T cell population. The genesis, selection, variation and

basis of this population heterogeneity are thus major new questions of relevance to

understanding T cell responses.

Progress of work during the current reporting year (2014-2015)

[note: Only those experimental systems in which notable progress has occurred are described

in detail here for lack of space. Progress in other systems will be discussed with RAP-SAC

members to provide a comprehensive overview.]

A. Genesis and consequences of metabolic distinctions between closely related

lymphocytic cell lineages

While B and T lymphocytes mediate very distinct functions, a number of parallels exist

between them with regard to lineage development and post-activation peripheral

differentiation. B and T cells use common molecular mechanisms for diversification of

antigen receptor genes via gene rearrangement for expression of a unique antigen receptor on

the surface, and undergo multiple rounds of proliferation both during lineage differentiation

as well as post-activation peripherally. These differentiation transitions requiring cells to

move in and out of proliferative programs are associated with modulations in energy

requirement and in handling stress. These metabolic modulations would be expected to occur

during differentiation transitions of lymphocytes and be crucial for their success. However,

little is known about stage-specific metabolic alterations in early stages of lymphocyte

lineage differentiation, although essential metabolic transitions have been identified during

post-activation differentiation for B and T cells. The known commonalities between the two

lymphocytic lineages lead also to the expectation that metabolic mechanisms modulated

during differentiation transitions would be similar between them. However, our recent

observations that mice hypomorphic for the constitutively and ubiquitously expressed

mitochondrial oxido-reductase, Apoptosis-inducing factor (Aif), show defects in T, but not B

65

cell lineage differentiation, indicate that that the two lineages may well use distinct metabolic

landscapes. We therefore hypothesize that B and T lymphocytic developmental transitions

would be accompanied and modulated by lineage-specific metabolic alterations. We intend to

investigate this possibility by characterizing comparative metabolic transitions associated

with early stages of mouse lymphocyte differentiation, by comparing developmentally-

equivalent stages of lymphocytic lineages, and testing the functional significance of these

metabolic states.

We have begun to approach this by characterising the major naive B and T cell populations in

peripheral lymphoid organs, which are expected to be in the same quiescent state. Yet, we

find, they show specific metabolic-functional differences. Thus, glucose uptake is higher in

naive CD4 and CD8 T cells than in naive B cells, while the baseline rate of protein synthesis

is higher in naive B cells than in naive T cells. Mitochondrial mass and cytochrome-c levels

are higher in naive B cells than in naive T cells. These differences are functionally

meaningful. Thus, naive B cells are more susceptible to cell death in response to inhibition of

protein synthesis, while naive T cells are more susceptible inhibitors of glycolysis. We

observe similar differences between immature bone marrow B cells and thymic SP cells,

indicating that they are imprinted during lineage differentiation in the primary lymphoid

organs. Further, we have also compared the pre-B and the pre-T stages of B and T lineage

cells undergoing light chain recombination, and find that these differences between glucose

uptake and susceptibility to glycolysis inhibition on the one hand and protein synthesis and

susceptibility to protein synthesis inhibition on the other, are detectable at that

developmentally comparable stage as well. Thus, it appears that metabolic landscapes are

differently wired even in closely analogous cell lineages. We intend to pursue the genesis and

consequences of these metabolic distinctions further.

B. Genetic basis of autoantibody response to anti-complement factor H (CFH) in renal

disease

Hemolytic uremic syndrome (HUS), characterized by microangiopathic hemolytic anemia

and renal failure, is a common cause of acute kidney injury in childhood. Typical HUS is

associated with diarrhoea, is likely caused by Shiga-like toxins and was thought to account

for most cases in children. However, non-diarrhoea-associated 'atypical' HUS (aHUS) is

becoming increasingly evident globally, has an unsatisfactory long-term course and outcome,

with risk of relapses and progressive kidney disease. Many if not most patients with aHUS

show genetic or acquired defects in the complement system that enhance endothelial damage

and favor development of disease. While genetic deficiencies in genes for various proteins

that regulate the complement cascade have been reported, 6-10% patients in HUS cohorts

from developed countries show autoantibodies to an important complement regulator,

complement factor H (CFH). Preliminary studies in Indian children at AIIMS New Delhi

have suggested that the frequency of anti-CFH autoantibodies in Indian children suffering

from aHUS is almost 6 times high. Further, the occurrence of these antibodies in studies

elsewhere is very strongly associated with mutations in genes encoding CFH-related (CFHR)

proteins, specifically, CFHR1 and CFHR3. However, whether this is the case in India has not

been clear. Also, the relationship of CFHR1/3 deletion and the development of anti-CFH

autoantibodies is an immunological enigma as yet.

We have therefore participated in a study on a nationwide scale being anchored at AIIMS

New Delhi for the presence of anti-CFH autoantibodies in Indian children with aHUS and

their relation to disease severity and course. We have begun by examining the genetic

66

association of the development of these autoantibodies with the deletion of complement

factor H-related genes CFHR1 and CFHR3 patients with aHUS, and comparing the frequency

of that deletion in normal healthy controls. In order to detect CFHR1 and 3 gene deletion and

copy number variation, we have used can a multiplex ligation-dependent probe amplification

(MLPA) methodology, and have designed and optimised cheaper, faster and easier PCR-

based assays, in both a quantitative real-time version that can detect copy numbers and

heterozygous deletion genotypes, and an in-gel version for routine diagnostic usage for

identifying homozygous CFHR1/3 gene deletion.

Our study so far shows that, unlike reports from the global North, between 50-60% of

paediatric aHUS patients in India show the presence of anti-CFH autoantibodies, and ~80%

of these show evidence of a homozygous deletion of the cfhr1/3 gene/s. On the other hand, in

a control set of healthy individuals, only ~10% showed evidence for a homozygous cfhr1/3

deletion, in agreement with healthy population reports from the global North. Thus, the

background frequency of the cfhr1/3 deletion genotype is likely to be the same in India as

elsewhere in the world; yet, its relative prominence, in association with anti-CFH

autoantibodies, in paediatric aHUS is much greater in India, creating a clinical-immuno-

pathological conundrum that we intend to continue to work on.

Remarkably, preliminary data show that ~30% of anti-CFH-negative aHUS cases have a

homozygous cfhr1/3 deletion, suggesting either low-level anti-CFH antibody presence or an

autoantibody-independent pathogenetic connection between the genotype and the disease.

This issue will be further examined as well. We have also begun to characterise these anti-

CFH antibodies in terms of concentrations, isotype, epitopic specificity and affinity. CFH

consists of 20 complement control protein (CCP) domains each of ~60 amino acids. The N-

terminus of CFH functions as a fluid-phase complement regulator mainly via CCPs 1-4 while

the C-terminus regulates host cell surface recognition through binding to C3b and other cell-

surface moieties. While anti-CFH autoantibodies in aHUS patients are thought to be mainly

directed at the C-terminus, there is some disagreement over this issue. We have begun

collaborating with Dr. Arvind Sahu, NCCS, Pune, whose group has created recombinant

fragments of CFH and made them available to us for testing aHUS sera. We find, remarkably,

that while the C-terminal epitope/s in CCP17-20 are the major targets of the autoantibodies,

they also show distinct low-affinity cross-reactivity with the CCP5-8 region. This finding is

being pursued further, and assays to determine the relative avidity of the anti-CFH

autoantibodies have now been developed for further use.

Future plans

[note: These plans are based on the current state of work, but it is difficult to predict the areas

in which the next year will see most notable progress. Therefore, these plans are only rough

guides to the intended efforts over the next future, and may be carried over for a variety of

reasons.]

1. The re-engineering of metabolic pathways during lymphocyte development will be further

dissected. We will examine metabolic profiles at earlier stages of B and T cell differentiation.

We will also examine the developmental-functional consequences of specific metabolic step

interruptions, using Aif-hypomorphic mice among other models.

2. We will pursue the characteristics and the genesis of anti-CFH autoantibodies in aHUS.

We will also begin to build appropriate animal models for examining the immunological

mechanisms involved connecting CFHR1/3 deletion and anti-CFH autoimmunity.

67

3. As reported in previous years, we are continuing to examine the basis of cellular

heterogeneity in apparently homogenous naive CD8 T cell populations, the functional

consequences of such heterogeneity and the contribution of metabolic decisions to the

variation.

4. Again, as reported in previous years, we are continuing to work on delineating the precise

role of Btk-dependent and Btk-independent signals in the regulation of peripheral maturation

of B cells. We will extend these studies to the analysis of Btk-dependence of other

hematopoietic lineages such as the eosinophilic lineage for which we have previously shown

preliminary data.

5. We will continue to examine the role of immunoproteasomal components in quantitative

regulation of signal transduction in myeloid and lymphoid cells.

6. We will begin to examine the evolutionary divergence of classical and alternate activation

of myeloid immunocytes from a variety of taxa.

7. Based on some preliminary data from both human and mouse systems, we will initiate a

collaborative study of the genetic regulation of quantitative immunological traits of

functional significance.

Action taken on the RAP/SAC 2014 recommendations

While the RAP/SAC had extensively discussed the data shown last year, no specific

suggestions had been made for changes.

Publications

Original peer-reviewed articles

1. Rane S, Das R, Ranganathan V, Prabhu S, Das A, Mattoo H, Durdik JM, George A,

Rath S, Bal V (2014) Peripheral residence of naïve CD4 T cells induces MHC class II-

dependent alterations in phenotype and function. BMC Biol 12: 106.

2. Rathore DK, Nair D, Raza S, Saini, Singh S, Kumar A, Tripathi R, Ramji S, Batra A,

Aggarwal KC, Chellani HK, Arya S, Bhatla N, Paul VK, Aggarwal R, Agarwal N, Mehta

U, Sopory S, Natchu UCM, Bhatnagar S, Bal V, Rath S, Wadhwa N (2015) Underweight

full-term Indian neonates show differences in umbilical cord blood leukocyte phenotype:

a cross-sectional study. PLoS One (in press)

68

Protease-catalyzed splicing of peptide bond

Principal Investigator RP Roy

Project Associate Anurag Mishra

Ph. D. Students Shikha Singh

S Vijay Kumar

Prity Yadav

Avinash Kumar

Shagun Shukla

Collaborators S Ramakumar, IISc, Bangalore

D Chowdhury, JNU, Delhi

Theme of research

We study the principles underlying peptide ligation reactions catalyzed by proteases and

transpeptidases with a view to apply them to the semisynthesis of proteins and assembly of

well defined bioconjugates that may be useful in a variety of biotechnological applications.

Transpeptidase sortase that catalyzes covalent anchoring of surface proteins to the cell wall in

gram-positive bacteria has turned out to be a wonderful enzyme in this endeavor. The

propensity of sortase to ligate LPXTG proteins to an amine nucleophile offers unprecedented

opportunities in synthetic protein chemistry. Currently, we are studying structural aspects of

substrate recognition to expand the synthetic ambit of Sortases.

Objectives

A. Studies on structure, dynamics and function of Sortases

B. Sortase-mediated protein labeling and conjugation

Work reported in 2013 - 2014

Objective A

Studies on structural aspects of substrate recognition by Sortase A (SrtA) of S. aureus was

reported. The sequence and conformational signatures of a minimalist bona fide substrate was

delineated. Our results established an acetylated and amidated LPNT tetrapeptide as the

minimalist recognition motif of SrtA and that the kinked conformation due to Pro residue in

LPXTG was obligatory for productive substrate binding. Our studies indicate that the LPETG

peptide earlier observed in the crystal structure of SrtA-substrate complex may not be bound

to the enzyme in a catalytically competent state.

Objective B

We had developed a bioorthogonal Sortase-Click reaction suite for defined assembly of

multivalent proteins by combining sortase-mediated ligation and azide-alkyne Huisgen

cycloaddtion reactions. We had utilized lysine-based multiple antigenic peptide (MAP) and

69

β-cyclodextrin (β-CD) scaffolds for this purpose. Several dendritic scaffolds representing a

variety of shapes and pattern were synthesized and characterized. The Sortase-click based

chemoenzymatic strategy was demonstrated with ubiquitin and an immunogenic domain (D3)

of RrgB, a major pilin protein of S. pneumoniae, using linear, branched and cyclic scaffolds.

Progress of work during current reporting period (2014-2015)

Objective A

Sortases are usually classified as A to F based on their membrane topology and substrate

specificity. The prototypical sortase A (SrtA) of Staphylococcus aureus has become a

wonderful tool for in vitro as well as in vivo protein labeling, engineering and

bioconjugation. The ability of this enzyme to ligate LPXTG pentapeptide motif in natural

bacterial surface proteins or engineered proteins/peptides to an aminoglycine derivatized

moiety has enabled myriad biotechnological applications such as, but not limited to, site

specific incorporation of fluorophores, photoaffinity probes, fatty acids, unnatural amino

acids and immobilization of proteins on solid surfaces.

The above applications are testimony to the prowess of sortase in synthetic protein chemistry

and engineering. Most of the applications however owe their allegiance to the housekeeping

SrtA of S. aureus. The utility of S. aureus SrtA is limited to LPXTG donors to Gly based

acceptors. The exception to this is our work demonstrating 6-amino sugars as surrogates for

Gly-derivatized acceptors and that the S. pyogenes SrtA can accept Ala-based nucleophiles in

addition to Gly. Further newer challenging applications of sortases would require availability

of enzymes with orthogonal specificities in both donor and acceptor polypeptides. Besides,

detailed structural elucidation of sortases of different types may be necessary for rational

design of substrate tolerance. In this connection it is pertinent to note that some bacteria

encode class E (SrtE) and F (SrtF) sortases which are annotated to process non-canonical

LAXTG or other hitherto unknown sorting signals as against LPXTG motif preferred by

archetypal SrtA. These enzymes are likely to enhance the synthetic ambit and expand the

sortase toolkit. However, SrtE and SrtF enzymes have not been characterized. Accordingly,

we have initiated studies of SrtE and SrtF from Streptomyces avermitilis and Thermobifida

fusca respectively. Contemporaneous with this, structural studies on SrtA of Streptococcus

pneumoniae has also been elaborated to glean further insights in to the action mechanism of

this enzyme.

The pneumoniae SrtA exists as a dimer in solution and forms intertwined domain swapped

dimers in crystals. Interestingly, active site residues (H141, C207 and R215) lie on an open

interface in 3D structure. The individual mutation to Ala results in abrogation of catalytic

activity. Curiously while H141A and C207A mutants exist as dimer, the R215A mutant

predominantly form monomers in solution suggesting that R215 may play a role in stablizing

the dimeric form of the enzyme. In the crystal structure, R215 forms a salt bridge with D209

and also engages L210 in van der Waal‟s interactions. We prepared D209A mutant to

evaluate the influence of the aforementioned interactions in dimerization vis a vis enzymatic

activity. Interestingly D209A mutation rendered the enzyme inactive but did not alter the

ability of the enzyme to assume a dimeric form implicating that D209 - R215 interaction may

have an important role in catalysis. Likewise mutation of L210 to Gly inactivated the

enzyme and shifted the equilibrium to a monomeric state. The overall results of mutational

studies of a cluster of residues located in the β7/β8 loop highlight the significance of this

region in catalysis and maintenance of the quaternary structure of pneumoniae SrtA.

70

Bioinformatic analysis of the recently published genome of S. avermitilis predicts the

presence of at least four Class E Sortases and 13 putative substrates, all of which contain a

LAXTG motif near the C- terminus. The truncated version (50 residues deleted from the N-

terminus) of one the predicted class E sortase (SAV4333) was cloned, expressed and purified.

The observed mass of the protein (22191 Da) matched well with its expected mass (22188Da)

indicating that the expressed protein was authentic. Preliminary biochemical characterization

revealed that SrtE efficiently transferred LAXTG peptide substrates to Gly- but not to Ala

nucleophiles. LPXTG substrates were poorly accepted by the enzyme.

Thermobifida fusca genome encodes for a single sortase (262 amino acids which is classified

as SrtF. Since it is the only sortase (accession no: YP_290439) present, it is expected to

perform the housekeeping function associated with SrtA in other gram-positive organisms.

For ease of expression and purification, a truncated version of this sortase (Δ64) was

expressed with a N-terminal His tag. The mass of the recombinant SrtF was found to be

24239 Da which fit to the calculated mass of the expressed protein minus a Met residue

suggesting that the N-terminal Met was cleaved by E Coli aminopeptidases during

expression. Preliminary transpeptidation assays revealed that SrtF transferred both LPXTG

and LAXTG peptides with almost equal efficiency to Gly-nucleophiles.

Objective B

We have been seeking strategies for semisynthesis of Ubiquitin (Ub) or SUMO conjugates

that can serve as substrate for deubiquitinating or desumoylating enzymes. Ubiquitylation and

Sumoylation of proteins is highly regulated reversible protein modification that is

accomplished by a sequential action of multiple enzymes in which ATP hydrolysis is utilized

to activate the C-terminal Gly residues of Ub or SUMO for isopeptide linkage to specific

epsilon Lys residues of protein substrates. The generation of Ub/SUMO conjugates in useful

amounts for in vitro studies is complicated by the involvement of multi-steps and the

occurrence of a myriad variants of associated ligases. Inspired by the fact that sortase-

catalyzed transpeptidation entails peptide ligation to a Gly-Gly motif, we have initiated

studies to explore if sortase can be useful in the semisynthesis of Ub/SUMO conjugates.

Toward this, we obtained Ub and SUMO clones from Addgene (MA, USA) and suitably

modified these for expression of a protein compatible with sortase specificity. The 76

residues Ub sequence has a flexible LRLRGG tail at its C-terminus. This sequence was

modified to LRLPNTGG, LRLPETGG, LPLTGG, LPMTGG and LPNTGG respectively.

The proteins were expressed with a hexa-His tag, purified and characterized by mass

spectrometry. Likewise, C-terminus sequence of SUMO-1 (VYQEQTGGHSTV) was

changed to VYLPQTGGHSTV and a hexa-His tagged sortase-recognizable pure protein

was obtained.

For ligating a bona fide peptide substrate to sortase-recognizable Ub and SUMO proteins, we

considered peptides of different chain length from residues 381-391 (KKLMFKTEGPD) of

p53 which contains the target site (K386) for ubiquitylation and sumoylation. The peptides

carrying isopeptide linked di-Gly residues to K386 were assembled by standard Fmoc

protocol exploiting an orthogonal [Fmoc - Lys(Dde) – OH)] protection at K386 site. After

final coupling of N-terminal (, -) BOC protected Lys, Dde group was removed by

hydrazine for installation of branched Gly residues. The peptides were cleaved and purified

by reverse-phase HPLC.

71

Future plans

Investigations involving basic and applied aspects of sortase enzymes will continue on the

following lines: (a) We will carry out rationale mutagenesis of S. pneumoniae sortase to

excavate the mechanism of substrate recognition and catalysis as well as to unequivocally

establish the fidelity/relevance of the domain swapped structure by generating hybrid dimers

composed of wild type catalytic Cys184 and inactive Cys184Ala mutants, (b) We have

recently obtained diffracting quality crystals of SrtE from S. avermitilis. This would be the

first structure of this class of sortase. We would carry out structure-based mutational studies

to understand as to why this sortase prefers LAXTG over LPXTG substrates, (c) The T. fusca

SrtF prefers both the aforementioned substrates equally well and therefore comparative

analysis of the substrate specificity will be carried out to analyze the mechanistic imperatives

of substrate recognition in sortase family of enzymes, and (d) We would like to generate

Ub/SUMO conjugates and evaluate if these could serve as potential substrates for respective

de-conjugating enzymes. The execution and follow up of one or the other point indicated

above would, of course, depend on results and their topical significance.

Action taken on the RAP/SAC 2014 recommendations

No specific recommendations were made.

Publications

Original peer-reviewed articles

1. Biswas T, Pawale VS, Choudhury D, Roy RP (2014) Sorting of LPXTG peptides by

archetypal Sortase A: Role of invariant substrate residues in modulating the enzyme dynamics

and conformational signature of a productive substrate. Biochemistry 53: 2515-24.

72

Cell Death Regulation

Principal Investigator Chandrima Shaha

Project Associate Rajeev Pandey

Ph. D. Students Dipankar Ash

Radhika Mathur

Ashish Kumar

Sagnik Giri

Durgesh Manohar Pitale

Collaborators K Werbovetz, Ohio State University, Ohio

R Madhubala, JNU, New Delhi

C Mandal, IICB, Kolkata

Theme of research

The overall theme of the research program is to elucidate the processes that influence cell

death programs under varying physiological conditions in diverse model systems.

Objectives

Regulatory networks driving cell fate decisions are important to investigate in the context of

understanding diseases. Broadly, our research programme explores the underlying

mechanisms of cell survival and death in diverse intracellular and extracellular conditions.

The model systems used by us are a lower eukaryotic cell, the Leishmania parasite and the

higher eukaryotic mammalian carcinoma cells. Study of both the cellular models is expected

to help us understand how complex eukaryotic regulatory systems evolved because some of

the cellular pathways may be universal features of eukaryotic cells. Using different

experimental approaches in these two models, we explore the precise mechanisms by which

cells die, how these processes are regulated by diverse signaling pathways, inter-relationship

between the various death processes and the evolutionary significance of cell death in a

lower eukaryote.

Work reported in 2013-2014

A. Cell death in protozoan parasites

This laboratory has been working on cell death mechanisms in Leishmania donovani, a

protozoan parasite and the causative agent of Kala-azar. As death processes are closely

related to cellular defense mechanisms, we use two defensive enzymes as models, the

mitochondrial tryparedoxin peroxidases (mTXNPx) and the cytosolic tryparedoxin

peroxidases (cTXNPx) to understand how and when they operate to help the cell to survive.

During the last reporting year, we showed that biochemical inhibition of calmodulin (CaM),

absence of CaM or Ca2+

prevents mitochondrial translocation of the mTXNPx both in vitro

73

and in vivo. This supported the requirement of CaM for mitochondrial transport as shown by

the earlier mutation based studies. Inference from the aggregation and unfolding assays

speculated that CaM was responsible for maintaining the translocation competence of

mTXNPx from cytosol to the mitochondria in the Leishmania parasite. This was a novel

demonstration of involvement of calmodulin in the intracellular trafficking of a protein by

regulating the signal peptide. In another study on macromolecules expressed by the

Leishmania parasite, we reported sterol composition of the promastigote forms of the

parasite with an idea to further investigate the possible role of sterols in cellular defense. We

showed that the major sterol in L. donovani was ergosterol and an increase in ergosterol

occurred in response to anti-leishmanial drug antimony.

B. Mechanisms underlying cell death in cancer

Complexes formed between molecules from apoptotic and autophagy pathways present

potential targets for chemotherapeutics design as disruption of such complexes could alter

cell survival. In the last reporting period we showed that p53 interacted with Beclin-1 in

embryonal carcinoma cells, an event that played an important role in the determination of

cell fate. Analysis of possible binding site of p53 to Beclin-1 showed the region of 1-150

amino acid of Beclin-1 as the BH3 domain essential for binding to other BH3 domain

proteins. Our findings provided evidence that p53 interaction with Beclin-1 facilitated

Beclin-1 ubiquitination through lysine 48 linkage, resulting in proteasome mediated

degradation. Cisplatin treatment disrupted the Beclin-1 p53 interaction resulting in higher

level of Beclin-1 due to lesser ubiquitination. This higher concentration of Beclin-1

increased autophagy and offered protection to the cells from cisplatin induced death. It was

concluded that Beclin-1 p53 interaction defines one additional molecular subroutine crucial

for cell fate decisions in embryonal carcinoma cells. Subsequently, we initiated studies with

the mTOR complexes of embryonal carcinoma cells to gain further insight into the control of

apoptosis and autophagy.

Progress of work during the current reporting year (2014-2015)

A. Cell death in protozoan parasites

Investigations in parasite defense mechanisms have been a long term goal of our work with

the idea that insights into such functions would enable us to eventually interfere with the

function of vital enzymes required for survival of the parasite without interfering with the

host system. The Leishmania parasites, not being naturally endowed with any canonical

peroxidases such as catalase or selenium type glutathione peroxidase rely on the

trypanothione dependent tryparedoxin peroxidase system for defense against peroxides.

Continuing our studies on the tryparedoxin peroxidases, we sought to answer yet another

unanswered question as to what are the relative functions of mTXNPx and cTXNPx in cell

death in the face of oxidative stress produced at different sites. We generated multiple

parasites expressing the peroxidases in different forms. Two mutant parasites with

compromised enzymatic activities of cTXNPx (cys52) and mTXNPx (cys81) were generated

along with parasites expressing normal enzymatic activities. These parasites were challenged

with mitochondrially generated oxidative stress through inhibition of respiratory chain

complexes and cytosolic oxidative stress through direct application of oxidative stress to the

74

cytosol. Parasites expressing competent mTXNPx survived mitochondrial oxidative

challenge better than those expressing mutant mTXNPx and were less capable of surviving

cytosolic oxidative stress. The parasites expressing competent cTXNPx survived cytosolic

oxidative challenge better than those expressing mutant cTXNPx and were less capable of

surviving mitochondrial oxidative stress (Fig. 1). This underscores the importance of local

functions of these enzymes that are important because the parasites are exposed to severe

cytosolic oxidative stress during infection. These studies will explain why there are

differential abilities of the parasites to combat stress and therefore higher or lesser sensitivity

to drugs. Studies are ongoing with function of these enzymes during infection.

Since our last report of ergosterol profile in Leishmania parasites, we now show that

ergosterol levels undergo a major increase when challenged with anti-leishmanial agent

potassium antimony tartrate (PAT). Interestingly, this is preceded by an increase in very long

chain polyunsaturated fatty acids (FA) namely 5,8,11,14-Eicosatetraenoic acid (c20) and

4,7,10,13,16,19-Docosahexaenoic acid (c22). When these fatty acids were administered

separately to wild type cells, they were able to induce increase in the levels of ergosterol in a

similar manner as that of PAT. We used allidochlor and metazochlor known as specific long

chain fatty acid inhibitors in plants because there are no known inhibitors for

trypanosomatids. Both the inhibitors were able to block PAT induced long chain fatty acid

increase successfully in the promastigotes. Inhibition of the fatty acids lowered ergosterol

increase causing an elevation of cell death. Two sterol biosynthesis inhibitors (SBI)

amorolfine and simvastatin, the former acting on ERG24 and ERG2 genes and the latter

operating on ERG13 gene of the ergosterol biosynthesis pathway were used to inhibit sterol

biosynthesis. Treatment of promastigotes with amorolfine or simvastatin during exposure to

PAT was able to reduce ergosterol levels by approximately 60% within 6 h. Both amorolfine

and simvastatin by themselves did not kill cells but potentiated the death inducing property of

PAT where a combination of amorolfine and simvastatin was more lethal.

We sought to investigate if reactive oxygen species (ROS) was linked to the increase in sterol

and FAs because our earlier studies have shown that PAT induced an ROS increase in

parasites after administration. ROS is particularly relevant for these parasites as they are

exposed to ROS while infecting the macrophages and in the secondary host gut, the sandflies.

The first barrier that ROS encounters while entering a cell is the membrane and one of the

primary components of the Leishmania parasite membrane is ergosterol. Interestingly, PAT

induced ROS and ergosterol increase could be blocked by antioxidants like trolox, a cell

permeable derivative of vitamin E and NAC, an antioxidant that raises the cellular pool of

scavengers of free radicals and vitamin C. To confirm that the changes observed in lipid

profiles by PAT was due to increased levels of intracellular ROS or not we used H2O2 as a

direct source of ROS. H2O2 could induce an increase in the levels of various sterols including

ergosterol. Like our results with PAT, this increase in ergosterol levels was also transient and

was inhibited in the presence of NAC and trolox. The above observations show that direct

application of ROS or drug induced production of ROS, both cause a transient increase in

ergosterol content of the cell. Further studies are ongoing to amalgamate the findings into a

model of sterol involved defense mechanism.

B. Mechanisms underlying cell death in cancer

Our models have been the embryonal carcinoma and acute myeloid leukemia cells used as in

vitro cultures as well as tumors in xenograft models. The inactivation of mTORC1 dependent

negative feedback loops is the main cause of rapamycin analogs in clinical trials. We used

75

niclosamide, a specific inhibitor of mTORC1 that caused a significant reduction of tumor size

suggesting that this drug do not have such effects and is able to cause cell death. Since the

role of p53 in mTORC2 inhibition was not known, we studied the effects and show that

presence of p53during inhibition of mTORC2 downregulates mTORC1 inhibition. In the

absence of p53, inhibition of mTORC2 has no effect on mTORC1. Further interactions are

now being explored.

Nitric oxide (NO) has been shown to be effective in cancer chemoprevention and therefore

drugs that help generate NO would be preferable for combination chemotherapy or solo use.

We have earlier reported a combination of cisplatin with a flavonoid fisetin being able to

inhibit tumor production in mice. We used fisetin to see its efficacy in regulating apoptosis in

acute leukemia cells. Tumors generated by these cells were regressed in the presence of

fisetin. Death induction in vitro was facilitated by nitric oxide resulting in the activation of

both the extrinsic and the intrinsic apoptotic pathways. Fisetin also inhibited the downstream

components of the mTORC1 through reduction of levels of p70 S6 kinase and inducing hypo-

phosphorylation of S6 Ri P kinase, eIF4B and eEF2K. Nitric oxide inhibition restored

phosphorylation of downstream effectors of mTORC1 and rescued cells from death. Fisetin

induced Ca2+

entry through L-type Ca2+

channels and abrogation of Ca2+

influx reduced

caspase activation and cell death. It was concluded that apoptotic death of acute monocytic

leukemia cells is NO dependent and elevated Ca2+

entry was essential for activating the

caspase dependent apoptotic pathways. Therefore, manipulation of NO production could be

viewed as a potential strategy to increase efficacy of chemotherapy in acute monocytic

leukemia.

Future plans

The future plans will be to complete our programme of efforts to understand the defense

mechanisms in the protozoan parasite Leishmania in relation to cell death. The importance of

these studies lies in their utility to determine situations where parasites behave as sensitive or

not sensitive to drugs or are infective or non-infective. Therefore, future studies will be

steered towards establishing an essential role of the cTXNPx and mTXNPx enzymes in

infection and response to drugs using a range of parasites generated through mutations. The

current information on the mTXNPx enzyme acting as a chaperone has added a new

dimension to the role of this enzyme. Current reports on the use of tryparedoxin peroxidases

as successful vaccine candidates, adds a new interest in the subject. It is possible that we may

try using domains of the enzymes non-homologous to human enzymes to generate a vaccine,

but this will depend on the time frame required for such an effort. In the other study, the

involvement of sterols as possible antioxidant molecule is a novel finding. Our endeavors will

be to understand how sterols change with drugs and determine their efficacy. This is

important from the point of view of understanding the rampant drug resistance occurring with

the various formulations and literature is very sparse on the subject. Our interests also lie in

the modulation of host pro and anti-apoptotic proteins by the parasite that determines the fate

of an infection. Studies will comprise of functional analysis of the spectrum of survival

proteins in the hosts in response to infection.

The programme of study of understanding testicular cancer, we will make limited efforts to

continue both in vivo and in vitro studies in our model of embryonal carcinoma cells. We will

continue our studies with mTORC1 and mTORC2 relationships in determining the fate of

embryonal carcinoma cells in vivo and in vitro. Both mTORC1 and mTORC2 being

76

important in cancer, mechanistic studies are expected to enrich the understanding of the role

of these complexes in testicular cancer.

Action taken on the RAP/SAC 2014 recommendations

Discussions at the RAPSAC meetings made some suggestions on the mechanistic aspects of

several findings. These have been incorporated.

Publications

Original peer-reviewed articles

1. Ash D, Subramanian M, Surolia A, Shaha C (2015) Nitric oxide is the key mediator of

death induced by fisetin in human acute monocytic leukemia cells. Am J Cancer Res 5:

481-497.

2. Singh AK, Pandey RK, Siqueira-Neto JL, Kwon YJ, Freitas-Junior LH, Shaha C,

Madhubala R (2015) A proteomic based approach to gain insight into reprogramming of

THP-1 cells exposed to Leishmania donovani over an early temporal window. Infect

Immun doi:10.1128/IAI.02833-14.

3. Bhattacharya K, Bag AK, Tripathi R, Samanta, SK, Pal BC, Shaha C, Mandal C (2014)

Mahanine, a novel mitochondrial complex-III inhibitor induces G0/G1 arrest through

redox alteration-mediated DNA damage response and regresses glioblastoma multiforme.

Am J Cancer Res 4: 5-22.

4. Tripathi R, Ash D, Shaha C (2014) Beclin-1 p53 interaction is crucial for cell fate

determination in embryonal carcinoma cells. J Cell Mol Med 18: 2275-2286.

5. Pandharkar T, Zhu X, Mathur M, Jiang J, Schmittgen T, Shaha C, Werbovetz K (2014)

Studies on the antileishmanial mechanism of action of the arylimidamide DB766: azole

interactions and role of CYP5122A1. Antimicrob Agents Chemother 58:4682-4689.

Reviews / Proceedings

1. Mathur R, Shaha C (2014) Cell death in a kinetoplastid parasite, the Leishmania spp. In

Leishmania: Genomics, Molecular Biology and Control. (Eds. Adak, S and Dutta R,

Horizon Scientific Press, UK) pp 79-92.

77

Characterization of Minisatellite tagged mRNA transcripts from the

human sperm genome

Principal Investigator Sher Ali

Project Associates Leena Rawal

Deepak Panwar

Ph.D. Students Md. Qudratullah

Priyank Singhavi

Suresh Kumar

Collaborators LC Garg, NII

J Ahmad, AMU, Aligarh

Theme of research

Using spermatozoal cDNA from different categories of the infertile males and two

minisatellite based primers 33.15 (CACCTCTCCACCTGCC) and MN3

(GAAAGAAAGAAAGAAAGAAAGAAA), we conducted minisatellite associated sequence

amplification. A total of fifty one 33.15 tagged mRNA transcripts and sixty, MN3 tagged

ones were uncovered. We analysed corresponding genes and detected copy number variation

in the infertile males both in blood and spermatozoal samples but not in the normal fertile

ones. All the transcripts showed high level of expression in spermatozoa compared to any

other somatic tissues. Sperm genome analysis enabled to capture elusive gene(s) implicated

in control and regulation of human male in/fertility.

Objectives

1. To uncover mRNA transcripts tagged with minisatellites 33.15

(CACCTCTCCACCTGCC) and MN3 (GAAAGAAAGAAAGAAAGAAAGAAA)

from the spermatozoa of different categories of the infertile males.

2. To assess quantitative expression of these mRNA transcripts in ten commercially

purchased human tissues cDNA using Real time PCR.

3. Copy number assessment of the MASA uncovered transcripts in the infertile males both

in blood and semen samples compared to that in the normal fertile ones.

4. Cloning, chromosomal and spermatozoal localization of C4orf26 and MPPE1 candidate

genes.

Work reported in 2013-2014

Fate of the human Y chromosome linked genes and loci in prostate cancer cell lines

DU145 and LNCaP

Prostate cancer is a known cause of mortality in men worldwide although the risk factor

varies among different ethnic groups. Loss of the Y chromosome is a common chromosomal

abnormality observed in the human prostate cancer although a total of 13 chromosomes have

been implicated. We screened 51 standard sequence tagged sites (STSs) corresponding to a

male-specific region of the Y chromosome (MSY), sequenced the coding region of the SRY

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gene and assessed the status of the DYZ1 arrays in the human prostate cancer cell lines

DU145 and LNCaP. The MSY was found to be intact and coding region of SRY showed no

sequence variation in both the cell lines. However, DYZ1 arrays showed sequence and copy

number variations. DU145 and LNCaP cells were found to carry 742 and 1945 copies of the

DYZ1, respectively per haploid genome. The DYZ1 copies detected in these cell lines are

much below the average reported in normal human males. Similarly, the number of

“TTCCA” repeat and its derivatives within the DYZ1 arrays showed variation compared to

those of the normal males. Work on additional cell lines and biopsied samples would

augment our understanding about the susceptibility of this region. Based on the present work,

we construe that copy number status of the DYZ1 may be exploited as a supplementary

prognostic tool to monitor the occurrence of prostate cancer in the biopsied samples together

with the possible markers originating from other chromosomes.

Progress of work during the current reporting year (2014-2015)

Minisatellite tagged mRNA transcripts from the human sperm genome

Human spermatozoa have mRNA transcripts in their cytoplasm representing different types

of genes. We used 33.15 (CACCTCTCCACCTGCC) and MN3 [(GAAA)6] primers

representing minisatellites and cDNA from the spermatozoa of normal males and infertile

patients of various categories (Oligospermic, Azoospermic and Infertile males with normal

spermiogram) to conduct Minisatellite Associated Sequence Amplification (MASA). In the

process, we uncovered 51 mRNA transcripts tagged with 33.15 and 60, tagged with MN3.

Further, we undertook full length characterization of Chromosome 4 Open Reading Frame 26

(C4orf26) and Metallophosphoesterase 1 (MPPE1) candidate genes and localized them onto

chromosomes 4 and 18, respectively and onto the spermatozoa employing FISH.

Mining of mRNA transcripts using 33.15 and MN3 mediated MASA Reaction

Cloning and sequencing of the resultant amplicons uncovered by MASA led to the

identification of 51 different 33.15 tagged and sixty MN3 tagged mRNA transcripts in the

spermatozoa from the normal, oligospermic, azoospermic and infertile males with normal

spermiogram as mentioned above. The cDNA sequences were submitted to GenBank.

Further, each individual sequence was used to search database to ascertain homology with the

known sequences. Among 33.15 tagged transcripts, 13 showed homology with the 12 known

genes in human. Others showed no homology with any of the known genes and therefore

were placed in novel category. Three transcripts pAKT-8, 13 and 46 were retrieved from

oligospermic and normal fertile males. Infertile with normal spermiogram and normal fertile

males were found to share two transcripts pAKT-9 and 20. Similarly, 3 transcripts pAKT-41,

43 and 45 were shared amongst oligospermic, infertile with normal spermiogram and normal

fertile males. Two transcripts pAKT-32 and 42 were common amongst oligospermic,

azoospermic, infertile with normal spermiogram and normal fertile males.

Amongst 60 uncovered MN3 tagged transcripts, none showed homology with known genes

and therefore were placed in the novel category. The transcripts pAST-4, 11, 12, 33 and 45

were common among oligospermic and azoospermic males. The transcript pAST-39 was

present in oligospermic and infertile males with normal spermiogram, whereas infertile males

with normal spermiogram and normal fertile males had one common transcript pAST-26.

Similarly, oligospermic, azoospermic and infertile with normal spermiogram males had two

transcripts pAST-7 and 37 in common.

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Expression levels of tagged mRNA transcripts in males representing different categories

using Real time PCR

The expression of 33.15 tagged transcripts was assessed across all the patients from different

categories and normal fertile males using spermatozoal cDNA and transcript specific primers.

The patients of a given category showed inter-individual differences in expression of repeat

tagged genes. The expression data for the transcripts in males belonging to one category is

shown as mean ± SEM (Figure 1). The expression profiles showed significant differences

between normal males and that of the patients. Several transcripts expressed in all the

categories of males, while others did not. The expression results are summarized in Figure 2.

Figure 1. Expression profile of 33.15 tagged transcripts in patients with different spermatogenic status. Here, O,

A, I and N denote oligospermic, azoospermic, infertile males with normal spermiogram and normal fertile

controls.

Figure 2: Expression Summary of Spermatozoal mRNA transcripts tagged with consensus of 33.15 repeat loci

Across Different Categories of patients and Normal Males

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Of the sixty MN3 transcripts, several of them expressed in more than one category of the

patients. However, transcript pAST-2 expressed exclusively in the azoospermic males (Figure

3). One transcript pAST-25 showed almost equal expression in oligospermic, infertile, males

with normal spermiogram and normal fertile males. Three transcripts (pAST-31, 48 and 52)

showed higher expression in the infertile males with normal spermiogram and in normal

fertile males compared to that in the patients with spermatogenic impairments (oligospermic

and azoospermic). Similarly, three transcripts (pAST-1, 16 and 32) showed higher expression

in azoospermic males compared to that in other categories. A total of five (pAST-4, 10, 40,

41, 42), eighteen (pAST-13, 15, 19, 20, 23, 34, 36, 48, 50, 51, 53-59) and twenty three

(pAST-3, 7, 9, 11, 12, 14, 17, 18, 21, 22, 24, 26, 27, 29, 33, 37-39, 44, 45, 47, 50, 60) MN3

tagged transcripts showed higher expression in oligospermic, infertile males with normal

spermiogram and normal fertile males, respectively. The expression results are summarized

in Figure 4.

Figure 3. Expression profile of MN3 tagged transcripts in patients with different spermatogenic status. The

samples coding is the same as mentioned earlier.

Figure 4: Expression summary of spermatozoal mRNA transcripts tagged with MN3 repeat specific to Normo

and Azoo patients

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Relative expression of 33.15 tagged transcripts in commercially purchased cDNA of

human tissues

Commercially purchased cDNA samples from human testis, prostrate, bone marrow, brain,

heart, kidney, lung, spleen and uterus were used as template for analyzing the expression of

33.15 tagged transcripts. The expression of all the sperm retrieved transcripts except one was

found to be highest in the spermatozoa than in any of the tissues studied. The remaining one

pAKT-29 showed 94% homology with the Family with Sequence Similarity 199, X-Linked

(FAM199X) gene (originating from the X chromosome). Surprisingly, this gene showed

highest expression in prostate and then testis cDNA and relatively reduced expression both in

adult and foetal brains. The expression profiles of some representative transcripts in tissues

are shown in Figure 5.

Figure 5. Expression profile of 33.15 tagged transcripts in different human tissues. A single

bar represents the mean expression of the transcript in a particular tissue cDNA.

Copy number variation of MASA uncovered candidate genes

Two copies of C4orf26 gene was present in blood samples of normal males. Accordingly, one

copy of this gene was detected in the sperm DNA of normal fertile males. However, copy

number of this gene in the infertile males varied from 1- 4. This gene was found to have one

copy in the sperm DNA (like that in normal fertile males) in about 14% oligospermic and

20% infertile males with normal spermiogram. Similarly, the copy number of MPPE1 also

showed variation ranging from 1-6. The copy number of several genes was not constant even

within a given category of the infertile males. Analysis of additional samples along this line

would throw light on the un/stable nature of such genes in varying categories of in/fertile

males.

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Chromosomal and spermatozoal localization of C4orf26 and of MPPE1genes

Using fluorescence in situ hybridization (FISH), the C4orf26 and MPPEI genes were

localized onto human metaphase chromosomes 4 and 18, respectively (Figures 6 and 7) and

on the spermatozoa. The FISH results corroborated with the qPCR data supporting copy

number variation of the genes as mentioned above.

Figure 6: Localization of C4orf26 on metaphases, interphases and spermatozoa using

Fluorescence in situ hybridization (FISH). (A) represents the chromosomal localization of

C4orf26 gene on the interphase nuclei, (B) blood metaphase and (C) spermatozoa. The

metaphase, interphase and sperm nuclei stained with DAPI. In the panel (B) probe for SRY

(which detects the presence of SRY on Y chromosome and X-centromere) probe was used as

an internal positive control. Patient IDs are shown in red text, where OL indicates

oligospermic and INS, infertile males with normal spermiogram.

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Figure 7: Localization of MPPEI on metaphase chromosome 18, interphases and

spermatozoa using Fluorescence in situ hybridization (FISH).

Future plans

Present study provides a road map to have a comparative profile of the satellite tagged

transcripts in different categories of infertile males narrowing the search of genes possibly

implicated in testicular dysfunction in general and impaired spermatogenesis in particular.

We propose to undertake characterization of other genes showing expression in a given

category of the infertile males. This approach is likely to enrich our understanding on the

spermatozoal genome.

Action taken on the RAP/SAC 2014 recommendations

No recommendation was received.

Publications

Original peer-reviewed articles

1. Sharma M, Rawal L, Panwar D, Sehgal N, Ali S (2014) Differential expression of

Homeobox C11 protein in water buffalo Bubalus bubalis and its putative 3D structure.

BMC Genomics 15: 638-349.

2. Panwar D,

Rawal L, Ali S (2014) Molecular docking uncovers TSPY binds more

efficiently with eEF1A2 compared to eEF1A1. J Biomol Struct Dyn 21: 1-12.

3. Rawal L, Pathak D, Sehgal N, Ali S (2015) Transcriptional dynamics of Homeobox C11

gene in water buffalo Bubalus bubalis. DNA Cell Biol (in press).

84

Cellular and molecular aspects of reproduction and viral infection

Principal Investigator Satish Kumar Gupta

Project Fellows Ananta Prasad Arukha

Pankaj Singh

Nripendra Nath Mishra

Vidisha Minhas

Ajay Kesharwani

Aakanksha Agarwal

Ph. D. Students Abhinav Shrestha

Sudha Saryu Malhotra

Ankita Malik

Piyush Chaudhary

Sonam Verma

Collaborators AK Panda, NII

R Varadarajan, IISc, Bangalore

RK Singh, University of Allahabad, Allahabad

SB Katti, CDRI, Lucknow

KP Suja, HLL Lifecare Ltd, Thiruvantapuram

DN Modi, NIRRH, Mumbai

Theme of research

One of the areas of scientific pursuit in our lab is to design and evaluate the recombinant

proteins based contraceptive vaccines and their delivery to generate long-lasting immune

response. To understand implantation biology during pregnancy, the regulatory mechanisms

associated with cytokines, growth factors and hormones mediated trophoblast cell migration,

invasion and differentiation are being investigated. In addition, attempts are being made to

develop microbicides for prevention of sexually transmitted infections of HIV-1 and HSV-2.

Objectives

To develop contraceptive vaccine for the management of wildlife population

To understand the molecular mechanisms associated with migration, invasion and

differentiation of trophoblast or trophoblast derived cancer cells

To develop microbicide for prevention of sexually transmitted HIV-1 and HSV-2

infections

Work reported in 2013-2014

Dvelopment of contraceptive vaccine

Last year, the immunogenicity and contraceptive efficacy of various gamete specific

recombinant proteins in FvB/J femlae mice was reported. Recombinant TT-KK-dZP3 showed

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dose dependent inhibition of fertility that correlated with anti-TT-KK-dZP3 antibody titers. In

addition, contraceptive potential of recombinant TT-KK-Sp17(C-terminal) in female mice

was also reported.

Molecular mechanisms associated with trophoblast migration, invasion and

differentiation

As part of a separate project, studies pertaining to the understanding of the molecular

mechanisms associated with trophoblast invasion and differentiation were reported.

Treatment of JEG-3 cells with LIF led to an increase in the invasion of the cells with

concomitant activation of STAT3 and ERK1/2 signaling pathways. Activation of the above

mentioned pathways was associated with elevated expression of various invasion associated

factors such as mucin 1, Fos, Jun, etc. Further, STAT3ser727 phosphorylation was found to

play an important role in LIF mediated JEG-3 trophoblastic cell invasion and gene

expression.

Using BeWo cells as experimental model to study trophoblast differentiation, decrease in

hCG/forskolin mediated syncitialization of α- or β-hCG knocked-down BeWo cells was

associated with decreased activation of p-PKA, p-CREB and p-β catenin. Supplementaion of

hCG in culture medium can partially recover syncitialization of α- or β-hCG knocked-down

BeWo cells. The studies revealed the importance of hCG in trophoblastic cell fusion.

Development of microbicide for prevention of sexually transmitted viral infection

In an effort to prepare herbal microbicide with an aim to prevent sexual transmission of HIV-

1, the potential of the extract prepared from the stem bark of Acacea catechu for inhibition of

HIV-1 infection was reported. The plant had potent HIV-1 protease inhibitory activity.

Progress of work during the current reporting year (2014-2015)

Development of contraceptive vaccine

To enhance the immunogenicity and contraceptive efficacy of recombinant TT-KK-dZP3

[encompassing promiscuous T cell epitope of tetanus toxoid (TT; aa residues 830-844)

followed by dilysine linker (KK) and ectodomain of dog zona pellucida glycoprotein-3

(dZP3; aa residues 23-348)] following two injections schedule, CpG motif was included

along with alum. It was observed that inclusion of CpG motif resulted in the increase of

serum IgG titers (53.90 ± 8.82 x 103

AU; intraperitoneal group) as compared to without CpG

formulation (26.5 ± 2.28 x 103

AU). Also inclusion of CpG had resulted in better

contraceptive efficacy both in terms of infertility as well as sub-fertility as compared to

without CpG formulation. The relevance of systemic versus mucosal immune response in

curtailment of fertility was also investigated. Upon comparing intraperitoneal (systemic) and

intranasal (mucosal) routes it was observed that, contraceptive efficacy achieved through both

the routes was identical, as 50% of the immunized animals failed to conceive in both the

groups. In intraperitoneal group, serum/vaginal IgG titers of non-pregnant mice was

significantly higher (p = 1.57 x 10-5

and 0.03 for serum and vaginal IgG, respectively) than

pregnant mice. While in intranasal route, vaginal IgA seemed to be primary determinant for

infertility as non-pregnant mice showed significantly higher vaginal IgA titer (p = 0.01) as

compared to that of pregnant mice (Fig. 1). These studies suggest that bio-availability of

86

antibodies against TT-KK-dZP3 in female genital tract is important for contraceptive

efficacy.

Fig. 1. Serum/vaginal IgG/IgA titer of pregnant/non-pregnant mice immunized with recombinant TT-KK-dZP3

supplemented with alum and CpG motif following two injection schedule: Panels A & B show serum IgG and

vaginal IgG and IgA titers for pregnant and non-pregnant mice immunized through intraperitoneal route with 50

µg of recombinant TT-KK-dZP3 supplemented with alum and CpG motif following two injection schedule.

Panels C & D show serum IgG and vaginal IgG and IgA titers for pregnant and non-pregnant mice immunized

through intranasal route in the same experiment.

With an aim to further optimize TT-KK-dZP3 based formulations to elicit sustained antibody

response, mice were immunized with poly-lactide (PLA) based microparticles incorporating

recombinant TT-KK-dZP3 supplemented with alum and CpG motif following one and two

injections schedule. Two injections schedule showed higher antibody titers. Splenocytes

isolated on day 7 after the second injection and stimulated in vitro with soluble recombinant

TT-KK-dZP3 secreted IFN-γ as well as IL-4 in the culture supernatant. Antibody isotyping

studies of immune sera revealed a predominantly Th1 type of antibody response.

This year, we have cloned and expressed in E. coli cDNA encoding a novel fusion protein

TT-KK-dZP3-GGG-bRNAse-GnRH-CSPII-GnRH, encompassing promiscuous T cell

epitope of TT, followed by a dilysine linker, a fragment of dog ZP3 (aa residues 306-346),

triglycine spacer (GGG), promiscuous T cell epitope of bovine RNase (bRNase; aa residues

120- 131), two repeats of gonadotropin releasing hormone (GnRH) sandwiched by a T cell

epitope of circumvalent sporozoite protein (CSP-II; aa residue 362-383). The

immunogenicity and contraceptive efficacy of the purified recombinant TT-KK-dZP3-GGG-

bRNAse-GnRH-CSPII-GnRH was evaluated in female FvB/J mice following a three

injection schedule. Analysis of the pre- and post-immunization sera by ELISA revealed high

antibody titers in the immunized animals against ZP3 and GnRH. Indirect

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immunofluorescence assay showed recognition of mouse zona pellucida matrix by the

antibodies. Ninety percent of the immunized female mice failed to conceive on mating with

the fertile males while in control group all animals conceived. Also, sera of the immunized

mice induced significant reduction in mouse in vitro fertilization. The high contraceptive

efficacy of recombinant TT-KK-dZP3-GGG-bRNAse-GnRH-CSPII-GnRH was confirmed

by another independent investigator.

To enhance the contraceptive efficacy of Sp17 C-terminal fragment, a recombinant fusion

protein bRNase-KK-Sp17(C)-KK-TT-GnRH-GnRH, comprising of promiscuous T cell

epitope of bRNase followed by dilysine linker, mouse Sp17 C-terminal (aa residues 76-126),

dilysine linker, promiscuous T cell epitope of TT and tandem repates of GnRH was expressed

in E. coli and purified by Ni-NTA affinity chromatography. We plan to immunize female as

well as male mice simultaneously with recombinant bRNase-KK-Sp17(C)-KK-TT-GnRH-

GnRH, to check whether inclusion of GnRH has increased the immunogenicity and

contraceptive efficacy of Sp17.

Molecular mechanisms associated with migration, invasion and differentiation of the

trophoblastic cells

i) Trophoblastic cell migration: Expression of various Wnt ligands such as Wnt2, Wnt3,

Wnt4, Wnt5a, Wnt5b, Wnt7b, Wnt10b, and Wnt11 was investigated during migration of

HTR-8/SVneo trphoblastic cells in a scratch wound assay. Quantitative RT-PCR analysis

revealed a significant increase in the expression of Wnt4 and Wnt11 at 24 h when cells were

grown either in 10 or 1% FCS. Treatment of HTR-8/Svneo cells with varying concentrations

of hepatocyte growth factor (HGF) showed a significant increase in the percent Migration

Index, which was highest at 50 ng/ml. Treatment of the cells with HGF also led to further

increase in the expression of Wnt4 and Wnt11 as compared to when cells were cultured in

absence of HGF. These studies showed the importance of Wnt4 and Wnt11 during the

migration of HTR-8/SVneo cells.

ii) Trophoblastic cell invasion: In addition to migration, invasion of HTR-8/SVneo

trphoblastic cell line in matrigel invasion assay was studied in presence of varying

concentrations of LIF, EGF and IFNγ. Treatment with LIF and EGF significantly increased

the invasion of HTR-8/SVneo cells whereas treatment with IFN-γ led to a significant

decrease in the invasion. A significant increase in Akt and Erk1/2 phosphorylation along with

the increase in STAT-1 and STAT-3 phosphorylation was observed in EGF treated HTR-

8/SVneo cells. Experiments with Erk1/2 phosphorylation inhibitor, U0126, suggested the role

of Erk1/2 phosphorylation in STAT-1 degradation and increased STAT-3 phosphorylation.

To understand the relevance of miRNA mediated modulation during trophoblast invasion

HTR-8/SVneo cells were treated with the optimized concentrations of LIF, EGF and IFN-γ.

Out of all differentially expressed miRNA, 14 upregulated and 2 downregulated miRNA

were common when cells were treated with EGF and LIF. We have identified target miRNAs

using bioinformatic tools, which might be playing an important role in the regulation of

invasion. Hsa-miR-7-5p which is down-regulated, is known to inhibit invasion in melanoma

cells and target insulin receptor substrate 2. Hsa-miR-1246 is upregulated and is known to

increase invasion and migration in hepatocellular carcinoma cells. Hsa-miR-125b and hsa-

miR-100-5p are downregulated and target SERPIN B3, which is known to induce eithelial to

mesenchymal transition in hepatocellular carcinomas. Hsa-miR-1297 is downregulated in

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cells treated with IFN-γ that targets guanylate binding protein-1 (GBP-1), which inhibits

invasion of endothelial cells by decreasing MMP-1 expression.

iii) Trophoblastic cell differentiation: This year, we investigated whether silencing of α- or

β-hCG in BeWo cells affects β-catenin expression via Wnt liagand(s) either in canonical or

non-canonical fashion during forskolin mediated syncytialization. We found that BeWo cell

fusion is associated with an increased expression of active β-catenin and that could be

associated with increased expression of wnt10b, independent of hCG regulation. We were

also able to rule out non-canonical activation and stabilization of β-catenin by

phosphorylation at serine-675 via PKA in case of BeWo cell fusion by using H89 mediated

PKA inhibition (Fig. 2).

Fig. 2 Effect of PKA inhibitor on BeWo cell fusion. BeWo cells were cultured in presence or absence of PKA

inhibitor (H89; 10 μM) and cell fusion was assessed by Desmoplakin I+II staining. In addition, expression

profile of P-CREB and P-β-catenin (Ser675) was assessed by Western blot.

89

Herbal formulation for prevention of HIV-1 and HSV-2 infection

Herbal formulation, comprising of 50% ethanolic plant extracts prepared from stem bark of

Acacia catechu, leaves of Lagerstroemia speciosa and Terminalia chebula, was evaluated for

its activity against HIV-1 and HSV-2. The herbal formulation showed dose dependent

inhibition of HIV-1 and the observed IC50 value and therapeutic index (TI) were 2.17 µg/ml

and 71.10 respectively, in a cell-free virus based assay using TZM-bl cells and HIV-1NL4.3

(X-4 tropic). The herbal formulation also showed potent inhibitory activity against HIV-1

integrase, reverse transcriptase and protease with the IC50 value of 38.61, 13.3 and 4.2 µg/ml

respectively. Apart from the activity against HIV-1, the herbal formulation also exhibited

anti-HSV-2 activity at pre-infection (IC50 = 17.91 ng/ml), post-infection (IC50 = 19.41

µg/ml), attachment (IC50 = 0.30 µg/ml) and penetration (IC50 = 1.93 µg/ml) stages of HSV-2

infection in plaque reduction assay in Vero cells. The herbal formulation up to 100 µg/ml did

not show any significant reduction in the viability of commonly found vaginal Lactobacilli

strains. Further, measurement of transepithelial resistance (TER) on the tight junctions

formed by Caco-2 epithelial cells in presence of the herbal formulation suggest that it did not

distrupt the monolayer formed by the epithelial cells.

Future plans

With respect to the contraceptive vaccines, next year, it is planned to immunize

simultanously male as well as female mice with recombinant proteins to explore if 100%

contraceptive efficacy can be achieved. Further, vaccine delivery optimization studies will be

undertaken so as to elicit long lasting immunity with minimum number of injections. If the

approval from Review Committee on Genetic Manipulation (RCGM), DBT, Government of

India is received, attempts will be made to complete acute toxicity studies in rodents and sub-

acute toxicity studies in beagle dogs. The role of Wnt4 and Wnt11 in the migration of

trophoblastic cells will be confirmed by their silencing. The importance of selected miRNA

in the invasion of trophoblastic cells will be investigated by using mimics or by silencing. In

addition to miRNA, transcriptomics studies will also be undertaken to study the relevance of

various proteins in the invasion of the trophoblastic cells. Role of NR4A, sub-family of

nuclear orphan receptors, will be studied during trophoblast cell fusion using BeWo cells as

model. Moreover, role of miRNA in regulating syncytialization, in addition to invasion of

trophoblastic cells, will also be investigated. The relevance of either canonical or non-

canonical Wnt signaling pathways during migration and differentiation of trophoblastic cells

will also be undertaken. The relevance of hypoxia in the context of migration and invasion of

trophoblastic cells will also be initiated. It is contemplated that next year we will complete

pre-clinical safety evaluation of herbal microbicide formulation and hand-over the product to

the industrial partner.

Action taken on the RAP/SAC 2014 recommendations

Keeping in view of the recommendations of the RAP/SAC 2014 to move forward with the

contraceptive vaccine for wildlife population management, an application to RCGM, DBT

has been submitted to get the approval to conduct acute toxicity studies using E. coli-

expressed recombinant TT-KK-dZP3.

90

Publications

Original peer reviewed articles

1. Kushwaha RN, Debnath U, Singh P, Saxena R, Gupta SK, Tripathi RK, Siddiqui HH,

Katti SB (2015) New piperazine-derived NNRTIs as anti-HIV agent: synthesis, biological

evaluation and molecular docking studies. Indo American Journal of Pharm Research

2015:5(01).

2. Shrestha A, Srichandan S, Minhas V, Panda AK, Gupta SK (2014) Canine zona pellucida

glycoprotein-3: up-scaled production, immunization strategy and its outcome on fertility.

Vaccine 33:133-140.

3. Shembekar N, Mallajosyula VVA, Chaudhury P, Upadhyay V, Varadarajan R, Gupta SK

(2014) Design and characterization of a humanized monoclonal antibody that potently

neutralize 2009 pandemic H1N1 virus. Biotechnol J 9:1594-1603.

Reviews/Proceedings

1. Gupta SK (2014) Role of zona pellucida glycoproteins during fertilization in humans. J

Reprod Immunol doi: 10.1016/j.jri.2014.08.006

2. Gupta SK (2014) Unraveling the intricacies of mammalian fertilization. Asian J Androl

16:801-802.

3. Gupta SK, Shembekar N (2015) Monoclonal antibodies. In Textbook of Biochemistry and

Human Biology (Eds. Talwar GP, Sarin SK, Hasnain SE). Prentice-Hall of India Pvt Ltd,

New Delhi, India. pp1233-1239.

91

Cellular and molecular biology of human cancer

Principal Investigator Anil Suri

Project Staff Scientist Nirmala Jagadish

Project Fellows Sumit Agarwal

Deepak Parashar

Rahul Dwivedi

Nainee Goyal

Rukhsar Fatima

Aditi Sharma

Sapna Purohit

Ph. D. Students Namita Gupta

Swarnendra Singh

Vikash Kumar

Amos Prashant Topno

Collaborators PK Julka, AIIMS, Delhi

GK Rath, AIIMS, Delhi

R Kumar, AIIMS, Delhi

A Seth, AIIMS, Delhi

A Thakar, AIIMS, Delhi

A Suri, AIIMS, Delhi

G Makharia, AIIMS,Delhi

A Chaturvedi, AIIMS, Delhi

V Suri, AIIMS, Delhi

A Bhatnagar, Safdarjung Hospital and VMM College, Delhi

A Gupta, VIMHANS Hospital, Delhi

TC Sadasukhi, MG Medical College and Hospital, Jaipur

NK Lohiya, University of Rajasthan, Jaipur

A Batra, Safdarjung Hospital and VMM College, Delhi

T Rajkumar, Cancer Institute (WIA), Chennai

Theme of research

Over the last three decades, knowledge about the molecular biology of human cancers has

vastly expanded. A host of genes and proteins involved in cancer development and

progression have been identified and many mechanisms at molecular, cellular and even tissue

level have been, at least partly, elucidated. In fact, cancer research has now reached a critical

stage, in which the accumulated knowledge on molecular mechanisms needs to be translated

into improved prevention, diagnosis, and treatment. Understanding the mechanisms involved

in tumorigenesis has wide ranging implications for targeting the treatment of cancer. Tumor

specific antigens (TSA) represent a unique class of tumor antigens, which are expressed in a

variety of cancerous tissues and are silent in normal tissues. Cancer testis (CT) antigens

represent a unique class of tumor antigens under this category, which are expressed in a

variety of cancerous tissues and are silent in normal tissues, except for the testis. A

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characteristic commonly shared by CT antigens is, aside from the highly tissue-restricted

expression profile, their likely correlation with tumor progression and immunogenicity in

cancer patients. Also the differential expression of germ cell specific genes in various cancer

tissues reveals the important link between the two complementary disciplines of cell survival

i.e. developmental and cancer biology.

Objectives

Numerous candidate cancer associated genes have been identified to date. However, for the

vast majority of these genes, neither the expression pattern of the protein product, nor its

localization and function in the tumor tissues has been investigated. The identification of

specific genetic markers that are associated with tumor progression and aggressiveness may

prove to be useful to assess the progression of disease. We are focusing on tumor associated

proteins for the assessment of disease risk, early detection of disease, prognosis, and response

to treatment as well as disease recurrence. The application of such gene products

(biomarkers) to cancer will lead the way because of the unique association of genomic

changes in cancer cells with the disease process. Most importantly, cancer biomarkers for

prognostic, prediction and pharmacodynamics may aid in the rational development of anti-

cancer drugs. In addition, our goal is to delineate in greater detail the gene-expression

pathways involved in cellular growth, cell migration, and invasion for the treatment of

cancer.

Work reported in 2013-2014

Breast cancer remains the major cause of death in women worldwide. Recent reports indicate

that majority of cancer related deaths occur in economically weak and developing countries,

such as India. The existing treatment modalities for breast cancer patients are based on

expression of ER, PR and HER2 molecules. However, a major challenge remains with breast

cancer patients with triple-negative tumors for which there are no or limited therapy available

and have poor prognosis. Therefore, in this regard we investigated the involvement of a well

characterized CT antigen, sperm associated antigen 9 (SPAG9) in breast cancer using various

breast cancer cell line models. Gene silencing approach was employed to study the

association of SPAG9 with early spread and metastasis in highly aggressive triple-

negativeMDA-MB-231 breast cancer cells which may lead to new therapeutic strategies.

To the best of our knowledge, this is the first report where we have put forth an evidence of

potential role of SPAG9 in cellular growth, migration, invasion and colony forming ability in

highly aggressive triple-negative MDA-MB-231 breast cancer cells. Furthermore we also

demonstrated that SPAG9 expression was higher in all breast cancer cell compared to normal

mammary epithelial cells. In addition, in vivo xenograft studies further strengthen the role of

SPAG9 in breast cancer. Our study provides an association between SPAG9 expression and

its potential role in breast cancer, and thus lays a foundation for developing a promising

therapeutic target for triple-negative breast cancer.

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Progress of work during the current reporting year (2014-2015)

Head and Neck Cancer: Cancer is the leading cause of death in economically developed

countries and the second leading cause of death in developing countries. In developing

nations, it has been reported that one in four cancers in male occur in head and neck region

and accounts for 30% of all cancers. Importantly, salivary gland tumor (SGT) accounts for 3-

5% of total head and neck cancer and of wide histological and biological diversity. Early

detection of SGT would be essential for more effective clinical management leading to

improved quality of life and increased survival rate. The present study was initiated to

undertake a more comprehensive analysis of SPAG9 gene and protein expression in SGT

specimens in the context of clinic-pathological parameters, i.e., histo-pathological

characteristics. We also investigated the humoral response against SPAG9 in various stages

and histotypes of SGT patients. Our results suggest that SPAG9 may be used as a novel

diagnostic biomarker for early detection of SGT thus, may be useful in better management of

SGT patients.

SPAG9 gene expression in SGT patients

The SPAG9 gene expression was investigated by RT-PCR in SGT tissue along with available

matched associated non-cancerous tissue (ANCT) specimens (Figure 1). The data revealed

that 80% (82 of 102) of tumor specimens showed SPAG9 gene expression irrespective of

benign, malignant tumor, stages and various histotypes (PA: pleomorphic benign tumors,

MEC: mucoepidermoid, AdCC: adenoid cystic, ACC: acinic cell, CC: clear cell, BCAC:

basal cell, ANOS: adenocarcinoma not otherwise specified, PLGA: polymorphous low grade

adenocarcinoma).

Figure 1. SPAG9 expression in various stages and histotypes of SGT specimens

Validation of SPAG9 gene and protein expression in SGT patients

SPAG9 gene expression was also determined in serial SGT tumor specimen sections by in

situ RNA hybridization studies using in vitro synthesized riboprobes. Our in situ RNA

hybridization studies employing antisense riboprobes confirmed SPAG9 gene expression in

SGT specimens. As expected sense riboprobes failed to show SPAG9 gene expression in any

of the serial tissue specimen sections as depicted in Figure 2.

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Figure 2. SPAG9 gene expression in various stages of SGT specimens

SPAG9 protein expression was further confirmed by immunohistochemistry (IHC) in serial

SGT tissue sections of benign tumors, various stages of malignant tumors and different

histotypes and available matched ANCT specimens. SPAG9 protein expression was found in

80% (>10% of cells found positive for SPAG9 protein expression) of SGT specimens,

whereas no expression was detected in 72 paired available matched ANCT specimens as

shown in Figure 3.

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Figure 3. SPAG9 protein expression in various stages of SGT specimens

SGT specimens were also probed with control IgG which failed to show any

immunoreactivity against SPAG9 protein (Figure 3). SPAG9 protein expression was found in

63% (10 of 16) of benign tumors, 93% (13 of 14) of malignant stage I, 88% (15 of 17) of

stage II, 75% (24 of 32) of stage III and 87% (20 of 23) of stage IV tumors. In addition, 81%

(25 of 31) of specimens found positive for lymph node involvement showed SPAG9 protein

expression as compared to 85% (47 of 55) of specimens negative for lymph node

involvement. Various histotypes of SGT specimens showed SPAG9 protein expression in

63% [(10 of 16); IRS=38.40 ± 5.36] of pleomorphic benign tumors, 90% [(27 of 30);

IRS=69.19 ± 3.19] of mucoepidermoid, 83% [(10 of 12); IRS=67.30 ± 6.30] of adenoid

cystic, 80% [(4of 5); IRS=70 ± 3.58] of acinic cell, 88% [(14 of 16); IRS=70.92 ± 4.86] of

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clear cell, 80% [(4 of 5); IRS=73.50 ± 11.30] of basal cell, 70% [(7 of 10); IRS=68.80 ± 7.27]

of adenocarcinoma NOS and 75% [(6 of 8); IRS=67.60 ± 6.07] of polymorphous low grade

adenocarcinoma (Figure 4). However, control IgG showed no SPAG9 immuno-staining in

Figure 4. SPAG9 protein expression (A, B) in various histotypes of SGT specimens

any of the SGT histotypes under investigation. Thus, our data showed no discrepancy within

the results obtained from RT-PCR and IHC studies.

Humoral response against SPAG9 protein in SGT patients

The circulating anti-SPAG9 antibodies were investigated in sera of 62 available SGT patients

employing ELISA. Our data revealed that 68% (42 of 62) of SGT patients generated humoral

response against SPAG9 protein. Interestingly, we found that these patients with anti-SPAG9

antibodies were also found to be positive for SPAG9 gene and protein expression. In Figure

5A, our data revealed that humoral response generated against SPAG9 was found in 60% (6

of 10) of benign, 90% (9 of 10) of malignant stage I, 80% (8 of 10) of stage II, 48% (11 of

23) of stage III and 89% (8 of 9) of stage IV tumors. It is important to note that 74% (14 of

19) of malignant SGT patients with positive lymph node involvement generated anti-SPAG9

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antibodies as compared to 67% (22 of 33) of SGT patients with negative lymph node

involvement. In addition, the patient‟s sera of different histotypes also revealed anti-SPAG9

antibodies which demonstrated 60% (6 of 10) of pleomorphic benign tumor patients, 72% (13

of 18) of mucoepidermoid, 63% (5 of 8) of adenoid cystic, 67% (2of 3) of acinic cell, 71% (5

of 7) of clear cell, 80% (4 of 5) of basal cell, 63% (5 of 8) of adenocarcinoma NOS and 67%

(2 of 3) of polymorphous low grade adenocarcinoma patients (Figure 5A).

Figure 5. (A) ELISA-based humoral response against SPAG9 protein in SGT patients. (B)

Western blotting analyses (C) Neutralization studies in serial tissue sections of benign and

malignant stages of SGT.

We observed a significant difference (P=0.0001) among benign and malignant tumor patient

sera found positive for circulating anti-SPAG9 antibodies using Mann-Whitney U-test.

However, no significant association was found between benign and malignant tumors

(P=0.302) by Pearson‟s χ2 test. Similarly, no significant difference was found between stage

I & II (P=0.413), stage II & III (P=0.509), stage III & IV (P=0.107) and between lymph node

involved (P=0.858) malignant tumors as assessed by Mann-Whitney U-test. However, a

significant association was found between stage III & IV samples (P=0.033) as determined

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by Pearson‟s χ2 test. Kruskal Wallis test revealed no significant difference in circulating anti-

SPAG9 antibodies among various malignant stages (P=0.505) of SGT and different

malignant histotypes (P=0.798).

Further the presence of anti-SPAG9 antibodies in the sera of SGT patients was also

confirmed by Western blot analysis as shown in Figure 5B. Immuno-reactivity against

SPAG9 protein was detected in the sera of benign and malignant SGT of tumor stages and

histotypes as compared to sera from 60 normal healthy donors which showed no immune-

reactivity. Furthermore, the specificity of immuno-reactivity of patient‟s sera against

recombinant SPAG9 protein was confirmed in neutralization assays which showed complete

loss of immuno-reactivity with SPAG9 protein (Figure 5B). Likewise pre-incubation of anti-

SPAG9 antibody with SPAG9 recombinant protein (15µg/ml) led to complete loss of

immune-reactivity as depicted in Figure 5C.

Collectively, our data demonstrated that majority of SGT patients exhibit SPAG9 expression

and elicit humoral response against SPAG9 protein in the sera irrespective of the stages and

the histotypes of SGT. SPAG9 expression was associated with malignant state of SGT

patients and exhibited significantly higher antibody response in malignant tumor patients as

compared to benign tumor patients. Here we are reporting the possibility of developing serum

based biomarker for better cancer management of SGT patients. Further studies are warranted

to investigate the association of SPAG9 in large number of SGT patients.

Future plans

1. Novel cancer associated candidate discovery platform for cancer therapeutics approaches

2. Investigate the immunotherapeutic modalities for cancer treatment.

3. Study the effect of gene silencing and discovery of novel candidate targets to understand

the underlying mechanisms and signaling pathways involved in various malignant

properties of cancer cells.

Action taken on the recommendations of 2014 RAP/SAC

As suggested in the previous RAP/SAC, the progress has been achieved as reflected in the

present year reporting.

Publications

Original peer-reviewed articles

1. Agarwal S, Parashar D, Gupta N, Jagadish N, Thakar A, Suri V, Kumar R, Gupta A,

Ansari AS, Lohiya NK, Suri A (2014) Sperm associated antigen 9 (SPAG9) expression

and humoral response in benign and malignant salivary gland tumors. Oncoimmunol

3:12, e974382.

2. Singh S, Suri A (2014) Targeting the testis-specific heat-shock protein 70-2 (HSP70-2)

reduces cellular growth, migration, and invasion in renal cell carcinoma cells. Tumor Biol

35:12695-706.

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Biology of T lymphocytes

Principal Investigator Vineeta Bal

Project Associates Dr Nidhi Jain

Mr Saptak Banerjee

Ph. D. Students Arundhoti Das

Neelam Oswal

Parna Kanodia

Sanket Rane

Collaborators A George, NII

A Bagga, AIIMS, Delhi

B. Ravindran, ILS, Bhubaneswar

G Medigeshi, THSTI, Faridabad

JM Durdik, University of Arkansas, USA

N Wadhwa, THSTI, Faridabad

S Rath, NII

S Sopory, THSTI, Faridabad

S Bhatnagar, THSTI, Faridabad

S Basak, NII

S Majumdar, NII

S Vrati, THSTI, Faridabad

UCM Natchu, THSTI, Faridabad

Theme of research

Two major areas are under investigation. Firstly, role of infection and inflammation in

immune response and secondly studies in T cell fate decisions. The first area has two

components, one focussing on mechanisms regulating podocyte mediated barrier function in

the kidney; and the second part on the role of T cells in Japanese encephalitis infection. In the

second area CD4 T cell aging, survival, proliferation and differentiation are analysed in

different systems.

Objectives

1. To study mechanisms associated with renal dysfunction and proteinuria.

2. To study the role of T cells in Japanese encephalitis infection in mouse model.

3. To study the Th1/Th2 differentiation fate of CD4 T cells ex vivo and in vitro.

4. To characterise the effects of in vivo aging on CD4 T cell function.

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Work reported in 2013-2014

A. To study mechanisms associated with renal dysfunction and proteinuria.

In continuation of the work in mouse model of minimal change nephrotic syndrome, we

demonstrated that TLR2, TLR3 and TLR4 ligands given systemically lead to albuminuria in

wild type B6 mice but not in CD80-/- mice. Using irradiation and reconstitution experiments

we showed that presence of TLRs on bone marrow (BM) derived cells is necessary whereas

their presence on radiation resistant cells which includes podocytes is not. We also showed

that, in contrast, expression of CD80 on BM-derived cells is dispensable whereas ability to

upregulate CD80 on radiation resistant podocytes is not. For this demonstration in addition to

B6 and CD80-/- mice we also used LPS resistant C3H/HeJ and LPS sensitive C3H/OuJ mice,

MyD88-/- mice. Using podocyte cell lines some of these observations were confirmed in

vitro. We also showed that chitohexaose, an M2 activator of macrophages can inhibit TLR-

mediated albuminuria in preliminary experiments.

B. To study the role of T cells in Japanese encephalitis infection in mouse model.

We reported some work on mouse model of Japanese encephalitis (JE). We observed that

adult mice lacking αβ-T cells are highly susceptible and die over 10-12 day period as

compared to the wild type (WT) mice which are resistant. This was associated with higher

plaque forming units and higher levels of proinflammatory cytokines in TCRβ-/- mice.

TCRβ-/- mice showed a breach in the blood brain barrier (BBB). Further dissection of

immune components involved in T cell function was done by adoptively transferring cells

into TCRβ-/- mice prior to infection. Our data showed that CD8 cells capable of efficient

granule mediated lytic potential are necessary.

C. To characterise the effects of in vivo aging on CD4 T cell function and phenotypic

features.

We have been working on this aspect for a long time and identified some components of

molecular signalling which might contribute to individual naïve T cell aging. We showed that

malfunctioning of dual specificity phosphatase (DUSP)-6 and lower levels of miRNA181a

might be responsible for T cell aging.

Progress of work during the current reporting year (2014-2015)

A. To study mechanisms associated with renal dysfunction and proteinuria.

In the mouse model of nephrotic syndrome, we primarily used LPS, a TLR4 ligand to induce

albuminuria. Since TLR expression on BM-derived cells and CD80 expression/induction on

podocytes appears critical we looked for the connecting link, a possible soluble mediator to

connect these two events. Serum cytokine levels following LPS injection showed a high

serum levels of IL-6, TNF and IL-10 with different time kinetics. This was associated with

increase in serum CD80 levels as well. While CD80-/- mice showed upregulation of serum

cytokines to the same extent, there was no CD80 detectable in serum. Based on published

reports we speculated that TNF might be the soluble factor responsible for albuminuria.

Direct injection of TNF in B6 mice led to albuminuria whereas CD80-/- mice were

resistant. We also used TNF antagonist Etanercept to block in vivo TNF action. Injection

of LPS along with Etanercept significantly inhibited the extent of albuminuria and CD80-uria

in B6 mice, confirming the role of TNF in the pathology.

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We also identified the potential signalling intermediates responsible for TNF mediated

upregulation of CD80 on podocytes in vitro. We found that p38 MAPK, MEK/Erk signalling

was involved which possibly led to NFB-mediated transcriptional upregulation of CD80 in

the nucleus. We confirmed that TNF mediated signalling in podocytes leads not only to

induction of CD80 mRNA but also protein expression.

IL-10 is an anti-inflammatory cytokine with a dominant role in alternate activation of

macrophages. IL-10-/- mice show a hyperinflammatory phenotype. LPS injection also leads

to upregulation of serum IL-10 levels. We therefore examined the role of IL-10 in our model.

IL-10-/- mice were more sensitive to TLR mediated upregulation of serum TNF as well as

albuminuria and CD80-uria. Since we have shown that chitohexaose (Chx) can inhibit LPS

mediated albuminuria, and it is an activator of alternate activation pathway of macrophages,

we further looked for its role in the pathology. Chx injection along with LPS led to lowering

of serum TNF level and enhancement of IL-10 level, suggesting that it may be acting

through IL-10 induction. This was confirmed when Chx was injected along with LPS in IL-

10-/- mice. While this treatment decreased albuminuria and CD80-uria in B6 mice, there was

no effect on albuminuria or CD80-uria in IL-10-/- mice, implying that IL-10 is necessary for

Chx action. Both LPS and Chx use TLR4 as the receptor and hence inhibition of LPS-

mediated induction of albuminuria and CD80-uria could be explained by competitive

inhibition of LPS action. We also confirmed it by using C3H/HeJ and C3H/OuJ mice, where

C3H/HeJ mice are resistant to Chx mediated effects. However, interestingly, when poly(I:C),

a TLR3 ligand, was used to induce albuminuria, Chx treatment of mice could still inhibit

albuminuria. These data suggest that Chx mediated alternate activation of macrophages

leading to IL-10 production can have inhibitory effects on cells activated by other signals.

Together, our data propose a pathway where TLR-mediated inflammation-induced TNF

secretion by BM-derived cells leads to p38 MAP kinase, MEK/Erk and NFB signalling

dependent CD80 induction on podocytes with consequent proteinuria. Etanercept blocks this

pathogenetic process by inhibiting TNF-mediated CD80 induction on podocytes, and Chx

blocks it by TLR4-mediated M2 macrophage activation leading to IL-10 production

negatively regulating TNF production, thus identifying potential therapies for use in

minimal change NS.

B. To study the role of T cells in Japanese encephalitis infection in mouse model.

We had shown by adoptive transfer approach that CD8 T cells are necessary to provide

protection from JE infection in our mouse model. We next characterised role for CD4 T cells.

Neutralising antibodies (Abs) are reported to offer protection from JE infection and hence it

is expected that JE-specific CD4 T cells would be necessary. First we estimated JE-specific

antibodies in WT and TCR-/- mice. While JE-specific IgM Abs were detectable in both

strains of mice on day 5 and day 12 post-infection, IgG Abs were detectable only on day 12

post-infection in WT mice, not in TCR-/- mice, as expected. Next, MHCII-/- mice were

infected in parallel with WT mice and mortality observed. MHCII-/- mice survived as well as

WT mice despite near absence of CD4 T cells in the periphery. We also examined whether

any of the cytokines commonly associated with effector CD4 T cell function influence the

outcome of JE infection. Thus, IL-4-/-, IL-10-/- and IFN-/- mice were infected with JE and

mortality compared with WT mice. To our surprise these mice were as resistant to JE

infection as WT mice. Transfer of T cells from these mice to TCR-/- mice prior to infection

also offered protection comparable to that observed with transfer of WT T cells. Together,

these data show that while CD4 T cells are needed for Ab production in JE infection, during

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primary exposure to JE CD4 T cells and Abs may not be critically important, unlike CD8 T

cells.

C. To study the Th1/Th2 differentiation fate of CD4 T cells ex vivo and in vitro.

In our efforts to understand how memory cells genrated in vivo over the lifetime of the

murine or human host behave in terms of cytokine production and transcriptional regulation,

we have been comparing ex vivo CD4 memory cell functions with naïve CD4 T cells

activated in vitro. We had observed that polyclonal memory population in vivo is capable of

producing either Th1 or Th2 type of cytokines despite co-expression of transcription factors

t-bet and gata-3. We confirmed these observations using dual colour Elispot assay for ex vivo

memory cells from C57Bl.6 as well as Balb.b mice, where we found near absence of T cells

capable of producing both IFN and IL-4. We extended these findings in two different ways.

Firstly, we examined ex vivo memory CD4 T cells from an additional mouse strain FVB and

also used memory CD4 cells from aged B6 mice. Both of them also showed near absence of

dual cytokine secreting T cells. Earlier we had reported that naïve CD4 T cells activated in

non-polarising conditions in vitro also show co-expression of transcription factors but

absence of dual expression of cytokines. These findings were extended using naïve CD4 T

cells from TCR transgenic mice. Antigen-specific activation of OT-II cells from Balb.b mice

in vitro also resulted in co-expression of transcription factors but absence of dual expression

of cytokines.

D. To characterise the effects of in vivo aging on CD4 T cell function.

After showing that naïve CD4 T cells expressing lower levels of CD4 are individually older

cells by extensive functional and phenotypic characterisation, and identifying DUSP6-

miR181a mediated signalling as a key event, we looked for potential differences in early

signalling events post-activation. We did not find any differences in calcium flux or

phosphorylation of proximal signalling kinases such as Zap70 or Lck between naïve CD4lo

and CD4hi cells.

Future plans

Following are the best possible directions of research on various themes as perceived today.

Depending upon the progress made and results obtained they may change over a period of

time.

A. Based on identifying a significant role for CD80 in barrier function, we would like to

extend the observations to look at the role of CD80 in other inflammatory conditions such as

inflammatory colitis.

B. Since T cells have a role in barrier tissues, in the context of JE infection also we will

look at the role of T cells and CD80 in JE infection.

C. Attempt will be made to identify potential epigenetic changes in memory CD4 T cells

capable of producing a single category of cytokine but co-expressing transcription factors on

one hand and polarised memory CD4 cells on the other.

D. We would like to explore whether there are any metabolic differences between naïve

CD4lo and CD4hi cells by establishing in vitro cultures and/or sorting cells ex vivo.

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E. We would also like to initiate work on healthy human donors to examine whether immune

cytome undergoes seasonal changes and if so which cellular subsets show more variation and

why.

Action taken on the RAP/SAC 2014 recommendations

There were no specific suggestions conveyed to change the proposed course of

investigations, though a lot of discussion had taken place.

Publications

Original peer reviewed articles

1. Rane S, Das R, Ranganathan V, Prabhu S, Das A, Mattoo H, Durdik JM, George A, Rath

S, Bal V (2014) Peripheral residence of naïve CD4 T cells induces MHC class II-

dependent alterations in phenotype and function. BMC Biol 12: 106.

2. Rathore DK, Nair D, Raza S, Saini, Singh S, Kumar A, Tripathi R, Ramji S, Batra A,

Aggarwal KC, Chellani HK, Arya S, Bhatla N, Paul VK, Aggarwal R, Agarwal N, Mehta

U, Sopory S, Natchu UCM, Bhatnagar S, Bal V, Rath S, Wadhwa N (2015) Underweight

full-term Indian neonates show differences in umbilical cord blood leukocyte phenotype: a

cross-sectional study. PLoS One (in press)

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Epigenetic regulation of the eukaryotic genome: Role of transcriptional

insulators in organizing chromatin

Principal Investigator Madhulika Srivastava

Project Fellow Ambica Paul

Ph. D. Students Manisha Jalan

Abhilasha Kanaujia

Faizan Uddin

Collaborators K Pfeifer, NIH, USA

Theme of research

The mechanisms by which cis-acting regulatory elements interact with each other in context

of chromatin are incompletely understood even though such interactions are crucial for

appropriate regulation of nuclear processes like transcription and VDJ recombination. CTCF

dependent insulators play an important role in the functional organization of the mammalian

genome as they can coordinate intrachromosomal and interchromosomal contacts and thus

influence cis-DNA interactions. A large number of CTCF binding sites have been identified

genome-wide suggesting their extensive involvement in governing cis DNA interactions

among regulatory elements. Our efforts are directed towards understanding how the

mammalian insulators influence chromatin domain organization and contribute to regulation

of nuclear processes. A combination of genetic, molecular and biochemical approaches are

being utilized for investigations.

Objectives

Transcription as well as RAG mediated recombination is exquisitely regulated during

development at antigen receptor loci like IgH, TCR/, TCR etc. This underscores the

importance of appropriate enhancer-promoter interactions. Further, recombination requires

physical interaction between RSS elements associated with the V, D and J segments. These

segments are located at large distances from each other on the chromosome and higher order

chromatin reorganization is necessary to bring them together prior to recombination. By

exhibiting long range interactions between different types of elements, the antigen receptor

loci present a useful framework to explore the nature of interactions amongst various

regulatory elements. Long range interactions of chromatin have been demonstrated to be

orchestrated by CTCF. Taking advantage of this, we are currently investigating the

chromatin structure and organization of the wild type and genetically manipulated TCR loci

to understand various aspects of CTCF dependent insulator function as well as of other

regulatory elements important for VDJ recombination.

Work reported in 2013-2014

Organization of an ectopic CTCF dependent insulator at the TCR locus was observed to

impair enhancer E dependent transcription and D-to-J recombination in the genetically

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manipulated TCR-ins allele. Consistent with the position dependence of insulator activity, in

TCR-ins allele, promoter PD1 regulated transcription and recombination of DJC1 gene

segments was severely curtailed but promoter PD2 driven transcription and recombination

of DJC2 gene segments was comparable to the wild type TCR allele. Further, ChIP-qPCR

analysis of the region encompassing the two DJC clusters was carried out to detect the

presence of activating and repressive histone modifications, Rag2, RNAPolII and CBP on the

wild type and genetically modified alleles that had the functional insulator (TCR-ins) and

non-functional insulator (TCR-mut). The chromatin structure discerned by ChIP analysis was

consistent with the functional observations made earlier with regard to the ability of the

ectopic insulator to curtail enhancer E activity. Ehas been proposed to regulate the locus

by “looping” with the promoters as well as by some form of “tracking.” Keeping this in view,

chromosome conformation capture (3C) analysis was initiated to investigate the interactions

between E, PD1, PD2 and the ectopic insulator.

We have earlier reported that in addition to blocking the enhancer activity at TCRlocus, the

inserted H19-ICR also altered the choice of V segments used for VDJ recombination in a

CTCF dependent manner suggesting the ability of CTCF to modulate interactions between

cis-regulatory elements other than promoters and enhancers. We have identified a few

regions of the TCR locus that bind CTCF and standardized chromosome conformation

capture (3C) assay to investigate the higher order chromatin organization defined by CTCF

binding. It was evident that the extent of CTCF occupancy on the identified CTCF binding

sites as well as the frequency of interactions amongst them was variable. Additionally, the

insertion of ectopic CTCF binding sites as H19-ICR (as created in TCR-ins mutant mice)

altered the pattern of interactions as ascertained by allele specific-3C-qPCR strategy. Each of

the interactions investigated i.e. C3-C5, C3-C6 and C6-C5 were reduced 2-3 fold in the TCR-

ins allele compared to the wild type allele. This effect was CTCF dependent as the TCR-mut

allele, unable to bind CTCF at the ectopic locations, did not show such a reduction. Our

analysis suggested that CTCF based chromatin loops are relevant for V-to-DJ recombination

at TCR locus and get altered under the influence of ectopic CTCF binding sites. The

alteration has a significant functional outcome as reported earlier.

Progress of the work during current reporting year (2014-2015)

To understand the interactions between E, PD1, PD2 and the ectopic insulator, we have

extended our previous year‟s analysis. Appropriate conditions for 3C-qPCR (allele specific)

assay have been established and the interactions between these elements in the wild type,

TCR-ins and TCR-mut alleles have been examined. We observe E-PD1 interaction to be

significantly reduced in TCR-ins allele compared to the wild type and TCR-mut alleles. This

suggests that an intervening insulator prevents looping of enhancers to the promoter. In

context of our chromatin structure analysis reported in the previous year, our results suggest

that within the framework of altered chromatin loopscape that segregates the enhancer and

promoter into separate loops, insulators also interact with the enhancer and promoter as well

as organize a barrier to the interactions and/or to the influence of the enhancer on the

insulated domain. Consequently, they may simultaneously appear to be an enhancer decoy,

promoter decoy as well as elements that prevent tracking of enhancers and/or of other

epigenetic changes in case the enhancers employ these mechanisms to activate the locus.

Several CTCF binding sites have been identified at various antigen receptor loci including

TCR locus. Our ChIP analysis indicated significant variation between the CTCF binding

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sites with regard to their occupancy by CTCF and in their ability to interact with each other.

To investigate this intriguing aspect, we have initiated characterization of CTCF binding

sites. The variation may be a consequence of DNA sequence of the core CTCF binding sites,

the regions flanking them, variation in the nucleosomal structure in the vicinity of CTCF

binding sites, presence of other proteins in the flanking regions etc. These variations are

likely to dictate the extent of binding/eviction of CTCF, interactions amongst them, ability to

act as insulators etc. and thus help to understand their relevance for recombination. We have

purified the CTCF protein (amino acids encompassing the Zn finger domains that bind the

DNA). This will be used in EMSA assays to determine the affinity of the protein to various

CTCF binding sites of the TCR locus in vitro. To complement these studies, an in vitro

enhancer blocking assay has been established that will be used to examine the ability of the

CTCF binding sites to act as insulators.

Even though the degree of binding of CTCF and cohesin to these sites does not vary

substantially between Double negative (DN) T cells and ProB cells, the interactions between

the CTCF binding sites were observed specifically in the DN cells suggesting that T cell

lineage specific factors might contribute to the locus organization. Since E is a critical

regulatory element that is active specifically in the DN cells, it is possible that E and/or the

proteins associated with it contribute to the TCR locus organization and thus impact V-to-

DJ recombination beyond generating the accessibility of 25kb region of DJC clusters. This

view is negated by a recent report which demonstrated the ability of V regions to interact

with DJC regions in the absence of E. The sufficiency of this interaction for V-to-DJ

recombination could not be tested as absence of the enhancer precluded even D-to-J

recombination. However, in TCR-ins allele, the enhancer activity is curtailed in a position

dependent manner but the enhancer is active and V-to-DJ recombination operative. We have

observed that functional curtailment of E activity by the ectopic insulator correlates with

impairment of V-to-DJ recombination in a position dependent manner. Does this suggest an

involvement of E in V-to-DJ recombination in some manner ? To dissect these issues, we

have initiated a study to examine interaction of V regions with the E and DJC clusters in

wild type TCR locus as well as in presence of functional or non-functional insulator. Our 3C

analysis so far indicates that V segments interact with Eto a variable extent which is not

correlated to their usage for VDJ recombination.

Future plans

The possible reasons underlying variation between CTCF binding sites of TCR locus will be

examined. Interaction of V regions with DJC regions and the enhancer E will be

evaluated to gain insights into the possible role of E in V-to-DJ recombination and efforts

will be made to evaluate the alterations in interactions amongst various regions of TCR

locus when CTCF is knocked down.

Action taken on the RAP/SAC 2014 recommendations

The projects being pursued were discussed in detail. It was suggested that the TCR locus

organization be investigated in cells where CTCF has been knocked down. These

experiments are in progress.

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Analysis of Salmonella typhi-host cell interaction

Principal Investigator Ayub Qadri

Ph. D. Students Farhat Parveen

Sonia

Jitender Yadav

Mohd. Anees Ahmed

Sana

Theme of research

Pathogenic Salmonella species produce different clinical manifestations depending upon

Salmonella serovar and the type of host. In humans, Salmonella Typhi causes systemic

infection, typhoid, in which bacteria spread to spleen, liver, bone marrow and gall bladder

after invading the intestinal epithelium; infection with non-typhoidal serovar Salmonella

Typhimurium produces only self-limiting localized gastroenteritis. In contrast, S.Typhi does

not establish infection in normal mice whereas Salmonella Typhimurium infection in mice

results in a systemic outcome that is analogous to human typhoid. The reasons for different

clinical outcomes produced by S.Typhi and S.Typhimurium and for the host specificity

exhibited by these two closely related Salmonella serovars are not understood. Further, how

pathogenic Salmonella subverts host immune responses in order to establish infection is also

not clear. Work in our laboratory focuses on these two aspects of host-pathogen interaction

during Salmonella infection.

Objectives

Broadly, our studies are aimed at

i) identifying differences in host-pathogen interactions which ensue during infection with

S.Typhi versus S.Typhimurium.

ii) understanding modulation of immune responses during infection with pathogenic

Salmonella. This also includes investigating changes that Salmonella might undergo upon

sensing host cues and studying relevance of these changes in inflammation and immunity.

iii) understanding the regulation of Toll-like receptor (TLR)-activated inflammatory and

innate immune responses.

Work reported in 2013-2014

We have been studying how the two-way host-pathogen cross-talk might regulate

inflammatory and innate immune responses during infection with pathogenic Salmonella. We

showed that the expression of a key TLR/NLR (Nod-like receptor) ligand from Salmonella

might be regulated through sensing of host-derived lipid(s). Last year we reported that this

flagellin expression-inducing activity might also be generated as a result of caspase-1

activation triggered early on through intracellular delivery of flagellin by Salmonella. These

results suggested that activation of caspase-1 by flagellin and induction of flagellin

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expression by caspase-1 - dependent host signal(s) might serve as an amplification loop for

caspase-1 - dependent pyroptosis, which would serve to bring about clearance of the

pathogen. Downregulation of flagellin expression that is seen during progression of infection

in mice would limit pyroptosis and in turn promote establishment of infection. The

mechanism responsible for the downregulation of flagellin expression is currently under

investigation in our laboratory.

Alongside understanding modulation of host immunity by Salmonella and the reasons for

host specificity shown by different Salmonella serovars, we have also been investigating

regulation of TLR-induced cellular responses which play a vital role in inflammation and

immunity. Last year, we showed data to establish that TLR responses might be amplified by

host lipids including lysophosphatidylcholine (LPC). The induction of cytokines by TLR

agonists was reduced when LPC - specific G-protein coupled receptor (GPCR), G2A, was

blocked with an antibody. These results unveiled a cross-talk between TLRs and G2A in the

activation of inflammatory and innate immune responses with microbial TLR ligands. We

also showed that in human T cells, engagement of TLRs with microbial TLR agonists readily

brings about secretion of neutrophil chemoattractant CXCL8. Interestingly, this response was

considerably reduced when T cells were first activated through the T cell receptor. The

reduction was seen at the mRNA as well as protein level. TCR activation did not however

alter activation of proximal signaling intermediates by these ligands.

Progress of work during the current reporting year (2014-2015)

A. T cell receptor - generated signals regulate innate immune responses from human T

cells

We reported previously that freshly isolated CD4 T cells from human peripheral blood

secrete neutrophil chemoattractant CXCL8 upon stimulation with TLR2 and TLR5 agonists

Pam3CSK4 and flagellin respectively. However, when these cells were first activated through

the T cell receptor with a cocktail of anti-CD3 antibody and anti-CD28 antibody, their ability

to produce CXCL8 was considerably reduced. TCR activation did not however affect

activation of NF-kB and MAP-kinase pathways of intracellular signaling in response to TLR

ligands. We now show that the reduction in CXCL8 secretion is also observed during

stimulation of T cells with the endogenous innate stimulus, IL-1. More importantly, while

freshly isolated CD4 T cells did not produce any IFN- upon incubation with Pam3CSK4 and

flagellin, TCR-primed cells began to secrete IFN- in response to these TLR agonists in the

absence of concurrent TCR engagement. These T cells showed increased activation of p38

and JNK MAP-kinases in response to TLR stimulation, and inhibition of p38 abrogated TLR-

induced IFN- secretion. The change of innate immune response from CXCL8hi

IFN-null

in

freshly isolated to CXCL8lo

IFN-hi

in activated T cells was also observed in response to IL-1.

Our results suggest that the innate immune response of human T cells might shift from a

proinflammatory to an effector type following activation of these cells through the T cell

receptor. These findings have significant implications for T cell-mediated antimicrobial

immunity.

B. Caspase-1 regulates replication of Salmonella in macrophages

Caspase-1 activation following infection with pathogenic Salmonella results in cell death

associated with release of IL-1 and IL-18 collectively termed pyroptosis. One of the

bacterial effectors that brings about caspase-1 activation is flagellin. Previous work from our

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laboratory showed that sensing of lysophospholipids derived from intestinal epithelial cell

(IEC) or pyroptotic macrophages activates release of proinflammatory flagellin from

Salmonella thereby revealing a novel mode of regulation of inflammatory responses during

infection with this pathogen. In addition to flagellin (and Salmonella pathogenicity island-1

effectors (SPI-1) which activate caspase-1 through activation of the Nlrc4 inflammasome,

Salmonella also brings about caspase-1 activation through activation of the Nlrp3

inflammasome by Salmonella pathogenicity island-2 (SPI-2) - dependent as yet unidentified

ligand. As the infection progresses, the expression of flagellin in Salmonella is

downregulated (Cummings LA et al., J Immunol. 174:7929, 2005; our unpublished data), a ploy that

abrogates Nlrc4 - dependent activation of caspase-1 and consequently promotes

establishment of infection.

On the other hand, expression of SPI-2 is required for the establishment of systemic infection,

which raises a possibility that Nlrp3-dependent caspase-1 activation might occur during later

stages of infection. We therefore sought to investigate if casapse-1 activation that might be

triggered through SPI-2 - dependent mechanism has a role to play in bacterial replication /

persistence. Interstingly, ex vivo treatment of splenic macrophages obtained from mice on day

5 post S.Typhimurium infection with caspase-1 inhibitor zYVAD resulted in reduction in

intracellular bacterial numbers in a concentration – dependent manner. Conversely, treatment

of these infected macrophages with casapse-1 activators such as alum increased intracellular

bacterial load. This effect was dependent on caspase-1 as treatment of S.Typhimurium –

infected bone marrow-derived caspase-1 deficient macrophages with alum did not affect

intracellular bacterial load. The effect was however not specific to alum as similar increase

was obtained when cells were transfected with flagellin protein or LPS, former, as mentioned

above, activates caspase-1 through Nlrc4 inflammasome and the latter through a non-

canonical casapse-11 - dependent pathway. Preliminary data suggests that casapse-1 might

regulate intracellular Salmonella replication through modulation of cellular acidification.

These results reveal a previously unappreciated role for caspase-1 in regulating bacterial

replication during establishment of infection with pathogenic Salmonella.

Salmonella Typhi infection in mice activates an anti-bacterial activity that limits bacterial

replication

Salmonella Typhi produces systemic infection (typhoid) in humans but does not establish

infection in mice. On the other hand, S.Typhimurium causes self-limiting localized

gastroenteritis in human but produces a systemic infection in mice that is similar to human

typhoid. To investigate reasons for this host specificity, we infected mice intraperitoneally

with S.Typhi and S.Typhimurium and analysed cellular influx and bacterial load in the

peritoneal cavity, and determined cytokines in serum. Infection with both these Salmonella

serovars resulted in recruitment of CD11b and Gr1 positive cells to the site of infection.

Serum IL-6, IL-12p40 and TNF-α levels were much higher in S.Typhimurium - infected

mice. On the other hand, bacterial load, both intracellular and extracellular, was (as expected)

considerably lower in S.Typhi-infected mice indicating that murine peritoneum not only

disallowed intracellular S.Typhi replication but it also did not permit its extracellular

replication. To analyse if that might be due to induction of an anti-bacterial activity with

S.Typhi, cell free peritoneal fluids were collected from Salmonella-infected mice and their

ability to inhibit bacterial replication in vitro was checked. The results showed a dose-

dependent reduction in growth of S.Typhimurium treated with cell free peritoneal fluid from

S.Typhi - infected mice. This reduction was severeal fold higher than that obtained with

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peritoneal fluid from S.Typhimurium-infected mice. The production of this activity was not

dependent on MyD88 indicating that it may not be induced through TLR activation. In vitro,

although this activity was not produced as efficiently as in vivo, its secretion required

infection with live bacteria; neither antibiotic- treated S.Typhi nor bacterial extract prepared

from S.Typhi led to production of this activity. These results suggest that induction of anti-

bacterial factor(s) with S.Typhi might be one of the reasons that prevents establishment of

systemic infection with this Salmonella serovar in mice.

Vi suppresses activation of Rac-1/Cdc-42 activation during Salmonella infection

Our previous findings showed that Salmonella Typhi can employ Vi to suppress

inflammatory and innate responses that are triggered in intestinal epithelial cells and

mononuclear phagocytes through activation of TLRs. It is however known that during

infection of IECs, inflammatory responses with Salmonella are not only produced through

ligation of membrane and endosomal TLRs but also through activation of Cdc42/Rac-1 by

effectors that are delivered intracellularly by pathogenic Salmonella. We therefore asked a

question if engagement of membrane prohibitin with Vi would also modulate this kind of

inflammatory response. The results showed that treatment with Vi inhibits activation of Rac-

1 and reorganization of actin cytoskeleton in Salmonella-infected cells. This modulation

suppressed activation of NF-kB and MAP-kinase pathways of intracellular signalling that are

required for the production of neutrophil chemoattractant CXCL8 and other immune

mediators. Consistent with this finding, infection of epithelial cells with Vi positive

Salmonella Typhi resulted in much less CXCL8 secretion as compared to Vi negative

Salmonella Typhi. Our results provide new insights into how targeting of membrane

prohibitin with Salmonella Typhi virulence polysaccharide might downregulate antimicrobial

epithelial responses and contribute to establishment of infection.

Future plans

Two way host-pathogen cross-talk in the modulation of immunity and establishment of

infection with Salmonella

It is clear from our findings that the establishment of infection with Salmonella is not a one-

way affair. Pathogen - sensing by the host brings about induction of inflammatory and innate

immune responses that are crucial for clearance of the pathogen from the host. Pathogens

also deliver effectors that have the ability to modulate a number of immune activities.

Importantly, in the course of this host-pathogen cross-talk, pathogen can also sense host-

derived cues and respond by upregulating or downregulating molecules, which might be

relevant to the establishment of infection. Recent findings from our laboratory suggest that

activation of caspase-1 with effectors from Salmonella might regulate how the pathogen

behaves intracellularly and this behavior seems to result from sensing of host cues which are

generated as a result of caspase-1 activation. Not only does this influence the expression of

one of the key TLR/NLR ligands, flagellin, which plays a crucial role in immunity against

Salmonella but our preliminary data suggest that caspase-1 - generated cues might also

modulate replication and fitness of the pathogen. In future studies we would like to

understand the nature of these cues and the mechanism by which these host signals might

regulate establishment of infection with Salmonella. Long term, we would also like to

investigate if host-sensing by Salmonella alters expression of molecule(s) that might be direct

targets of the adaptive immune system.

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Understanding host specificity of S.Typhi infection, and analysis of S.Typhi-specific

host-pathogen interactions

Mouse model of Salmonella infection

Our data with this infection model suggests that induction of an innate immune activity might

limit extracellular replication of S.Typhi. Studies have been initiated to establish the identity

of this activity and investigate its relevance in intracellular replication of Salmonella.

Role of Vi in host-pathogen interaction

Our results from in vitro infections with epithelial cells show that S.Typhi can engage Vi to

inhibit anti-microbial responses that are produced as a result of activation of GTPases, Rac-1

and Cdc42, following sensing of bacterial effectors by cells. We would like to understand the

mechanism by which Vi-prohibitin interaction inhibits Salmonella-induced activation of

GTPases and suppresses inflammatory / immune responses.

Elucidating IEC-DC interaction during infection with Salmonella

The eliciting of different clinical manifestations by Salmonella Typhi and Salmonella

Typhimurium in humans might be determined early on as a result of different responses

produced by intestinal epithelial cells following infection with these two Salmonella serovars.

These responses might also influence how immune cells such as dendritic cells underlying

epithelial cells handle these two pathogens. We have therefore initiated studies on elucidating

IEC-DC interaction during infection with Salmonella.

We will also continue our studies on understanding the cross-talk between TLR and TCR in

human T cells.

Action taken on the RAP-SAC 2014 recommendations

The suggestions given by the members during the last RAP-SAC interactive meeting were

very valuable in our studies.

Publications

Original peer-reviewed articles

1. *Santhanam SK, Dutta D, Parween F, Qadri A (2014) The Virulence polysaccharide Vi

released by Salmonella Typhi targets membrane prohibitin to inhibit T cell activation. J

Infect Dis 210: 79-88.

*in press last year, since published

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Molecular Basis of B cell Responses

Principal Investigator Devinder Sehgal

Project Associate Preetika Arya

Ph. D. Students Jaya Bhushan

Ruchika Dehinwal

Hina Jhelum

Sujata Kumari

Ajay Kumar

Manoj Kumar Rajak

Collaborators AK Panda, NII

K Natarajan, University of Delhi

RP Roy, NII

Theme of research

The theme of research is to decipher the molecular and cellular basis of immune response

against protein and polysaccharide antigens present on the surface of the human bacterial

pathogen Streptococcus pneumoniae (also called pneumococcus). The other research interest

is to find out how pneumococci cause disease and what interventions can be made to stop this

from happening. The research is focused on the pneumococcal products and strategies that

allow the pathogen to avoid being destroyed by the mammalian immune system, and the

types of immune response that can circumvent these strategies and products.

Objectives

The main objectives are (a) molecular analysis of immune response to pneumococcal cell

surface protein and polysaccharide antigens, (b) identification and characterization of

virulence factors such as toxins and adhesins from S. pneumoniae that are or may be related

to pathogenesis, (c) how these virulence factors interact with the immune system and host

cell to alter its cellular and molecular processes, and (d) evaluating the vaccine potential of

pneumococcal cell surface proteins.

Work reported in 2013-2014

Identification and functional characterization of secreted nuclease(s) from S.

pneumoniae

Neutrophils, a part of the innate immune system, play an important role during pneumococcal

infection. They clear the pathogen by deploying strategies like phagocytosis followed by

killing and releasing antimicrobial peptides upon degranulation. Apart from these, a novel

defense mechanism has been reported (referred to as NETosis) wherein neutrophils release

neutrophil extracellular traps (NETs). These traps are a framework of chromatin, histones and

antimicrobial proteins. NETs entrap the bacteria in the chromatin network and thereby

113

prevent their dissemination in the host. Pneumococcus has evolved a strategy to counter

NETs by expressing either membrane localized or secreted nuclease(s). The endonuclease

EndA from S. pneumoniae has been reported to be involved in clearing NETs by degrading

chromatin. The presence of additional nuclease(s) was inferred from published studies done

using endA deficient pneumococci. We are interested in identifying and characterizing these

nuclease(s). Towards this end, we constructed an autolysin (lytA) deficient strain of

pneumococci. We observed nuclease activity in the pneumococcal culture supernatant,

indicating the presence of nuclease(s) in the secretome. Further, the nuclease activity was

proteinase K sensitive, heat-labile and the activity was lost in the presence of EDTA

suggesting the requirement of divalent cations for its activity.

Functional characterization of lipoproteins from S. pneumoniae

Lipoproteins constitute one of the most abundant classes of surface proteins of S.

pneumoniae. Several lipoproteins have been demonstrated to serve as the substrate binding

protein of ABC transporters. Given their surface localisation, and importance in

pneumococcal fitness and virulence we focussed our study on the functional characterization

of pneumococcal lipoproteins. We constructed a pneumococcal strain deficient in lipoprotein

biosynthesis by deleting lipoprotein diacylglyceryl transferase (lgt) by inframe gene

replacement mutagenesis. The mutant was confirmed by nucleotide sequencing and flow

cytometry. To study the role of lipoproteins in modulation of alveolar macrophage function

we prepared Triton X-114 extract from wildtype and lgt deficient S. pneumoniae. We have

used a model murine alveolar macrophage cell line MH-S for our study. We observed that

deletion of lgt abrogated adhesion of S. pneumoniae to MH-S cells by >50%. The amount of

IL-6 and IL-12 induced in response to live and heat-inactivated lgt deficient pneumococci,

and Triton X-114 extract from lgt deficient pneumococci was significantly reduced compared

to the corresponding preparation of wildtype pneumococci. We quantified nitrite production

by MH-S cells in presence of live wildtype and live lgt deficient S. pneumoniae, and also

heat-inactivated wildtype and lgt deficient S. pneumoniae by Griess assay. Nitrite production

in the presence of live and heat-inactivated lgt deficient S. pneumoniae was significantly

reduced in comparison to the nitrite produced in presence of live and heat-inactivated

wildtype strain. To confirm our result we incubated the MH-S cells with Triton X-114 extract

from the wildtype and the lgt deficient pneumococci. We observed that incubation of MH-S

cells with extract from wildtype S. pneumoniae augmented the production of nitric oxide

(NO) while extract from lgt deficient S. pneumoniae did not. The production of nitrite by

MH-S cells was abrogated in the presence of inducible nitric oxide synthase (iNOS) inhibitor

L-NAME.

Progress of work during the current reporting year (2014-2015)

Identification and functional characterization of secreted nuclease from S. pneumoniae

One of the ways neutrophils kill bacterial pathogens is by releasing what are referred as

neutrophils extracellular traps (NETs). Pathogens like S. pneumoniae get trapped in NETs

and their dissemination is restricted in the host. Other workers have demonstrated that

endonuclease EndA helps S. pneumoniae evade NETs by degrading the DNA present in

NETs. Evidence suggests that additional nuclease(s) may be present. Last year, we presented

preliminary data in this regard.

114

To test whether pneumococcal culture supernatant degraded NETs, neutrophils were isolated

from the peritoneal cavity of mice and stimulated with PMA (phorbol 12-myristate 13-

acetate) to induce NET formation. Culture supernatant from lytA deficient pneumococci was

overlaid on the neutrophils. The cells were fixed, stained for myeloperoxidase and DNA, and

visualized by confocal microscopy. Pneumococcal culture supernatant degraded NETs in a

dose dependent manner. To further identify putative DNase, in-gel activity assay was

performed with pneumococcal culture supernatant. In this assay, the secretome was resolved

by SDS-PAGE containing calf thymus DNA. The gel was washed with water to remove SDS

and further incubated in renaturation buffer. The gel was stained with ethidium bromide and

counterstained with silver stain. The gel slices corresponding to the 3 DNA degradation

bands observed were analyzed by mass spectroscopy. Mass spectrometry data revealed the

presence of multiple proteins from each band. Each protein was analyzed on the basis of

MASCOT scores, molecular weight, unique peptides, conserved domains and literature. We

identified a protein SPD_1788 as a probable candidate. The gene encoding SPD_1788 was

PCR amplified from S. pneumoniae D39 genome, cloned and expressed in E. coli. SPD_1788

was purified to homogeneity from inclusion bodies.

Functional characterization of lipoproteins from S. pneumoniae

Lipoproteins are a major class of biomolecules present on the surface of S. pneumoniae.

Lipoproteins have been shown to be an important component of ABC transporters involved

in the uptake of nutrients. Last year, we reported that absence of lipoprotein diacylglyceryl

transferase (Lgt) resulted in the loss of lipoproteins from the surface of S. pneumoniae and

deletion of lgt abrogated adhesion of S. pneumoniae to model murine alveolar macrophage

cell line MH-S. We also reported that lipoproteins augment the production of

proinflammatory cytokines (IL-6, IL-12 and TNF-α) and nitric oxide (NO) from MH-S cells.

Lipoprotein mediated NO production was inhibited in the presence of inducible nitric oxide

synthase (iNOS) inhibitor L-NAME.

Alveolar macrophages were isolated from C57BL/6 mice and stimulated with Triton X-114

extract from the wildtype and lgt deficient S. pneumoniae. We quantified the production of

proinflammatory cytokines IL-6, IL-12 and TNF-α in the culture supernatants by ELISA. We

observed a significant increase in the production of proinflammatory cytokines in the

presence of Triton X-114 extract from wildtype S. pneumoniae. However, the amount of

these cytokines produced in presence of Triton X-114 extract from the lgt deficient strain was

comparable to the untreated control. The amount of NO produced by ex vivo alveolar

macrophages from C57BL/6 mice was also significantly enhanced in presence of Triton X-

114 extract from wildtype S. pneumoniae compared to the corresponding extract from the lgt

deficient strain. Incubation of MH-S cells with Triton X-114 extract from wildtype S.

pneumoniae resulted in the upregulation of iNOS. There was no induction of iNOS in control

samples treated with Triton X-114 extract from the lgt deficient S. pneumoniae. These data

confirmed our previous observations with MH-S cells and suggested that lipoproteins

modulate innate effector functions of alveolar macrophages. We were further interested in

dissecting the molecular mechanisms that are involved in modulation of alveolar macrophage

function. Since, modulation of host cell functions by lipoproteins is reported to be dependent

on recognition of lipoproteins by TLR2 present on host cells, we were interested in finding

out whether this was also true for pneumococcal lipoproteins. We stimulated MH-S cells with

Triton X-114 extract from wildtype and lgt deficient S. pneumoniae in presence of anti-TLR2

blocking antibody, and quantified cytokines and NO produced in the supernatant. We

observed significant abrogation in the production of TNF-α, IL-6 and IL-12 as well as NO in

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wildtype Triton X-114 extract stimulated samples in presence of anti-TLR2 blocking

antibody. This led us to conclude that recognition of pneumococcal lipoproteins by TLR2 is

important for modulation of alveolar macrophage function by S. pneumoniae. We next

explored the signalling pathways that are activated by lipoprotein-TLR2 interaction in

alveolar macrophages. We stimulated MH-S cells with an extract from the wildtype

pneumococci for various durations and analyzed the phosphorylation status of ERK, JNK and

p38 and, degradation of IκB by immunoblotting. We observed that lipoproteins activated

MAPKs and NF-κB pathways. We used signalling pathway specific inhibitors to identify

which of the MAPKs are responsible for the production of IL-6, TNF-α and NO. Our data

suggested that both ERK and JNK governed production of proinflammatory cytokines.

However, NO production was largely influenced by JNK pathway.

Future plans

The role of SPD_1788 in host-S. pneumoniae interaction would be assessed using ex vivo

neutrophils and by infecting mice. In addition, SPD_1788 would be biochemically

characterized.

To gain a better understanding of host-S. pneumoniae interaction, we screened pneumococcal

surface proteins on the basis of their conservation across various serotypes. One such protein

that showed up in our screen was CbpL. Bioinformatic analysis of CbpL suggested that it has

an excalibur domain at its N-terminus and a glucan-binding domain (YG repeat) in the

middle portion of the protein. Some workers have predicted CbpL to be a choline binding

protein. Pneumococcal surface proteins have been documented to serve as virulence factors,

and to have a role in nasopharyngeal colonization and invasive disease. We propose to study

CbpL in the context of host-S. pneumoniae interaction. The excalibur domain present in

CbpL shows similarity with the calcium binding motif of EF-hand Ca2+

-binding domain. We

propose to check whether CbpL binds calcium and whether calcium binding has any

implication for its functional activity. Similarly, we would like to test whether CbpL binds

glucans and/or choline. We plan to evaluate the role of CbpL in pneumococcal virulence/

pathogenesis by generating cbpL deficient pneumococci. We will also genetically

complement the cbpL deficient strain. The wildtype, cbpL deficient and genetically

complemented strains will be used for studying the role of CbpL in adhesion, invasion,

complement deposition and phagocytosis.

Action taken on the RAP/SAC 2014 recommendations

The work was presented before the RAP-SAC members. The scientific and technical

clarifications sought were provided. There was no specific recommendation from the

committee members.

Publications

Original peer-reviewed articles

1. Anish C, Upadhyay AK, Sehgal D, Panda AK (2014) Influences of process and

formulation parameters on powder flow properties and immunogenicity of spray dried

polymer particles entrapping recombinant Pneumococcal surface protein A. Int J Pharm

466: 198-210.

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2. Khan N, Qadri RA, Sehgal D (2015) Correlation between in vitro complement deposition

and passive mouse protection of anti-Pneumococcal surface protein A monoclonal

antibodies. Clin Vaccine Immunol 22: 99-107.

3. Saxena S, Khan N, Dehinwal R, Kumar A, Sehgal D (2015) Conserved surface accessible

nucleoside ABC transporter component SP0845 is essential for pneumococcal virulence

and confers protection in vivo. PLoS One 10: e0118154.

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To develop strategies for making sensors and actuators for biological

processes

Principal Investigator PK Upadhyay

Ph. D. Students Srikant Iyer

Barun Das

Alaknanda Mishra

Preeti Sahay

Kshama Jain

Research Associates Jashdeep Bhattacharjee

Collaborators S Bhaskar, NII

AK Panda, NII

Asok Mukhopadhyay, NII

P Nagarajan, NII

P Khanduri, St. Stephen‟s Hospital, Delhi

JM Puliyel, St. Stephen‟s Hospital, Delhi

R Juneja, AIIMS, Delhi

S Ramakrishnan, AIIMS, Delhi

R Lodha AIIMS, Delhi

S Varghese, Christian Blind Mission, Bengaluru

A Bhatnagar, INMAS, Delhi

S Khushu, INMAS, Delhi

Theme of research

To develop systems for monitoring biological processes.

Objectives

To develop tools for needle free immunization and cell therapy.

To study the biological processes like differentiation, hybridization etc. and to develop

devices and sensors based on such studies.

Work reported in 2013-2014

Differentiation of PBMCs to endothelial-like cells

Peripheral blood mononuclear cells (PBMCs) were differentiated to endothelial like cell by

culturing them in an appropriate angiogenic medium. Different approaches were investigated

for differentiating human PBMCs into endothelial cells.

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In the first approach, they were isolated by magnetically labeled antibody against CD133.

The CD133+ cells were then cultured in IMDM medium and characterized by the

incorporating DiI labeled acetylated low density lipoprotein (DiI-acLDL). In the second approach, the PBMCs were trans-differentiated into endothelial cells by

culturing them in EGM-2 medium supplemented with angiogenic cytokines like VEGF, FGF,

IGF in addition to hydrocortisone and heparin. The characterization of the cells was done by

RTqPCR, western blotting as well as immunocytochemistry.

In the third approach, a two steps procedure was followed for the differentiation of

monocytes into endothelial cells. In the first step, the monocytes were de-differentiated into

stem cell like cells called reprogrammed monocytes (RM). In the next step, the

reprogrammed monocytes were again re-differentiated into endothelial cells by culturing

them in medium containing 10% serum and 100 ug/ml endothelial cell growth supplement

(ECGS) for 15 days. The endothelial-like cells obtained at the end of the culture term were

characterized by RTqPCR and western blotting.

Hepatocytes like cells from PBMCs

The reprogramming of PBMCs was further investigated. It was observed that the

concentration of serum played a critical role in the degree of „acquired plasticity‟ in the

Reprogrammed Monocytes (RM). Non activated monocytes were isolated from the peripheral

blood of healthy volunteer and cultured with four different types of serums namely,

autologous human serum (AHS), human cord serum (HCS), embryonic stem cell grade fetal

bovine serum (ESC) and fetal bovine serum (FBS). The extent of „acquired plasticity‟ in

RM, as measured by the percentage abundance of CD34 and CD117, was highest by HCS

followed by ESC, AHS and FBS.

Engrafting RM and hepatocyte like cells

Partial hepatectomy of left liver lobe of SCID mouse were performed to generate the liver

injury model in immune-compromised mouse. Cells were transplanted intraspleenically

immediately after the partial hepatectomy. 24 days after partial hepatectomy the presence of

human specific Glyceraldehyde-3-phosphate dehydogenase (hu-GAPDH) was observed in

RT-PCR of different liver lobes.

Progress of work during the current reporting year (2014-2015)

A. Hepatocytes like cells from PBMCs

To evaluate whether the in vitro generated hepatocyte like cells (NeoHeps) can be a good

candidate for cell based therapy, NeoHeps were transplanted through spleen in a partially

hepetectomised SCID mouse. Homing and engrafting of NeoHeps was determined in the

regenerated liver tissue section by immunohistochemistry 10 days post transplantation.

The immunocytochemistry for human albumin and human connexin 32 in the tissue section

of the NeoHeps transplanted hepatectomized SCID mouse liver confirmed the engraftment of

human monocyte derived NeoHeps in the liver cortex (Figure 1).

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Figure 1. Confocal Image at 63X magnification of liver tissue section of (A) hepatectomized

SCID mouse without cell transplantation and (B) hepatectomized SCID mouse injected with

healthy NeoHeps. Connexin32 (Alexa Fluor 488), (2) Albumin (Alexa Fluor 594), (3)

Nucleus (DAPI) and (4) merge picture.

In order to confirm the functionality of the engrafted human NeoHeps in the mouse liver, the

presence of human albumin was checked in mouse serum 10 days post transplantation by

human albumin ELISA. The functional activity of transplanted NeoHeps, derived from

monocytes isolated from peripheral blood, was evident from the presence of human albumin

in the serum of the recipient mouse detected by ELISA .

To confirm the engrafted cells in the mouse liver tissue section were of human origin,

fluorescence in situ hybridization (FISH) was performed against the probe of human

centromeric region of chromosome 9. Binding of DNA probe specific to human chromosome

9 centromeric regions in the nucleus of the liver tissue section of NeoHep transplanted

hepatectomized SCID mouse further confirmed the engraftment of human origin cell in the

mouse liver cortex (Figure 2).

Figure 2. Fluorescence In Situ Hybridization on NeoHep transplanted 1/3rd partial

hepatectomized SCID liver section by probe specific to human chromosome 9 centromere.

The probe was biotinylated and streptavidin AlexaFluor 594 was used for detection.

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B. Differentiation of PBMCs to endothelial-like cells

In this project we aim to construct a small diameter (2-5mm diameter) tissue engineered

vascular graft which is immune friendly as well as long lasting as compared to the presently

available grafts.

The peripheral blood mononuclear cells (PBMCs) were reprogrammed to stem cell like cell

followed by its re-differentiation into endothelial–like cells and vascular smooth muscle like-

cells. These cells express prominent endothelial and smooth muscle cell markers as well as

show functional activity.

The re-differentiated vascular cells were then uniformly seeded onto a decellularized

xenogeneic vessel matrix using a bioreactor setup such that endothelial cells form the luminal

layer while smooth muscle cells form the outer layer of the graft.

Decellularization of rat inferior vena cava

A blood vessel is composed of a collagen matrix on which are overlaid the vascular cells i.e.

endothelial cells on the luminal side and smooth muscle cells on the external surface. An

allogenic or xenogenic transplantation of this vessel can lead to acute rejection of the graft.

Decellularization removes the cellular component of the graft leaving behind a collagen

scaffold. The structure of collagen is conserved through species making this acellular

collagen scaffold non-immunogenic.

The inferior vena cava of rat was selected for decellularization and subsequent

recellularization. The harvested rat inferior vena cava was decellularized with a 1% solution

of Triton-x 100 in MQ water using a continuous perfusion pump setup. The time period of

decellularization was standardized by a kinetic study, in which the vessel was decellularized

for different time periods and characterized to check the efficiency of decellularization.

Reseeding of the decellularized graft

The decellularized vessel was reseeded with the adherent population of PBMCs using a

bioreactor setup and in situ two step differentiation to endothelial cells was attempted. The

vessel was first coated with human fibronectin (HFN) by running HFN incorporated medium

overnight through the vessel. The PBMCs were then seeded and the culture continued for 21

days. Media change was done every 3 days and at the end of the culture term, the vessel was

harvested and stained for endothelial markers such as vWF, CD31 and VE-Cadherin.

C. Differentiation of PBMCs into retinal neuron like cells

The retina is one of the most complex high cell density tissues with high metabolic activity

and has a perfect integrity despite all its randomness. The retina has numerous types of cells

with varying morphologies and functions but altogether forms a unit that performs visual

transduction and imaging. Although most of the animals are capable of retinal regeneration at

specific developmental stages, only a few are known to carry regenerative properties for their

retina at maturity. The photoreceptor degeneration is the most common which can be caused

by even a small mutation in any part of the gene resulting in photoreceptor dystrophy.

Gradual loss of photoreceptors or other retina related cells like retinal pigment epithelium

(RPE) is the leading cause of retinal degeneration and leads to blindness, partial loss of vision

121

or severe visual impairment. Thus it is desirable to have an in vitro system of generating

retinal cells that may be further utilized for repopulating the retina to subside the level of

degeneration.

We utilized the similar two steps procedure, in the first step, the monocytes were de-

differentiated into stem cell like cells called reprogrammed monocytes (RM). In the next

step, the reprogrammed monocytes were were-differentiated into retinal neuron like cells by

culturing them in medium containing specific growth factors such as Fibroblast growth

factor-4, Epidermal growth factor, Retinoic Acid, Insulin growth factor-1, Stem cell factor,

Taurine with 0.5% Embryonic stem cell grade serum for 15 days. The retinal neuron like

cells (RNLC) obtained at the end of the culture term was characterized by scanning electron

microscopy (SEM), RTqPCR and immunocytochemistry.

The SEM images revealed a kind of morphological switch from Day 0 to reprogrammed

monocytes. Additionally, the pictures below confirms morphological similarities between

RNLCs and retinal cells.

Figure 3. SEM structure of monocytes at day 0.

Figure 4. The reprogrammed monocytes showing a change in morphology as compared to

monocytes.

Figure 5. Monocyte derived retinal cells showing various morphologies similar to the retinal

cells at Day 15. The red arrows highlight the typical retinal cell like morphologies.

The re-differentiated RNLCs express markers corresponding to diverse group of retinal cells

thus indicating a composite culture. The RNLCs were stained for (a) Glial Fibrillary Acidic

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Protein (GFAP) for glial cells (b) Cellular Retinaldehyde Binding Protein (CRALBP) for

retinal pigment epithelial cells and muller cells (c) Rhodopsin for rod photoreceptors (d)

PAX-6 as common retinal marker and (e) Protein Kinase C-alpha for bipolar cells;

suggesting that the marker for photoreceptor cells are expressed more evidently than the

others.

Development of a NOD SCID mouse model of retinitis pigmentosa

In this study, it would be required to test and validate the function of retinal neuron like cells

derived from human PBMCs in retinitis pigmentosa model of mouse so as to confirm whether

or not the generated cells are useful for the cell based therapy during retinal degeneration. For

this purpose, an immuno-compromised model for retinitis pigmentosa, NOD scid-CBA/J

pde6b rd1 is needed to avoid immuno rejection during transplantation and to study the role of

immunity in retinal degeneration.

The CBA/J mice homozygous for rd1 were crossed with female homozygous NOD SCID

mice. F1 off springs were intercrossed to generate F2 Progeny. Animals with appropriate

genotype were selected to obtain mice carrying homozygous alleles for both rd1 and SCID

genes .This clone of mice served as a foundation breeder for obtaining mice of the desired

genotype.

Future plans

To develop a small animal model of acute liver failure and to establish the utility of NeoHeps

for cell based therapy.

In the current study the NeoHeps cells have been produced from PBMCs. These cells have

been transplanted in immunocompromised mice models by injecting them in the spleen after

partial hepatectomy. Further research has shown that after transplantation these cells are able

to move to the injured liver and perform their basic functions such as albumin synthesis. The

next step in the current study is to try the procedure in higher animals (i.e. rats, rabbits) Acute

Liver Failure (ALF) models. This step is vital to check the contribution of NeoHeps cells in

supporting liver functions and the regeneration of liver of larger animals.

To make the xenogeneic implant of a small diameter vascular grafts by using decellurised

vessels and differentiated PBMCs.

The immunogenicity of the decellularized vessel would be evaluated by implanting it in an

immune competent mouse (BALB/CByJ) while the patency of the constructed tissue

engineered vascular graft (TEVG) would be studied by implanting this seeded graft in

continuum with the inferior vena cava of an immune compromised (NOD.CB17-Prkdcscid)

mouse. The implanted TEVG would be harvested from the mice and checked for thrombosis,

wall thickening and intimal hyperplasia.

To further characterize the retinal neuron like cells (RNLCs) differentiated from PBMCs.

To investigate aerogenic route immunization technology on non-human primates.

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Action taken on the RAP/SAC 2014 recommendations

No specific recommendation was made.

Publications

Original peer-reviewed articles

1. Iyer S, Arindkar S, Mishra A, Manglani K, Kumar JM, Majumdar SS, Upadhyay P,

Nagarajan P (2015) Development and evaluation of transgenic nude mice expressing

ubiquitous green fluorescent protein. Mol Imaging Biol (in press)

2. Tyagi P, Gupta N, Jain A, Upadhyay P, Puliyel J (2015) Intra-gastric pressures in neonates

receiving bubble CPAP. Indian J Pediatr 82:131-135.

Reviews/Proceedings

1. Jain Y, Upadhyay P (2014) An inexpensive and 'do it yourself' infant warmer without

electricity. Natl Med J India 27: 55.

124

Role of cell signaling in eukaryotic development

Principal Investigator Pushkar Sharma

Project Associates Prashant Kumar Modi

Anuj Tripathi

Ph. D. Students Praveen Kumar

Surbhi Jaiswal

Sudhir Kumar

Priyanka Bansal

Roseleen Ekka

Collaborators K Prasad, IOB, Bangalore

A Pandey, IOB, Bangalore

T Gilberger, BNI, Hamburg

C Doerig, Monash University, Melbourne

M Duraisingh, Harvard School of Public Health

D Soldati-Favre, University of Geneva

Theme of research

It is well known that extracellular signals control biological responses in most eukaryotic

cells by regulating specific intracellular signaling and trafficking cascades. We are interested

in signaling and trafficking events in two diverse cell types: 1) Apicomplexan parasites like

Plasmodium falciparum and 2) mammalian neurons.

Objectives

I. Dissection of intracellular signaling and trafficking cascades in Apicomplexan

parasites like Plasmodium falciparum.

Characterization of signaling pathways that operate in malaria parasite may help unravel

novel mechanisms involved in its development. We are interested in the role and regulation

of parasite signaling by second messengers like calcium, phosphoinostides and their effectors

in the life cycle of Plasmodium falciparum. Since Toxoplasma gondii is an excellent model

for studying obligate intracellular parasitism, we have recently started investigating some of

the signaling and trafficking pathways in this parasite, which may be relevant to

Apicomplexans at large.

II. Molecular mechanisms that regulate Cell Cycle Related Neuronal Apoptosis (CRNA)

While apoptosis of neurons is critical for brain development, it also results in neuronal loss

which leads to several neurodegenerative disorders like Alzheimer‟s Disease (AD). A subset

of neurons upon encountering neurotoxic insults attempt to re-enter the cell cycle, which is

reflected by the aberrant modulation of cell cycle proteins like cyclins and cyclin dependent

kinases (cdks). We are interested in molecular mechanisms that regulate Cell Cycle Related

Neuronal Apoptosis (CRNA).

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Work reported in 2013-2014

I. Dissection of intracellular signaling and trafficking cascades of Plasmodium

falciparum.

a. cAMP and calcium signaling in the blood stage development of malaria parasite.

Calcium Dependent Protein Kinases (CDPKs) are important calcium effectors that regulate

diverse parasitic processes. Some CDPKs are essential for the parasite, therefore, are

refractory to gene disruption. In order to unequivocally establish the role of PfCDPK1 in

asexual blood stage development, we used an approach which involved a FKBP or DD

domain. We reported that PfCDPK1 knock down resulted in a significant decrease in

erythrocyte invasion by the parasite providing a strong evidence for a role of this kinase in

this process. Preliminary proteomics studies were carried out on PfCDPK1-DD parasite line.

b. Role of phosphoinositides in parasite signaling and trafficking

CDPK7 is an atypical CDPK as it does not possess the classical CDPK architecture and in

addition has a PH domain. CDPK7 is conserved in Apicomplexans Plasmodium and

Toxoplasma. PfCDPK7 interacts with PI(4,5)P2 via its PH domain. PfCDPK7 gene

disruption was carried out and a PfCDPK7-KO line was generated, which was used to study

its function. PfCDPK7 regulates parasite maturation as well as parasite division. The

formation of Tubulovesicular networks (TVNs) and nutrient uptake was impaired in

PfCDPK7-KO parasites.

Toxoplasma gondii, an apicomplexan parasite, shares several similarities with Plasmodium in

processes like host cell invasion. Due to its simplicity and versatility for in vitro and in vivo

studies and its accessibility to genetic manipulation, Toxoplasma gondii ranks among the best

experimental models to study obligate intracellular parasitism. A conditional knockout of

Toxoplasma gondii CDPK7 orthologue, TgCDPK7, was performed to study its function in

this parasite.

II. Molecular mechanisms that regulate Cell Cycle Related Neuronal Apoptosis (CRNA)

We investigated the role of miRNA in neuronal cell cycle. Our studies suggested that miR34a

expression is elevated during neuronal differentiation and may promote this process. The

knock down of miR34a caused terminally differentiated neurons to de-differentiate and re-

enter the cell cycle a resulting in CRNA. In contrast, miR34a over expression caused

apoptosis, which was independent of cell cycle re-entry. We reported that miR34a may

promote neuronal differentiation and suppress the neuronal cell cycle by targeting cyclin D1.

miR-34a was deregulated in neurons treated with A42 and in a transgenic mouse model for

AD.

Progress of work during the current reporting year (2014-2015)

A. Dissection of intracellular signaling and trafficking cascades of Plasmodium

falciparum.

a. cAMP and calcium signaling in the blood stage development of malaria parasite.

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Calcium Dependent Protein Kinases (CDPKs) are major effectors of calcium signaling in

Apicomplexan parasites like Plasmodium falciparum and Toxoplasma gondii. Since

PfCDPK1 is essential for parasite growth, it is refractory to gene disruption. We used a

parasite line in which PfCDPK1 is fused to FKBP Death Domain (DD). Therefore, it is stable

in the presence of its ligand Shield-1, which when removed causes its “knock down”. Using

this parasite line, we were able to demonstrate the role of PfCDPK1 in host cell invasion. To

gain further insights into how it regulates this process, efforts were made to identify its

substrates using quantitative proteomics. For this purpose, lysates were prepared from

parasite with normal or reduced PfCDPK1 expression. After tryptic digestion and iTRAQ

labeling, peptides were separated by RPHPLC. Subsequently, phosphopetides were enriched

by using a TiO2 column prior to LC-MS/MS analysis. These studies lead to identification of

~3000 phosphopeptides and several of them were differentially phosphorylated upon

PfCDPK1 knock down. The phosphorylation sites on these peptides represented direct or

indirect PfCDPK1 targets, which included proteins from signalling modules, IMC and other

organelles. Some of these proteins were chosen for validation studies, which was performed

by performing in vitro PfCDPK1 kinase assays. Furthermore, several target phosphorylation

site were confirmed by LC-MS/MS analysis and site directed mutagenesis. The

physiological relevance of their phosphorylation by PfCDPK1 is being elucidated.

b. Role of phosphoinositides in parasite signaling and trafficking

The role of phosphoinositides (PIPs) in the biology of Apicomplexans Plasmodium and

Toxoplasma can be deciphered by studying their effectors in this parasite. We are studying

PfCDPK7, which interacts with PI(4,5)P2. As indicated above, PfCDPK-KO parasites exhibit

stalled TVNs and abrogated nutrient uptake. Since TVNs are generated by the action of

parasite enzymes like Sphingomyelin Synthases (SMS), we explored if lipid metabolism and

trafficking may be affected in these parasites. We found that proteins, which are targeted to

the Food Vacuole membrane and the Inner Membrane Complex, were mislocalized in a

significant number of KO parasites. It is possible that PfCDPK7 may regulate trafficking of

these proteins and/or the biogenesis of these organellar membranes was altered in KO

parasites. Preliminary metabolomic studies suggested significant alterations in phospholipids

like phosphatidylcholine (PC) in PfCDPK7-KO parasites. The malaria parasite can import

choline, which it utilizes via the Kennedy/CDP-Choline pathway. Alternatively, it can use

ethanolamine for PC synthesis as it codes for phosphoethanolamine (PE) methyltransferase

(PMT), which facilitates this process. Using radiolabelled choline, we could demonstrate that

indeed PC synthesis was significantly abrogated in PfCDPK7-KO parasites. Importantly,

when PfCDPK7-KO parasites, which show growth defects, were cultured at high choline

concentration an increase in growth phenotype was observed. Based on these results, it is

likely that PfCDPK7 may contribute to parasite development by regulating phospholipid

metabolism and/or trafficking.

TgCDPK7 is essential for the growth of Toxoplasma gondii. Therefore, we needed to

generate an inducible knockout (iKO) for PfCDPK7 in T. gondii. As reported recently,

TgCDPK7-iKO parasites exhibited growth defects and alteration in division, which was also

the case with PfCDPK7-KO parasites. Toxoplasma gondii salvages sphingolipids from the

host and also synthesizes them de novo. We explored if TgCDPK7-iKO exhibits defects in

lipid metabolism and trafficking. While exogenously provided BODIPY-Ceramide was

scavenged by the parasite via host golgi, significant difference in its incorporation in parasite

membrane was observed in the case of TgCDPK7-KO parasites. In addition, differences were

also found in phospholipid profile, which will be further evaluated.

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B. Molecular mechanisms that regulate Cell Cycle Related Neuronal Apoptosis

(CRNA)

We had previously reported that miR-34a levels increase during neuronal differentiation. It

regulates this process by targeting cyclin D1, as suppression of cyclin D1 is important for cell

cycle exit. miR34a down regulation in terminally differentiated neurons may promote

unwarranted neuronal cell cycle re-entry and apoptosis. On the other, overexpression of miR-

34a also caused cell death but importantly without altering the cell cycle. Therefore, we

proposed that miR34a needs to be expressed at optimal levels for neuronal survival. We

observed that miR34a, after an initial increase, was down regulated in response to prolonged

treatment with neurotoxic A42. Moreover, the expression of miR-34a undergoes significant

changes in the cortex of a transgenic (APP/PS1 Tg) mouse model of Alzheimer‟s disease

(AD); a significant decrease was observed in the APP/PS1 animals, which were >12m old.

Corroboratively, cyclin D1 levels were significantly higher in these situations. Since the

increase in cyclin D1 results in cell cycle re-entry and neuronal apoptosis (CRNA), the role of

miR34a in this process was investigated. A42 mediated CRNA was prevented by

overexpression of miR34a. Importantly, cyclin D1 overexpression neutralized the effect of

miR-34a suggesting that it may prevent CRNA via its ability to suppress cyclin D1. The

mechanisms via which miR34a is deregulated is being investigated. We found that aberrant

activation of MEK-ERK pathway caused by A42 causes deregulation of miR34a, which may

result in CRNA. These studies provide insights into a novel mechanism via which CRNA

may be regulated.

Future plans

We plan to use a multidisciplinary approach, which combines modern proteomics, systems

biology with traditional molecular and cell biology studies to dissect and delineate the

sisgnalling/trafficking pathways of Plasmodium. Our proteomics studies have resulted in

identification of novel targets of PfCDPK1. It is important to address how phosphorylation of

these proteins by PfCDPK1 and other kinases may be relevant to parasite processes like host

cell invasion. On similar lines, attempts to identify substrates of other kinases like CDPK7

will be made. Subsequently, parasite lines expressing the phosphosite mutants of substrate

proteins will be generated. The analysis of these transgenic parasites may reveal the effect of

phosphorylation on the function of target proteins. To further pursue our interest in the role of

phosphoinositides in parasite signaling/trafficking, role and regulation of other PIP

interacting proteins in various parasitic processes will be studied. One of our interests is in

how PIPs regulate the process of autophagy, which is necessary for parasite survival.

Further studies to understand the deregulation of miR-34a in neuronal cell cycle re-entry and

death are in progress. We need to address how MEK-ERK and possibly other pathways

regulate miR-34a expression during CRNA. The role of other miRNAs and cell cycle

proteins in neuronal differentiation and CRNA is also being investigated.

Action taken on the RAP/SAC 2014 recommendations

No specific suggestions were made.

128

Publications

Original peer-reviewed articles

1. Jacot D, Frénal K, Marq J, Sharma P, Soldati-Favre D (2014) Assessment of

phosphorylation in Toxoplasma glideosome assembly and function. Cell Microbiol 16:

1518-1532.

2. Kumar P, Tripathi A, Ranjan R, Halbert J, Gilberger T, Doerig C, Sharma P (2014)

Regulation of Plasmodium falciparum development by calcium-dependent protein kinase

7 (PfCDPK7). J Biol Chem 289: 20386-20395.

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Disorders of proliferation: Analysis of novel pathways and targets

Principal Investigator Rahul Pal

Project Associates Ruchi Sachdeva

Sonia Jain

Ph. D. Students Poonam Singh

Beneeta Kalha

Hritika Sharma

Collaborators I Huhtaniemi, Imperial College, London

A Jain, National Institute of Pathology, Delhi

Theme of research

The lab focuses on two disorders characterized by proliferative aberrance - systemic

autoimmune disease and tumorigenesis. In particular, the work seeks to investigate the

consequences of aberrant cell death in systemic autoimmune disease as well as to delineate

mechanisms and pathways by which human chorionic gonadotropin (hCG) can impact upon

the progression of cancer.

Objectives

Systemic Lupus Erythematosus (SLE) is a prototypical non-organ specific autoimmune

disease in which over a hundred distinct autoreactive antibody specificities have been

documented. The pathological consequence of such autoreactive immune responses are the

subject of intense investigation. Impairment of the uptake of apoptotic cells results in lupus-

like symptoms in mice. While apoptotic debris is believed to constitute the original antigenic

insult, elucidation of mechanisms by which an increasing number of moieties are targeted is

critical to understanding systemic pathology. The influence of apoptotic debris on innate and

adaptive immune responses is therefore being investigated. Erythrocyte lysis is a frequent

consequence of systemic autoimmunity; the immunological and physiological sequelae of the

release of sequestered hemoglobin also form a focus of current investigations.

hCG, while critical for the sustenance of pregnancy, is also secreted by a variety of cancers;

its presence has been associated with poor patient prognosis. Understanding the molecular

events and pathways by which hCG impacts on tumor progression forms a focus of the

laboratory, as is the development of novel immunotherapeutic anti-hCG vaccination

strategies.

Work reported in 2013-2014

A. hCG and tumorigenesis

Studies in βhCG transgenic (TG) mice

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Implantation of syngeneic tumor cells in mice transgenic for hCG permitted study of the

effects of exogenous CG in an in vivo environment. Higher tumor incidences and larger

tumor volumes were recorded in TG male and female C57BL/6 x FVBhCG/-

F1 mice upon the

implantation of Lewis Lung cancer (LLC) cells. Markedly higher transcript levels of IL-6,

IL-8, TNF, VEGF, MMP-2, MMP-9, Bcl-2, Bcl-xl and survivin were observed in tumors

isolated from TG mice.

hCG and chemoresistance

hCG has been linked with chemoresistance and poor patient prognosis. hCG reduced the loss

of viability induced by several chemotherapeutic drugs while also reducing the extent of

apoptosis. Tumor cells incubated with hCG demonstrated increases in the levels of cIAP-1,

XIAP, survivin HIF-1α, HO-1, PON2 and Hsp27. Silencing of survivin, HO-1 and Nrf-2

decreased hCG-induced chemoresistance.

Increasing evidence links toll-like receptors (TLRs) with the development of

chemoresistance. hCG, in combination with several TLR ligands, mediated synergistic

increases in chemoresistance. Intra-cellular signaling pathways activated upon the combined

addition of hCG and TLR ligands in the presence of drug were investigated. In many

instance, evidence for a tri-molecular synergy (involving hCG, several TLR ligands and

chemotherapeutic drugs) was obtained in the phosphorylation of JNK, p38, ERK and AKT.

Mice implanted with syngeneic LLC cells were immunized with a βhCG-based vaccine

formulation and also received either curcumin or tamoxifen. Co-administration of drugs and

immunotherapy resulted in further decreases in tumor volume and incidence, and in animal

survival.

B. Systemic autoimmunity

The immunobiology of hemoglobin (Hb)

The immunobiology of Hb formed a focus of ongoing investigation. Increased anti-Hb B cell

precursor frequencies were observed in autoimmune-prone mice. Along with the elucidation

and description of endogenously-generated anti-Hb autoantibodies, the effects of

immunization of autoimmune-prone mice with Hb were also assessed; accelerated

autoantibody sequestration in the kidneys resulted, with associated glomerulosclerosis.

Similarly-immunized non-autoimmune-prone mice remained unaffected. Ferric (but not

ferrous) murine Hb induced a preferential increase in maturation markers on bone marrow-

derived dendritic cells (BMDCs) derived from autoimmune-prone mice. The addition of

apoptotic blebs to splenocytes derived from autoimmune-prone (but not from non-

autoimmune-prone) mice led to the generation of antibodies to Hb. These results indicated

that the propensity towards generation of anti-Hb responses in autoimmune-prone mice may

contribute to pathology.

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Progress of work during the current reporting year (2014-2015)

A. hCG and tumorigenesis

Studies in βhCG transgenic (TG) mice

Previous work from our lab demonstrated that TG female FVB x FVBhCG/-

F1 mice

expressed age-related increases in serum hCG/CG and prolactin, with associated weight

again and a disruption in estrous cyclicity; ovarian hyperplasia was observed at three months

and pituitary adenomas at six months. TG female C57BL/6 x FVBhCG/-

F1 demonstrated

similar phenotype, save for the fact that histological aberrations in the ovaries and pituitaries

were significantly delayed. These results suggest that the progression to tumorigenesis

mediated by hCG/CG is not critically dependent on a specific murine genotype.

C57BL/6 x FVBhCG/-

F1 mice are also being employed to study the effects of circulating

hCG/CG on implanted tumors. Previous work had revealed higher tumor incidences and

larger tumor volumes in TG male and female C57BL/6 x FVBhCG/-

F1 mice implanted with

Lewis Lung cancer (LLC) cells. In addition, markedly higher levels of IL-6, KC, TNF,

VEGF, MMP-2, MMP-9, Bcl-2, Bcl-xl, surviving and versican transcripts were observed in

tumors derived from TG mice versus non-transgenic littermates. While serum levels of TGF

were higher in the sera of TG animals, levels of KC were heightened in TG animals

subsequent to the implantation of tumor cells. These results are in concordance with results

obtained upon the incubation of LLC cells with hCG in vitro and suggest that hCG/CG

increases levels of molecules known to have critical roles in tumor progression in vivo.

B. Systemic autoimmunity

The immunobiology of hemoglobin (Hb)

Previous studies had revealed the presence of serum and kidney-adhered anti-Hb antibodies

in aging autoimmune-prone mice. Further, the disease-enhancing effects of Hb immunization

were described; amongst other effects, enhanced autoantibody sequestration was observed in

the kidneys and the kinetics of disease onset was greatly accelerated. In further confirmation

of these results, methenamine–silver staining revealed that Hb immunization in lupus-prone

mice resulted in split basement membranes in the capillary loops at the edges of the

glomerulii; electron photomicrographs confirmed thickening of basement membranes,

accompanied by electron dense deposits. Along with previous data, these results further

validate Hb both as an autoantigen and as a pathogenesis-inducing immunogen in systemic

autoimmunity.

The differential effects of Hb on the maturation of BMDC derived from autoimmune-prone

and non auto-immune prone mice were previously elucidated. Current work assessed the

functional and mechanistic aspects of Hb-induced BMDC maturation. While BMDCs derived

from both FVB and NZM mice, induced to mature by ferric Hb, induced the proliferation of

allogeneic splenocytes, BMDCs derived from NZM mice were significantly better

stimulators. NZM BMDCs matured in presence of ferrous Hb also induced allo-responses

(albeit much more poorly), while similarly-matured FVB BMDCs were unable to do so.

BMDCs derived from NZM mice and matured in the presence of hemin were unable to

induce allo-responses, suggesting a requirement of intact ferric Hb for the elicitation of such

132

responses. Inhibition of Stat3 most effectively down-modulated ferric Hb-induced phenotypic

maturation of BMDC derived from both strains, however NZM-derived BMDCs were more

sensitive to its neutralization. Hb-induced cytokine secretion, while also maximally sensitive

to Stat3 inhibition, was equivalently suppressed in both strains. These observations contrast

with reports indicating that Stat3 negatively affects DC maturation. While the association of

aberrant Stat3 activation and autoimmunity has also been recognized, the molecular events

arising upon Hb-mediated activation of Stat3 in dendritic cells in the context of autoimmunity

deserve further attention.

Using solid phase assays and surface plasmon resonance, previous studies had indicated that

ferric Hb can associate with a number of autoantigens at physiologically-relevant affinities.

The implications of Hb-autoantigen interaction on the BMDCs maturation were assessed.

Expression of CD80 and CD83 was increased on the surface of BMDCs derived from NZM

mice when ferric Hb was added along with apoptotic blebs, compared to when Hb or blebs

were added alone; such effects were not observed in FVB mice. Co-incubation of Hb with

freeze-thawed cellular lysate did not result in such increases in either murine strain. Further,

upon co-incubation with Hb and apoptotic blebs, increases in CD80, CD83 and CD40 levels

on BMDCs derived from NZM mice were significantly higher than on BMDCs derived from

FVB mice under identical conditions. A variety of inflammatory cytokines were secreted at

significantly higher concentrations by BMDCs upon stimulation with Hb when added along

with apoptotic blebs; significantly lower levels were achieved when freeze-thawed cellular

lysate was added along with ferric Hb. These results imply that the interaction of Hb

(particularly as ferric Hb) with self-antigens (particularly as apoptotic blebs) results in

enhanced immune-stimulatory and inflammatory effects on dendritic cells derived from

lupus-prone mice. The fact that levels of free Hb were observed to be higher in lupus-prone

animals, and the fact that apoptotic blebs are believed to constitute the original antigenic

insult in SLE, make these observations additionally relevant.

Several TLRs are suggested to play a role in systemic autoimmunity. Given that many RNA-

and DNA- containing autoantigens in apoptotic blebs act as endogenous TLR ligands,

whether Hb could induce up-regulation in TLRs levels was explored. Ferric Hb enhanced

transcript levels of TLR3 and TLR8, along with more modest increases in TLR7 and TLR9

message levels, in THP-1 cells. Transcripts of TLR4 and TLR9 were increased in response to

ferric Hb in BMDCs derived from NZM mice while TLR4 and TLR8 transcripts were

increased in FVB BMDCs. The physiological relevance of these observations is under

investigation.

The influence of apoptotic blebs and apoptotic cell-specific antibodies on immune function

Aberrant apoptosis is a hallmark of SLE. In previous work, apoptotic blebs potently inhibited

the generation of BMDCs, an effect more severe in autoimmune-prone animals. Blebs were

also more effective than cell lysate in inducing BMDC maturation. Previous work had also

described the generation and characteristics of monoclonal apoptotic cell-specific antibodies

generated from lupus-prone mice and SLE patients. Much like apoptotic blebs, such

autoantibodies also induced BMDC maturation. In ongoing studies, the effect of

immunization of such antibodies was evaluated. Hyper-gammaglobulinemia and expansion of

the autoantibody repertoire was observed in syngeneic, autoimmune-prone mice; immunized

animals also exhibited severe glomeruosclerosis and increases in the mesangial matrix in the

kidneys. Non-autoimmune prone mice did not exhibit these effects. These results suggest that

the consequences of excessive apoptosis (or the deficient clearance of apoptotic cells) include

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effects on innate as well as adaptive immune responses, specifically in an autoimmune

milieu.

Future plans

In exogenous tumors arising upon the implantation of LLC cells in TG female C57BL/6 x

FVBhCG/-

F1 mice, whether increases in tumor-associated transcripts are paralleled by

increases in protein levels will be investigated. Immuno-histochemical analysis will enable

the identification of source cells, thereby confirming or negating the premise that tumor cells

and non-transformed resident cells collaborate in the elaboration of tumor-promoting factors

in an hCG/CG-driven manner. Further, tumor implantation studies employing

ovarectomized TG female C57BL/6 x FVBhCG/-

F1 mice, and/or drugs which interfere with

the secretion of prolactin, will permit the elucidation of individual contributions of

gonadotropin, steroids and prolactin to tumorigenesis.

Whether immune complexes comprising of two “self” components shown to be inflammatory

in an autoimmune milieu (apoptotic blebs and anti-apoptotic cell specific antibodies) are

endowed with synergistic activity will be evaluated. Hb-mediated signaling events in BMDCs

derived from autoimmune-prone mice will be investigated in greater detail, and intra-cellular

pathways further elucidated. The premise that the existence of autoreactive antibodies in

patients of malaria and leishmaniasis (conditions of infectious etiology associated with

erythrocyte lysis) is the consequence an Hb-mediated break in tolerance will be considered.

Whether Hb and autoantigens (when added sequentially or together) can synergistically

enhance TLR transcript and expression levels will be assessed, and the consequences of any

such increases evaluated. Whether Hb can influence the phenotype and function of

plasmacytoid dendritic cells will also be assessed.

Work in previous years had demonstrated the chemoprotective effects of hCG on tumor cells.

While up-modulating several genes associated with chemoresistance, hCG was also shown to

increase transcript levels of several TLRs in tumor cells. Previous work had also provided

evidence of the synergy between hCG and synthetic TLR ligands on chemoresistance. In an

effort to further the physiological relevance of these findings, whether packaged endogenous

autoantigens can mediate similar collaborative cyto-protective effects along with hCG will be

determined.

Action taken on the RAP/SAC 2014 recommendations

No specific recommendations were received.

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Study of genetic and immune factors associated with autoimmune

disorders : Type1 Diabetes and vitiligo

Principal Investigator Rajni Rani

Project Associate Vandana Panwar

Project Fellow Samrina Mehtab

Ph. D. Students Bhukya Naik

Varkhande S Risha

Anshu Sharma

Utpraksha Vaish

Ankita Dabla

Collaborators R Goswami, AIIMS, Delhi

RS Gokhale, NII/IGIB, Delhi

K Natarajan, JNU, Delhi

A Kamra Verma. DU, Delhi

SK Sarin, ILBS, Delhi

HK Kar, RML Hospital, Delhi

VK Sharma, AIIMS, Delhi

S Vijaya, IISc, Bangalore

I Nath, IOP, Delhi

R Begum, MSU of Baroda, Vadodara

P Stastny, Southwestern Medical Centre, Dallas, USA

Theme of research

The project aims to decipher the immunogenetic and autoimmune factors involved in the

destruction of pancreatic beta cells and melanocytes in Type 1 diabetes (T1D) and vitiligo

respectively. We aim to device ways to inhibit autoimmune responses in T1D.

Vitiligo, is a multifactorial disease etiology of which is not precisely understood. While

several hypotheses have been proposed including autoimmunity, it is not clear how the

pigment producing melanocytes are destroyed by the autoimmune responses. So, the theme

of this project is to understand the aetiopathogenesis of vitiligo with an aim to develop

therapeutic approaches for the disease.

Objectives

1. To study the role of Human leukocyte antigens (HLA) in aetiopathogenesis of both T1D

and vitiligo.

2. To study other Immune function related genes which may have a role in manifestation of

T1D and Vitiligo.

3. To study the autoimmune factors associated with T1D and vitiligo.

4. To design and use peptides in-vitro to inhibit autoimmune T-cell responses.

5. To encapsulate the peptides which inhibit Th1 immune responses in-vitro, in nano-sized

carriers for slow and targeted release.

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6. Study delivery of peptide/vector complexes in Balb-C and C57Bl6 mice followed by

NOD mice.

7. To differentiate mouse Mesenchymal stem cells into insulin producing cells.

8. To study the role of MHC restricted auto-antigen specific CD4+/CD8

+ T cells in

autoimmune destruction of melanocytes in vitiligo.

9. To study the role of Cytokines increased in vitiligo patients in aetiopathogenesis of

vitiligo.

10. To translate the knowledge and techniques developed from bench to bedside.

Work reported in 2013-14

A. Type 1 diabetes

Genetic basis of Type 1 diabetes

We had reported last year that LMP7 exon 2 SNP that results in an amino acid change from

Glutamine to Lycine (Q49K) at codon 49 showed a significant increase of CC genotype and

this increase was independent of the MHC alleles associated with T1D.

Mesenchymal stem cell treatment of non-obese diabetic mice

We also reported the results of four injections of MSCs treated with IFN-γ, TNF-α or IL-1β at

6 weeks, 8 weeks, 10 weeks and 12 weeks given to NOD mice. 100% of the mice injected

with IFN-γ and TNF-α treated MSCs remained non-diabetic. 60% of the NOD mice injected

with IL-1 β treated MSCs remained non-diabetic while 40% of the control mice remained

non-diabetic at the end of 32 weeks.

To compare the results of MSCs grown in high glucose media and MSCs induced to become

insulin producing cells, passage 9, 10, 11, 12 and 13 cells grown in high glucose media were

injected in mice at 9th

. In the set where differentiated insulin producing cells were given to

mice at the 9th

week 60% remained non-diabetic at the end of 28 weeks compared to 50% in

the control group. However, in the groups where passage 11, 12 and 13 MSCs were given to

NOD mice, 100% in the treated group and 40-50% in the control groups remained non-

diabetic.

B. Vitiligo

Validation of Microarray results:

We had reported validation of enriched pathways connected with cell cycle and keratinocyte

differentiation in keratinocytes treated with IFN-γ, IL-17A and a combination of the two

cytokines using real time PCR. Comparison of the cell cycle profile of normal human

keratinocytes using propidium iodide revealed a significant decrease in number of cells in the

S phase of the cell cycle in the keratinocytes treated with IFN-γ and IFN-γ IL-17A combined

treatment. Keratinocyte differentiation and epidermal development were other pathways that

were upregulated and we confirmed upregulation of genes like Involucrin (IVL), K16

(KRT16.) Integrin alpha 2 (ITGA2) and integrin beta 1 (ITGB1).

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Translational efforts

30 vitiligo patients were transplanted with autologous epidermal cells and pure melanocyte

cultures bilaterally and the results show significantly better pigmentation in the sides treated

with pure melanocyte cultures.

Progress of work during the current reporting year (2014-2015)

A. Type 1 diabetes (T1D)

Genetic basis of Type 1 diabetes: We analyzed the data of 239 T1D subjects and 752 normal

healthy controls from North India for LMP2 codon 60 G/A (R/H), LMP7 codon 49 of C/A

(Q/K) and LMP7 Intron 6 G/T polymorphism and HLA class-II alleles. The G (R) allele

(p<0.009) and homozygous GG (RR) genotype (p<0.01) of LMP2 codon 60, C (Q) allele

(p<0.0098) and homozygous CC (QQ) (p<0.03) of LMP 7 codon 49 and LMP7 Intron 6 G

allele (p<0.01) were significantly increased in T1D subjects compared to controls. Haplotype

analysis showed that haplotypes GCG and ACT (LMP2 codon 60- LMP7codon 49- LMP7

intron 6) (p < 5.9 X 10-13

, p < 1.9 X 10-8

respectively) were significantly increased and

haplotypes GCT and ACG (p<1.9 x10-12

, p<1.9 x10-13

respectively) were significantly

reduced in T1D patients irrespective of the gender, age at onset of T1D and the predisposing

HLA DRB1*03:01. These results suggest that association of LMP2 and LMP7 haplotypes

GCG and ACT with T1D may have a role in processing of auto-antigens to be presented by

MHC class-I molecules to cytotoxic T cells in T1D.

We further studied a gain of function mutant at nucleotide position 1858 C>T in PTPN22

(Protein tyrosine phosphatase non-receptor, type 22) gene encoding lymphoid tyrosine

phosphatase (LYP) in 250 T1D patients and 480 healthy controls. PTPN22 is a negative

regulator of T cell signaling. In spite of reports of absence of 1858T allele in Asians, we

observed this allele to be present in North Indians, albeit with low frequency (1.98%).

However, T1D patients from the same ethnic background showed significantly higher

frequency of the allele and heterozygous genotype 1858CT as compared to controls. Patients

with both 1858CT and 1858CC genotypes had predisposing MHC alleles. The association of

PTPN22 1858CT genotype with Type 1 diabetes was independent of the predisposing Human

leukocyte antigen (HLA) alleles DRB1*03:01, DRB1*04:01, DRB1*04:05 in North Indian

patients, suggesting their integrated roles in manifestation of T1D. Based on the reported role

of PTPN22 1858CT genotype in defective innate immune responses against viral infections,

and defects in early T cell signaling, it is tempting to speculate that it may be detrimental for

the destruction of pancreatic beta cells in the present scenario.

Mesenchymal stem cell (MSC) treatment of non-obese diabetic: We had planned to use to

two-pronged approach to treat T1D in NOD mice, by suppressing autoimmune responses

using MSCs treated with cytokines and replenishing with insulin producing cells. Our initial

efforts to differentiate MSCs into insulin producing cells (IPCs) gave variable results i.e.

sometimes we could differentiate them into IPCs and at times we could not. So, we decided

to sort the MSCs based on their surface markers CD29, CD73 and CD44 using FACS sorter

in early passages and then differentiated them into IPCs using stage specific differentiation

protocol. Briefly, MSCs that are from mesodermal lineage were first differentiated into

definitive endodermal lineage followed by pancreatic endodermal lineage and finally

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pancreatic hormone secreting cells and stained for inulin using Immunocytochemistry which

shows islet cell like cluster as shown in the figure 1. These IPCs along with cytokine treated

MSCs are being tested in NOD mice for their potential to secrete insulin in-vivo and inhibit

autoimmune responses respectively, to take care of diabetes in them.

Figure1. MSCs were differentiated into insulin producing cells. Control uninduced (a) and

induced cells (b and c) are shown by immunocytochemistry for insulin, islet-like clusters

were observed in the induced MSCs.

B. Vitiligo

Genetic basis of Vitiligo: Aberrant presentation of self antigens is the hallmark of several

autoimmune disorders. We have earlier shown association of MHC class-I and II alleles with

vitiligo which have a role in antigen presentation. However, for the antigen to be presented

by MHC molecules, it needs to be processed and broken down into small peptides which is

done by immunoproteasomes LMP2 and LMP7. Selected peptides are loaded onto the MHC

class-I molecule by transporters associated with peptide loading (TAP1 and TAP2). So, we

have studied genotype, allele and haplotype frequencies of single-nucleotide polymorphism

(SNPs) in LMP2 and LMP7 in 1320 vitiligo patients and 752 healthy controls. TAP1 and

TAP2 SNPs have been studied in 746 vitiligo patients and 748 healthy controls. While LMP2

exon 3 G/A SNP was in Hardy Weinberg equilibrium in both patients and controls, LMP7

exon2 SNP A/C and LPM7 intron 6 G/T SNP were not in Hardy-Weinberg equilibrium in

both patients and controls. The G to A substitution in LMP2 exon 3 leads to an amino acid

change from arginine (R) to histidine (H) at codon 60 (CGC to CAC). There were no

significant differences between vitiligo patients and controls in terms of either genotype or

allele frequencies for LMP2 exon 3 R or H alleles.

In LMP 7 exon 2 substitution of C to A results in amino acid change from glutamine (Q) to

Lysine (K) at codon 49 (CAG/AAG). Allele A (K) and genotypes AA (KK) and AC (KQ)

were significantly increased in all vitiligo patient, generalized vitiligo and localized vitiligo

compared to healthy controls. Allele C and homozygous CC (QQ) were significantly reduced

in all vitiligo, generalized and localized vitiligo patients. However, the significance was much

more in the generalized group.

LMP2 and LMP7 genes are localized on human chromosome 6 in the MHC class-II region

very close to DRB1 gene. So, to check whether this association of LMP7 exon 2 is due to its

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being in LD with predisposing MHC allele, we checked for the simultaneous presence of

predisposing HLA-DRB1*07:01 along with LMP7 exon 2 SNPs. Interestingly, both the

alleles A and C of LMP7 exon 2, along with predisposing HLA-DRB1*07:01 were

significantly increased in the patients. Significant increase of both alleles A and C of LMP7

exon 2 along with the predisposing HLA allele suggests the dominant effect of the HLA allele

irrespective of the LMP7 exon 2 allele.

For TAP1 intron 5 C/T, exon 10 G/A (D63G), TAP2 exon 5 A/G (V379I) and TAP2 exon 11

A/G (T665A) were studied. Significant increase in heterozygous GA in TAP1 exon 10 and AG

in TAP2 exon 11 with relative risk of 1.9 and 4.1 were observed in vitiligo compared to

healthy controls. Further analysis with type of vitiligo, age onset and gender bias is going on.

Comparison of lesional vs non-lesional skin of vitiligo: The literature has implicated

increased oxidative stress in the etiology of depigmented patches in vitiligo and we have

earlier shown that the detoxification pathways regulated by transcription factor, nuclear factor

E2–related factor 2 (Nrf2) and its downstream genes NAD(P)H:quinone oxidase-1 (NQO-1),

g-glutamyl cystine ligase catalytic subunit (GCLC), and g-glutamyl cystine ligase modifying

subunit (GCLM), GSTT1 and GSTM1 were upregulated in the lesional skin of vitiligo

patients. Mitochondria have been implicated in generation of ROS. Majority of ROS is

produced by oxidative phosphorylation (OXPHOS) or mitochondrial respiration. The

OXPHOS system consists of five multimeric enzyme complexes, Complex I to V which are

involved in generation of ATP. Since mitochondrial dysfunction is associated with several

human diseases, we checked for the expression of genes involved in OXPHOS in lesional and

non-lesional skin of vitiligo patients. Expression of 14 genes involved in complex I, 2 genes

involved in complex II, 7 genes involved in complex III, 6 genes involved in complex IV and

eight genes involved in complex V were studied and were found to be upregulated in the

lesional skin compared to the non-lesional skin of vitiligo. The implications of the

upregulation of OXPHOS in vitiligo are being investigated.

Future Plans

We would further like to explore different stem cell treatment strategies for prophylactic and

therapeutic effect of differentiated and undifferentiated MSCs in NOD mice with a view to be

translated in humans with cord blood derived MSCs. We would like to explore the possibility

of using two-pronged approach for treatment of Type 1 diabetes, where peptides encapsulated

in microparticles and mesenchymal stem cells will be used for treatment of diabetes in NOD

mice.

For vitiligo, implication of up-regulation of OXPHOS in the lesional skin will be explored.

We have generated micro-array data from melanocytes and keratinocytes treated with

different cytokines, while some data has been validated a lot needs to be validated at both

transcript and translational levels and their role, if any, in selective destruction of

melanocytes needs to be elucidated. There are no studies reported so far on the role of

miRNA in vitiligo. We carried out a genome-wide array for the expression of micro RNAs in

lesional (vitiligenous) and non-lesional (normal) skins of 18 patients for differential

expression of 328 micro RNAs using the FlexmiR platform (Luminex). 28 microRNAs were

found to be regulated.

To study the role of miRNAs, potential targets for the upregulated miRNAs will be obtained

from 2 databases- Miranda and RNA Hybrid using the program GomiR. The micro-array data

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of keratinocytes transfected with miRNAs of interest will be analyzed using DAVID. This

way the enriched pathways will be obtained and the genes that belong to those pathways will

be picked up as potential targets. Of the targets identified using bioinformatic tools, those that

are regulated in the vitiligo microarray dataset will be determined and considered as the final

set of putative targets. HaCaT cell cultures will be transfected with pre-miRNA (Ambion)

and the expression levels of these putative target genes will be determined by real-time

analysis.

It is also possible that the miRNAs are affecting only the translational levels of the protein

and not the transcript levels. To verify this, transfections will be done to obtain native protein

which can be used in proteomic analysis to determine differentially regulated proteins using

MALDI-TOF analysis. Expression of target proteins regulated by different miRNAs will be

checked for their role in aetiopathogenesis of vitiligo.

Additionally, our translational efforts seem to be giving good results and extending it further

will not only strengthen the treatment strategies for vitiligo but also give a ray of hope to the

severely depressed patients going through psychological trauma.

Action taken on the RAP/SAC 2014 recommendations

Queries raised by the RAP/SAC members were satisfactorily answered and their

recommendations have been followed.

Publications

Original peer-reviewed articles

1. Saida B, Dani P, Patnaik N, Agrawal B, Rajaratna T, Jaiswal A, Singh AK, Rani R

(2014) Haplotypes of polymorphic antigen processing genes for low molecular mass

polypeptides (LMP2 and LMP7) are strongly associated with Type 1 diabetes in North

India. J Diabetes Metab 5: 451.

2. Rani R, Israni N, Kumar A, Vasudevan S, Singh J (2014) Association of protein tyrosine

phosphatase non-receptor, type 22 (PTPN22) C1858T polymorphism with Type 1

diabetes in north India : A replication study. J Diabetes Metab 5: 342.

Review/Proceedings

1. *Rani R, Singh A (2014) Functional implications of MHC associations in autoimmune

diseases with special reference to Type1 diabetes, Vitiligo and Hypoparathyroidism. In

HLA & Associated Human Diseases, (Ed. Yongzhi Xi, inTech Publisher, Rijeka, Croatia)

pp 201-221.

*in press last year, since published

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Understanding the regulation of DNA replication

Principal Investigator Sandeep Saxena

Project Associates Akhil Varshey

Md. Muntaz Khan

Sheetal Uppal

Project Fellows Nasir Imam

Vijay Kumar M.J

Ph. D. Students Manpreet Kaur

Tanushree Ghosh

Ritu Shekhar

Raksha Devi

Praveen Kumar

Theme of research

DNA replication is a vital process of life and must be completed precisely during each cell

cycle. When mammalian cell experiences DNA damage, it activates checkpoint mechanisms

to stall the progression of cell cycle and DNA replication. Our laboratory is working towards

understanding the mechanisms by which microRNA and checkpoint proteins stall the cell

cycle, preventing genomic instability and cancer.

Objectives

We are studying the regulation of replication machinery during stress in order to identify

underlying mechanisms responsible for inhibition of essential replication factors during

stress. We are trying to understand the role of ubiquitination machinery in regulating

replication proteins under normal and stressed conditions. Further, we are investigating the

cellular response to aberrations in replication complexes. The objective is to identify yet

unknown checkpoint pathways that monitor the replication apparatus. Emerging evidences

suggest that microRNAs target genes that regulate DNA replication and cell cycle

progression and we aim to determine the role of microRNA in regulating the DNA replication

machinery as the cell progresses from one phase to the next. This would provide an insight

into the mechanisms by which microRNAs regulate mammalian cell cycle and DNA

replication during normal and pathological conditions. Summing up, we are attempting to

unravel the protective regulatory control of mammalian cells, failure of which is likely to

cause genomic instability.

Work reported in 2013-2014

Role of alternate single-stranded DNA-binding proteins in checkpoint signaling

Single-stranded DNA generated at stalled replication forks and during DNA repair serves as

an intermediate for activating the Ataxia telangiectasia and Rad3-related protein (ATR)

checkpoint kinase which phosphorylates Chk1, initiating a signal transduction cascade. It is

believed that binding of Replication protein A (RPA) to the single-stranded DNA is essential

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for the recruitment of ATR-ATRIP complex to the sites of DNA damage facilitating the

initiation of checkpoint response. We have previously proposed that ATR can phosphorylate

Chk1 independent of RPA. We had reported that in the absence of RPA complex, an alternate

single-stranded DNA-binding protein complex, hSSB1-INTS3, associates with the single-

stranded DNA generated at the sites of damage.

GINS subunit is required for centrosome integrity during mitosis

We have previously identified that depletion of a GINS subunit results in multipolar spindle

formation and increased centrosome number in mitotic cells, indicating that it is required for

mitotic progression.

Role of non-coding RNAs in regulation of cell-cycle

During the last reporting year we analyzed the role of non-coding RNAs in regulating the

transition between proliferation and quiescence. We standardized two microRNA assays in

our laboratory: hemi-nested and poly-A-tailing assay and utilized them to confirm the levels

of microRNAs in asynchronous growing and serum starved cells. On the basis of our results,

we had predicted that microRNAs regulate the cell cycle genes during transition between

proliferation and quiescence.

Progress of work during the current reporting year (2014-2015)

Role of alternate single-stranded DNA-binding proteins in checkpoint signaling

We have reported that in the absence of RPA complex, an alternate single-stranded DNA-

binding protein complex, hSSB1-INTS3, associates with the single-stranded DNA. We

observed that hSSB1 formed punctate foci in RPA70-depleted cells similar to what has been

reported after gamma-irradiation, indicating the localization of hSSB1 at the sites of genomic

stress (Figure 1A). The foci were absent after co-depletion of hSSB1 along with RPA70,

confirming that the observed immunofluorescence signal was from hSSB1. HSSB1 is part of

the single-stranded DNA-binding complex, wherein its partner protein, INTS3 serves as a

central adaptor required for assembly of the complex and recruitment of hSSB1 to the sites of

DNA damage. We observed that after RPA70 depletion INTS3 also formed punctate foci,

similar to hSSB1 (Figure 1B). Therefore, we demonstrate that alternate single-strand binding

protein complex, hSSB1-INTS3, forms punctate foci after RPA depletion. In order to assess

the requirement of hSSB1-INTS3 complex in Chk1 phosphorylation, hSSB1 or INTS3 were

silenced along with RPA and observed that co-depletion of hSSB1 or INTS3 suppressed the

RPA70 depletion-induced Chk1 phosphorylation (Figure 1C and D). Thus, Chk1

phosphorylation after RPA70 depletion-induced genomic stress is dependent on hSSB1-

INTS3. In summation, we report that the single-stranded DNA-binding protein complex,

hSSB1-INTS3 can recruit the checkpoint complex to initiate ATR signaling.

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Figure 1. Chk1 phosphorylation in the absence of RPA70 is dependent on single-strand binding protein complex,

hSSB1-INTS3. (A and B) HeLa cells transfected on three consecutive days with GL2 or RPA70 siRNA in

combination with HSSB1 or INTS3 siRNA as indicated were visualized for hSSB1 and INTS3 foci by

immunofluorescence with rabbit anti-hSSB1 and goat anti-INTS3 antibodies respectively. Right panel displays the

DAPI staining for each sample. Co-depletion of hSSB1 (A) or INTS3 (B) along with RPA70 confirms that the

immunofluorescence signal is from the respective proteins. (C) HeLa cells were transfected on three consecutive days

with GL2 or RPA70 siRNA in combination with INTS3 siRNA as indicated and the levels of RPA70, INTS3, total and

phosphorylated-Chk1 were assayed. (D) HeLa cells were transfected on three consecutive days with GL2 or RPA70

siRNA in combination with HSSB1 and/or HSSB2 siRNAs as indicated and the levels of RPA70, hSSB1, hSSB2, total

and phosphorylated-Chk1were assayed. * points to a cross-reactive band while LC refers to the protein loading

control. The numbers in parts C and D indicate phosphorylated-Chk1 levels following RPA70 depletion alone or in

combination with INTS3 or hSSB1 & 2 after normalization with the protein loading control.

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GINS subunit is required for centrosome integrity during mitosis

In eukaryotes, the GINS complex constituting of four subunits, Sld5, Psf1, Psf2 and Psf3, is

essential for both the initiation and elongation stages of the DNA replication process. GINS

in a complex with Cdc45 and MCMs, serves as the replicative helicase, unwinding duplex

DNA ahead of the moving replication fork. We have observed that depletion of a GINS

subunit results in mitotic aberrations. GINS depleted cells arrest with abnormally condensed

chromosomes, multiple centrosomes and failed chromosome congression (Figure 2A). Using

live cell imaging, we have established that GINS depleted cells do not complete mitosis. It is

known that cellular stress caused by DNA damage or replication stalling results in

centrosome amplification during interphase (Figure 2B). However, we have observed that a

controlled depletion of GINS did not trigger a DNA damage checkpoint response or result in

centrosome amplification during interphase (Figure 2B). Reduced levels of cenexin, CENP-E

and Cep170 were observed at the centrosome after GINS depletion (Figure 3). We have

previously demonstrated that GINS localizes to the centrosomes and our present work shows

that it is essential for centrosome integrity.

Figure 2. GINS depletion results in mitotic aberrations. (A) HeLa cells were transfected with control GL2 or two

different GINS siRNA (si1 or si2) followed by co-immunofluorescence with mouse anti-alpha tubulin and rabbit anti-

gamma tubulin antibodies in combination with anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 555 antibodies

respectively. Chromosomes were stained with DAPI. (B) HeLa cells were transfected with PCNA or GINS siRNA

followed by co-immunofluorescence with mouse anti-alpha tubulin and rabbit anti-gamma tubulin antibodies in

combination with anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 555 antibodies respectively. Nuclei were

stained with DAPI. Arrows mark the centrosomes as identified by gamma-tubulin staining. Note that mitotic

aberrations are observed in PCNA depleted but not in GINS depleted interphase cells.

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Figure 3. Loss of cenexin at centrosomes after GINS depletion. (A) HeLa cells were transfected with control GL2 or

GINS siRNA followed by co-immunofluorescence with mouse anti-cenexin and rabbit anti-gamma tubulin antibodies

in combination with anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 555 antibodies respectively. The

arrangement of immunofluorescence images obtained from each sample is illustrated at the top of the figure. Arrows

mark the centrosome position. Chromosomes were stained with DAPI. (B) Quantitation of cenexin and gamma

tubulin signal in GL2 or GINS siRNA transfected cells. P value was calculated using two tailed t test while error bars

indicate standard errors of the mean (P=0.02).

Role of non-coding RNAs in regulation of cell-cycle in osteosarcoma

Deregulation of cell cycle is frequently observed during oncogenesis and we are trying to

understand the role of non-coding RNAs in regulating cell cycle and its loss osteosarcoma.

We have classified 9 different osteosarcoma cell lines on the basis of rate of proliferation, in

vivo tumorigenicity, in vitro colony-forming ability and migratory potential. We have then

compared the mRNA and microRNA expression patterns of these different cell lines, which

were obtained by pan-genomic microarray hybridization. We observed that cell lines with

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Figure 4. MicroRNA control of cell cycle in osteosarcoma. (A) The table displays examples of cell cycle genes that

are deregulated in osteosarcoma. Based on TargetScan, miRWalk and miRanda, the microRNAs that target these

genes are predicted (denoted by •). Relative levels indicate the levels of mRNA and microRNAs in cell lines

displaying aggressive osteosarcoma phenotypes in comparison to slow growing control osteosarcoma cell lines. (B)

Flow cytometry of propidium iodide stained DNA from HeLa cells demonstrates a G1 block after overexpression of

specific microRNAs. Cells were transfected with microRNA mimics (# referred as per DRCC laboratory index)

followed by treatment with nocodazole (which blocks in G2/M phase).

high rate of proliferation showed the overexpression of the following cell cycle genes: CDK6,

GINS2, RPA3, CDT1 and Cyclin D. Expression of 20 genes which displayed overexpression

on genomic microarrays was tested by individual Real-Time PCR gene assays and we

observed that around 15 genes displayed more than 2 fold overexpression (Figure 4A).

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We assembled a list of around 30 downregulated microRNAs that are likely to target the

overexpressed cell cycle genes. Utilizing individual Real-Time PCR microRNA assays, we

observed that 12 microRNAs were downregulated by more than 75%, including miR-503,

miR-424, miR-628 and miR-363. Overexpression of many downregulated microRNAs

caused a cell cycle block in osteosarcoma cell lines, implying that their downregulation is

linked to rapid proliferation (Figure 4B). We are now ascertaining the target genes that were

downregulated after overexpression of these microRNAs which resulted in a cell-cycle block.

We will confirm the targets of microRNAs by assaying the activity of firefly luciferase

expressed in fusion with 3‟ UTR of predicted target genes. We will also address if

manipulating of microRNA alters the rate of proliferation, in vitro colony-forming ability and

invasive/migratory potential of the osteosarcoma cell lines. Therefore, in this part we aim to

determine the role of non-coding RNAs in control of cell-cycle during osteosarcoma.

We have previously identified microRNA whose expression changes during transition from

proliferation to quiescence. We observed that overexpression of these microRNAs blocked

the cell cycle in G1 phase, though the predicted target cell cycle genes were not

downregulated. To identify the targets of these microRNAs we will now determine the

changes in expression of transcriptome by pan-genome microarray hybridization. Thus, we

will determine the role of non-coding RNAs in regulating cell-cycle and DNA replication

genes during transition from proliferation to quiescence.

Future plans

RPA is the primary single-stranded DNA-binding protein in eukaryotic cells with vital roles

in DNA replication and repair. Mutations in RPA that impair its function have been reported

to cause chromosomal instability and linked to cancers. In the future we will assess how cells

respond to deficiencies in RPA activity. We have observed that RPA depleted cells

accumulate in S phase maintaining active DNA replication dependent on Chk1 activity. Thus,

we will determine the cellular response to decrease in RPA70 levels. We have observed that

GINS subunit localizes at the centrosomes and it essential for its integrity. In the future, we

will ascertain the physiological function of GINS subunit during mitosis. Emerging evidences

suggest that microRNA target genes that regulate DNA replication and cell cycle progression

and our objective is to determine how microRNA regulate the DNA replication machinery as

cell progresses from one phase to the next. We have then identified mRNA and microRNAs

that are deregulated in osteosarcoma. We will now confirm the targets of microRNAs by

luciferase assays. We will also address if manipulating of microRNA alters the rate of

proliferation, in vitro colony-forming ability and invasive/migratory potential. Summing up,

we are trying to understand the mechanisms by which microRNAs regulate mammalian cell

cycle and DNA replication in normal and pathological conditions.

Action taken on the RAP/SAC 2014 recommendations

No specific comment received.

147

Determining the signaling and repair pathways that are altered in human

cancer

Principal Investigator Sagar Sengupta

Project Associate Vivek Tripathi

Project Fellows Vinoth Madhavan

Mansoor Hussain

Arun Prasath D

Ph. D. Students Jyoti Kumari

Raina Priyadarshini

Swati Priya

Himanshi Agarwal

Preeti Attri

Collaborators V Nandicoori, NII

A Bajaj, RCB, Faridabad

G Legube, LBCMCP-CNRS, Toulouse, France

Arnab Mukhopadhyay, NII

S Chowdhury CSIR-IGIB, Delhi

Theme of research

Our research program evolves around the understanding of the biology of tumour

suppressors. One of the families of tumour suppressor being studied is the RecQ helicases.

BLM and RECQL4 are members of this family of DNA helicases. Germline mutations in both

BLM and RECQL4 helicase result in autosomal-recessive disorders, Bloom syndrome (BS)

and Rothmund-Thomson syndrome (RTS) respectively. BS afflicted individuals are

predisposed to almost all types of cancers while RTS individuals are predominantly

predisposed towards osteosarcomas. Since RecQ helicases are intimately involved in the

many vital cellular processes, they are ideal candidates to investigate the reasons for

neoplastic transformation.

Objectives

In the current year the work in the lab was aimed to dissect the mechanism of turnover of

BLM helicase.

Work reported in 2013-2014

Work in the lab had previously demonstrated how phosphorylated BLM interacts with 53BP1

and thereby maintains the genomic stability (Tripathi et al., J Cell Biology, 2007; Tripathi et

al., Carcinogenesis, 2008). The mechanism how BLM stimulates the ATPase and chromatin

remodeling activities of RAD54 has also been elucidated (Srivastava et al., J Cell Science,

2009). Specific phosphorylation and ubiquitylation events what determine the subcellular

localization of BLM under unstressed conditions and its subsequent recruitment to the lesion

sites after DNA damage were also elucidated (Kaur et al., Mol Cancer Res, 2010; Tikoo et

148

al., EMBO J, 2013). Finally the lab had also reported earlier how BLM enhances the

degradation of c-Myc by enhancing the binding of c-Myc to its cognate E3 ligase, Fbw7

(Chandra et al., J Cell Science, 2013). All these results signify the different functions

associated with BLM. To attain greater perspective about BLM function, it was essential to

determine the mechanism of BLM turnover. The work done on this project is being reported

for the first time.

Progress of work during the current reporting year (2014-2015)

BLM helicase is a substrate of Fbw7 during mitosis

We had recently reported that BLM interacts with Fbw7 isoforms and enhances the Fbw7-

mediated degradation of c-Myc. To understand the cell cycle phase(s) where BLM undergoes

turnover, double thymidine block of A-15 cells (BS fibroblast GM03509 cell line

complemented with chromosome 15) was carried out, followed by the release of the cells in

normal growth medium for defined time periods. BLM is induced in the early S-phase (3 hrs

post-release), its level persist upto G2 phase of the cell cycle (8 hrs post-release), after which

the steady state of the protein decreases progressively in the G2/M (9 hrs post-release) and

G1 phase (12 hrs post-release) of the cell cycle. Immunoprecipitation with K48-linked anti-

ubiquitin antibodies reveal that BLM undergoes enhanced ubiquitylation in G2/M compared

G1 phase. The ubiquitylation of BLM was also detected after immunoprecipitation with K48-

linked ubiquitin-linkage specific antibodies post-nocodazole treatment. Reciprocal

immunoprecipitations demonstrate that while BLM constitutively interacts with Fbw7 and

isoforms in all phases of the cell cycle, the association of the Fbw7 isoforms with the poly-

ubiquitylated BLM occurs only in G2/M phase (9 hrs post-release).

Hence we hypothesised that Fbw7 degrades BLM specifically during mitosis. Indeed in

asynchronously growing cells expressing GFP-tagged BLM, the levels of BLM in the

interphase cells were substantially more than in the mitotic cells. To study this effect in more

detail, Fbw7 null cells (HCT116 Fbw7 KO) or cells in which Fbw7 was ablated by siRNA

were used. Lack of Fbw7 stabilized mitotic BLM. This effect was specific for Fbw7

isoform as the lack of Fbw7 or Fbw7 could not rescue the levels of BLM post-nocodazole

treatment.

The E3 ligase function of Fbw7 is mediated via its WD40 and F box. The decrease of BLM

levels only by Fbw7 indicated a specific role of the N-terminal region of the E3 ligase in the

degradation process. Indeed, Fbw7N mutant did not decrease BLM levels. Neither

Fbw7F nor Fbw7WD40 mutants were able to decrease BLM levels, confirming that

degradation of BLM requires both these regions. The decrease in the BLM levels due to

overexpression of Fbw7 was reversed by the treatment with MG132, confirming the role of

the E3 ligase in the proteasomal degradation of BLM. Indeed, in vitro ubiquitylation

reactions reveal that recombinant BLM is poly-ubiquitylated by Fbw7 in a F-box and

WD40 domain dependent manner.

Degradation of BLM by Fbw7 depends on GSK3 and CDK2/CyclinA2 mediated

phosphorylation events at Thr171 and Ser175

The WD40 repeats in Fbw7 bind to its substrates which have been phosphorylated in a

conserved phospho-epitope, known as the phosphodegron. We found a single stretch of

149

sequence in BLM that matched the consensus phoshodegron motif 169

FVTPPQSHF177

. We

hypothesized that this putative BLM phosphodegron should also be phosphorylated by GSK3

as it matches with the GSK3 consensus (S/TxxxS/T). Using nuclear extracts obtained at

different stages of the cell cycle as the source of kinase, the phosphorylation of BLM during

mitosis was found to be GSK3-dependent. GSK3 phosphorylates BLM in the N-terminal

212 amino acids, which harbours the putative phosphodegron. Further, GSK3-dependent

phosphorylation of BLM T171A mutant was diminished during in vitro kinase assays. A

specific phosphopeptide was absent when phosphopeptide mapping was carried out with the

phosphorylated products, indicating that Thr171 in BLM is phosphorylated by GSK3.

To decipher the priming kinase that phosphorylates BLM at Ser 175 (+4 position), we

compared the core phosphodegron sequence (171

TPPQS175

) in BLM with all validated

phosphodegron sequences in Fbw7 substrates. We found that this core sequence exactly

matches with one of the two phosphodegrons in Cyclin E (380

TPPQS384

). Cyclin E Ser384 is

phosphorylated by Cdk2. Since BLM undergoes turnover during G2/M phase, we

hypothesized that CDK2 with its corresponding cyclin partner could be the candidate kinase

for the phosphorylation at Ser175. CDK2/Cyclin A2 complex has been shown to have mitotic

functions. Indeed, in vitro kination reveals that CDK2/Cyclin A2 phosphorylates BLM at

Ser175. Consequently, both BLM T171A and S175A mutants completely lose the ability to

bind to Fbw7, indicating conformation dependent interaction between BLM and Fbw7.

To determine whether lack of GSK3 and CDK2/Cyclin A2 phosphorylation events at

Thr171 and Ser175 affect Fbw7-dependent BLM ubiquitylation, in vitro ubiquitylation

reactions were carried out. BLM ubiquitylation was decreased by the presence of either

GSK3 inhibitor or Cdk2/Cyclin A2 inhibitor, roscovitine. Similar decrease in the levels of

Fbw7dependent BLM ubiquitylation was observed for both BLM T171A and S175A

mutants. Consequently, GSK3 inhibitor or roscovitine stabilized BLM levels even in the

presence of overexpressed Fbw7 or in the mitotic cells. Hence overexpression of Fbw7

could not decrease the protein levels of BLM phosphodegron mutants (T171A and S175A).

These results indicate that during mitosis BLM is phophorylated by GSK3 and

CDK2/Cyclin A2 at Thr171 and Ser175 respectively for subsequent K48-linked

ubiquitylation and degradation by Fbw7.

Chk1/Chk2 dependent mitotic BLM phosphorylation at Thr182 is a prerequisite for its

degradation

While BLM enhances Fbw7 dependent degradation of c-Myc, it (i.e BLM) is targeted by

Fbw7for degradation during mitosis. We hypothesized that a mitosis specific

phosphorylation event maybe the required switch, which allows BLM itself to be targeted by

Fbw7. In an earlier study by us, it was proposed that Chk1 may phosphorylate BLM at

Thr182. Using recombinant proteins and in vitro kinase assays we verified that both Chk1

and Chk2 kinases phosphorylate BLM at Thr182. Phosphopeptide map analysis using either

Chk1 or Chk2 indicate a loss of a specific phosphopeptide in the BLM Thr182 mutant. A

phosphospecific antibody which recognizes phosphorylated BLM at Thr182 (pThr182-BLM)

was generated which recognized only wildtype BLM phosphorylated by enzymatically active

Chk1/Chk2. Immunoprecipitation reactions carried out with pThr182-BLM antibody,

indicates that it recognizes BLM in vivo, specifically at Thr182 residue. pThr182-BLM

antibody specifically stains only the mitotic cells in celllines expressing BLM. Thr182

phosphorylation of BLM initiates in prometaphase and continues till mid to late anaphase.

150

Co-treatment of nocodazole with either Chk1 inhibitor (UCN-01) or Chk2 inhibitor II did not

appreciably decrease the level of Thr182 phosphorylation. However presence of both

Chk1/Chk2 inhibitors appreciably decreased the extent of the staining detected by pThr182-

BLM antibody, as revealed by a drastic drop in the colocalization factor (pThr182-BLM vs

BLM signal ratio). Indeed western analysis indicated that even after nocodazole treatment,

Thr182 phosphorylation on BLM was abrogated in presence of both Chk1/Chk2 inhibitors.

Interestingly, presence of both Chk1/Chk2 inhibitors rescued Fbw7 dependent degradation

of BLM due to lack of BLM ubiquitylation, as shown both in vitro and in vivo. The absence

of BLM ubiquitylation was because the mutation in Thr182 on BLM completely abrogated its

binding to Fbw7. These results indicate that Chk1/Chk2 dependent Thr182 phosphorylation

on BLM was required for its subsequent degradation during mitosis.

To further understand the functional consequences of Thr182 phosphorylation in BLM during

mitosis, we generated isogenic stable lines in BS patient cell line, GM03505, expressing

either GFP-tagged wildtype BLM (Clone 4.3) or the Thr182Ala mutant (Clone 50.1). A

stable cell line expressing the corresponding GFP (Clone 100) was also generated. Post-

nocodazole treatment, BLM degradation during mitosis was affected in cells expressing BLM

Thr182 mutant, indicating that this mitosis specific phosphorylation had an effect on BLM

stability. Co-staining of Thr182 phosphorylated BLM with multiple ubiquitin antibodies

revealed a high degree of colocalization in the nucleoplasm of the mitotic cells. Specifically,

the colocalization of pThr182-BLM staining with K48 ubiquitin antibody indicated that BLM

phosphorylated on Thr182 was targeted for degradation via K48-linkage. Hence

overexpression of Fbw7 failed to degrade BLM Thr182 mutant, indicating that Chk1/Chk2

mediated phosphorylation of BLM directly controlled Fbw7-dependent BLM degradation.

Future plans

Some of the future directions of the ongoing research projects in the lab are:

A. Understanding of the biological consequences of tumour suppressor BLM degradation on

chromosomal stability.

B. Determining the changes in the mitochondrial bioenergetics due to the absence of tumour

suppressor RECQL4 and elucidating how such changes may contribute to the cancer

predisposition seen in Rothmund-Thomson patients.

C. Dissecting the biological relevance of multiple E3 ligase mediated degradation of tumour

suppressor p53.

Action taken on the RAP/SAC 2014 recommendations

The data presented was appreciated by the RAP/SAC committee members.

Publications

Original peer-reviewed articles

1. Singh M, Bansal S, Kundu S,

Bhargava P,

Singh A,

Motiani RK,

Shyam R,

Sreekanth V,

Sengupta S, Bajaj A (2015) Synthesis, structure-activity relationship, and mechanistic

151

investigation of lithocholic acid amphiphiles for colon cancer therapy. Med Chem

Commun 6: 192-201.

2. Singh M, Kundu S, Reddy MA, Sreekanth V,

Motiani RK, Sengupta S,

Srivastava A, Bajaj

A (2014) Injectable small molecule hydrogel as a potential nanocarrier for localized and

sustained in vivo delivery of doxorubicin. Nanoscale 6: 12849-55.

3. Rajanala K, Sarkar A, Jhingan GD, Priyadarshini R, Jalan M, Sengupta S, Nandicoori VK

(2014) Phosphorylation of nucleoporin Tpr governs its differential localization and is

required for its mitotic functions. J Cell Sci 127: 3505-20.

152

Role of carbohydrates in modulating the structure and function of

glycopeptides

Principal Investigator Kanwal J Kaur

Project Fellow Vartika Anand

Ph. D. Students PRV Shabareesh

Rohini Dwivedi

Rahul Gupta

Collaborators DM Salunke, NII / RCB, Faridabad

M Sundd, NII

Theme of research

The project is aimed for understanding the role of carbohydrate domains in modulating the

structure and function of glycopeptides by involving different model systems such as

antimicrobial and thrombin-inhibitory glycopeptides.

Objectives

1. Synthesis and structural characterization of glycosylated amino acids

2. Structure-function analysis of the synthetic glycoconjugates

Work reported in 2013-2014

Antimicrobial peptides

In glycoproteins, usually Thr and Ser amino acids are O-glycosylated on their side chain

hydroxyl groups. It has been reported that the conformational impact of -GalNAc attached

to Thr residue differs significantly from that attached to Ser residue. To obtain insights into

the effect of glycosylated amino acid variation on the structural and functional properties of

drosocin, an analogue was synthesized by substituting its glycosylated-Thr at 11th

position

with glycosylated-Ser and named as S11

--D-GalNAc-Drosocin. For synthesizing this

glycosylated peptide, first critical building block, N-Fmoc-Ser (Ac3--D-GalNAc)-OH was

synthesized and then used for peptide synthesis. To compare the antibacterial activity of S11

-

-D-GalNAc-Drosocin with its non-glycosylated variant, the S11

-Drosocin was also

synthesized. Finally, all the synthesized peptides were characterized by mass spectrometry.

In order to analyze the antibacterial effect of Thr substitution with Ser in Drosocin analogues,

MIC of each peptide was determined against different Gram negative bacterial strains of E.

coli and Salmonella. Substituting Thr with Ser in non-glycosylated Drosocin did not affect

the MIC significantly, indicating that Ser or Thr alone did not determine differential

antimicrobial behaviour of Drosocin peptides. The comparison of antibacterial activity of S11

-

-D-GalNAc-Drosocin and Drosocin (T11

--D-GalNAc-Drosocin) demonstrated that the

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MIC for S11

-α-D-GalNAc-Drosocin was higher than T11

--D-GalNAc-Drosocin against E.

coli and Salmonella strains. With just a difference of -methyl group, S11

--D-GalNAc-

Drosocin could not display lethal action comparable to that of T11

--D-GalNAc-Drosocin.

Interestingly, S11

-α-D-GalNAc-Drosocin showed higher MIC than its non-glycosylated

counterpart, S11

-Drosocin, against bacterial strains of E. coli and Salmonella. This result was

intriguing, suggesting that the addition of N-acetyl galactosamine was not the only reason for

higher antimicrobial activity of T11

--D-GalNAc-Drosocin. These observations further

confirmed that there was a difference between the conformational impact of glycosylated-Thr

and glycosylated-Ser on the peptide backbone.

To analyze the structural effects of substitution of glycosylated-Thr, the comparative

conformational properties of Drosocin and its analogues were analyzed by CD spectroscopy

in PB (10mM, pH7.4), 90% TFE, and 10mM SDS.

Thrombin-inhibitory glycopeptides

With an aim to study the impact of glycosylation on the inhibitory potential of active site

directed, short peptidyl inhibitors of thrombin, two non-glycosylated tetra peptides were

synthesized. The results indicated that D-ChaPβhomoRG was more efficient than

fPβhomoRG in inhibiting thrombin‟s activity.

The inhibitory potential of C-terminal 23 residue peptide of Hirudin P6 and its cognate

glycosylated analogue (α-GalNAc-HP6) was estimated by fibrinogen competitive assays. It

was observed that the glycosylated HP6 (-GalNAc-HP6) had higher inhibitory activity than

its non-glycosylated form, suggesting the effect of sugar in modifying the functional activity

of thrombin-inhibitory peptide.

Progress of work during the current reporting year (2014-2015)

Antimicrobial peptides

Continuing the studies for understanding the role of glycosylation in modulating the structure

and function of peptides, we have undertaken the model system of cathelicidin class of

antibacterial peptides. Indolicidin (ILPWKWPWWPWRR-NH2) which belongs to the

cathelicidin family, has anti-endotoxic activity and a broad spectrum of antimicrobial

activity. However, at the bactericidal concentrations, indolicidin exhibits substantial toxicity

towards mammalian erythrocytes and other cells. It is a short, 13 amino-acids peptide and

contains the highest proportion of tryptophan residues ever seen with 39%. It has been

reported that the hydrophobic core of this peptide consisting of tryptophan residues, is

important for interaction with bacterial membrane as well as responsible for its cytotoxicity.

Studies have also shown that the overall structure of the peptide is most important factor in

interacting with membrane rather than only hydrophobic core. Peptide-carbohydrate mimicry

studies carried out from our laboratory earlier had suggested possible correlation of aromatic

residues such as Tyr and Trp to resemble the carbohydrate moieties. Interestingly, in in-silico

studies when the tryptophans present at different positions in indolicidin were substituted

sequentially with glycosylated amino acid and the resulted glycosylated analogues of

indolicidin were docked in the endotoxin structure, the calculated total potential energies for

all the complexes of endotoxin with glycosylated analogues of indolicidin were comparable

154

to that of the endotoxin complex with native indolicidin. Therefore we sought out to design

glycopeptides by incorporating glycosylated amino acid in place of the critical tryptophans in

indolicidin for investigating the sugar potential in reducing its cytotoxic property and detailed

structural and functional effect of glycosylation on this antibacterial peptide.

We have synthesized the glycosylated analogues of indolicidin by substituting W6 and W

11

with α-GalNAc-T. The designed analogues, α-GalNAc-T6-Indolicidin and α-GalNAc-T

11-

Indolicidin, were compared for their antibacterial potential, haemolytic and cytotoxic

activities with that of native indolicidin. The antimicrobial activities were analyzed by their

Minimum Inhibitory Concentrations (MIC) against different Gram-negative and Gram-

positive bacterial strains. All the analogues exhibited strain specific antimicrobial activities.

Both the glycosylated analogues displayed higher MIC in comparison to that of indolicidin.

However, both the glyco-analogues were found to be non-toxic to erythrocytes and

macrophage cell lines. The above results suggested that substitution of W at 6th

and 11th

positions were indeed resulting decrease in the cytotoxicity significantly but also

compromising the antimicrobial potency and indicating the important role of W6 and W

11 in

the determination of both antimicrobial and cytotoxic properties of indolicidin.

It has been reported that in indolicidin, tryptophane residue at 9th

position plays an important

role in its haemolytic activity and proline substitution with Lys results an increase in its

therapeutic index. With this background, the strategy for further designing of the analogue

involved the substitution of W9

with glycosylated threonine along with the replacement of P7

to K7 resulting designed peptide, α-glyco-Thr

9K

7-Indolicidin. The designed glycosylated

analogue exhibited antimicrobial activity comparable to native indolicidin against all the

tested different bacterial strains and it was found to be non-haemolytic at the concentration as

high as 100M and non-toxic to macrophage cell line even at the concentration of 100g/ml.

To confirm that the change in antimicrobial and cytotoxic activities of α-glyco-Thr9K

7-

Indolicidin was not due to the absence of W9 and sugar indeed was playing some role, the

synthesis of its non-glycosylated analogue, Thr9K

7-Indolicidin, was carried out. The non-

glycosylated analogue displayed MIC against the bacterial strains comparable to that of

native indolicidin and also displayed toxicity against rat erythrocytes and macrophage cell

lines similar to indolicidin. These observations confirmed that introduction of sugar affects

on the antibacterial, haemolytic and cytotoxic activities of the peptide.

Thrombin-inhibitory glycopeptides

For investigating the effect of carbohydrates on the structure and function of thrombin

inhibitory peptides, we had reported the inhibitory potential of the C-terminal 23-mer of HP6

and its corresponding glycosylated analogue (-GalNAc- HP6). With an aim to gain structure

based understanding for observed kinetic differences between non-glycosylated HP6 and α-

GalNAc-HP6, we have attempted co-crystallization of these peptides with human α-

thrombin. X-ray diffraction data was collected on home source x-ray diffractometer where

the co-crystals of α-GalNAc-HP6 diffracted to 2.3A resolution and HP6 diffracted to 2.4A

resolution. Data processing and refinement are in progress.

As it was known that the C-terminal of HP6 inhibited thrombin function by interacting

weakly with the exosite-1 alone, we conceived the designing of a HP6 derived bivalent

thrombin inhibitor which could interact both with exosite-1 and active site. Towards this end,

155

we have tethered an active site binding motif (D)fPRP to the C-terminal HP6 with and

without glycine linker. We have synthesized the non-glycosylated (BiG0HP6 and BiG1HP6)

and glycosylated (α-GalNAcBiG0HP6 and α-GalNAcBiG1HP6) forms of these designed

bivalent peptides to understand the impact of glycosylation on their inhibitory efficacy. All

the peptides were purified to homogeneity and characterized by mass spectrometry.

All the synthesized analogues of HP6 were assayed for their thrombin inhibitory efficiencies

by monitoring the release of p-nitro-aniline (pNA) at 405nm from s-2238 – a thrombin

specific chromogenic substrate, in presence of varying concentrations of inhibitors. Inhibitory

parameters like IC50 and Ki values were determined by measuring the initial rates of pNA

release. All the bivalent HP6 inhibitors exhibited inhibitory constant (Ki) in nano-molar

range. Non-glycosylated bivalent analogue bearing one glycine residue in the linker region

(BiG1HP6) displayed the higher inhibitory activity than that of BiG0HP6. Eventhough

glycosylation was found to enhance the inhibitory potential of HP6 analogue ; the same was

not observed in case of bivalent HP6 inhibitors. On the contrary, α-GalNAc bearing bivalent

HP6 peptides turned out to be weaker inhibitors in comparison to their corresponding non-

glycosylated forms.

To get a further insight into the kinetics of association and dissociation of these inhibitors

with thrombin, the surface plasmon resonance studies were carried out. The detailed kinetic

parameters based on the sensograms generated at different concentrations of inhibitory

peptides, were determined. Unimmobilized treated flow cell and trypsin immobilized flow

cell served as negative controls to monitor non-specific interactions of the peptides.

Reference subtracted sensograms were analyzed by Biacore T200 Evaluation software by

fitting the sensograms to 1:1 Langmuir kinetic model. The KD value obtained for

glycosylated bivalent HP6 inhibitor α-GalNAcBiG1HP6 was found to be higher than that of

the KD value obtained for non-glycosylated analogue, BiG1HP6. This observation

compliments our inhibition constants (Ki) derived from enzyme inhibitory kinetics data.

Besides this, a comparative analysis of the SPR derived association and dissociation rate

constants between glycosylated and non-glycosylated bivalent inhibitors revealed additional

information for the impact of glycosylation. Glycosylated bivalent HP6 exhibited slower rate

of dissociation than that of its non-glycosylated form. This slow rate of dissociation of

glycosylated bivalent HP6 possibly points out to the additional new interactions that the

inhibitor is capable of making with thrombin. However, this slow dissociation of α-

GalNAcBiG1HP6 has not lowered its KD value (KD = kd/ka) due to its slower rate of

association when compared to that of non-glycosylated BiG1HP6 which may reflect possible

conformational changes taking place during its association with thrombin.

Future plans

1. Indolicidin not only exhibits antimicrobial activity but is known to have other properties

such as endotoxin neutralizing activity, DNA binding and calmodulin binding. It would be

interesting to investigate whether designed glycosylated analogues of indolicidin which

display antibacterial activity equivalent to that of native indolicidin but non-cytotoxicity

towards eukaryotic cells, have these other properties. The designed glycosylated analogues of

indolicidin would be evaluated for their binding to LPS (entotoxin) by surface plasmon

resonance studies and polymyxin B displacement assay and comparison will be made with

native indolicidin. The anti-endotoxin activity of active analogues would be confirmed by

assaying their interference in NO release and B-cell proliferation, the known markers

156

associated with endotoxin activity. Moreover, these studies would help us in exploring the

effect of sugar on different properties of glycosylated indolicidin.

2. The previous structural studies of native indolicidin in two different micelle systems have

shown that it contains elements of both poly-L-proline type II helix and -turn. The detailed

structural studies of glycosylated indolicidin analogues will be undertaken to understand the

effect of glycosylation on their conformation.

3. Earlier we have shown the differential effect of sugar variation on the antibacterial activity

of Proline rich class of unstructured antimicrobial glycopeptides. The effect of sugar variation

and glycosidic linkage in the designed glycosylated analogues of indolicidin will be

investigated for their antimicrobial and other properties.

4. Studies related to the mechanism of action of designed antibacterial glycosylated

indolicidin analogues will be explored and comparison will be made with native indolicidin.

5. In case of thrombin-inhibitory peptides, detailed comparative structure-function correlation

studies of glycosylated, non-glycosylated HP6 peptides and their bivalent analogues will be

continued.

6. With an aim to provide structural understanding of the mode of interactions of previously

synthesized thrombin-inhibitory variegin glycopeptides with thrombin, either molecular

docking studies or co-crystallization of these peptides with α-thrombin would be attempted.

Action taken on the RAP/SAC 2014 recommendations

The scientific and technical queries were answered to the satisfaction of the RAP/SAC

members. No specific recommendation was made.

Publications

Original peer-reviewed article

1. Lele DS, Talat S, Kumari S, Srivastava N, Kaur KJ (2015) Understanding the

importance of glycosylated threonine and stereospecific action of Drososcin, a proline rich

antimicrobial peptide. Eur J Med Chem 92: 637-647.

157

Study of immunotherapeutic potential of Mycobacterium indicus pranii

(MIP) and the underlying mechanisms in animal models of tuberculosis &

tumor

Principal Investigator Sangeeta Bhaskar

Co-Investigator Pramod Upadhyay

Project Assistant Rahul Khatri

Ph. D. Students Mohd. Saqib

Bindu Singh

Ananya

Arvind Kumar

Theme of research

Whole bacterial vaccines rely on multiple antigens and built-in-adjuvanticity. Mycobacterial

strains which share cross-reactive antigens with M.tuberculosis are being considered as

alternatives to M.bovis for vaccine use. MIP shares antigens with M.tuberculosis and initial

studies had shown that vaccination with killed MIP induces protection against tuberculosis.

Hence, we further studied the protective potential of MIP and the underlying immune

responses.

Generation of antitumor immunity is often difficult in the tumor-bearing host because of

various negative regulatory mechanisms which can be overcome by activation of innate and

Th1 immune response. There were indications from some open labeled clinical studies that

MIP could be useful as an immunomodulatory adjunct in some cancers. In animal models of

tuberculosis we had found that MIP induces Th1 response which is also important for

antitumor activity. Hence, we had started this study to evaluate the immunotherapeutic

activity of MIP in mouse model of tumor.

Objectives

To investigate the protective efficacy of MIP immunization in live or killed form, through

parenteral route / by aerosol route, against subsequent infection with M.tuberculosis in animal

models. Study of immune response to M.tb in animals immunized with MIP as compared to

those immunized with BCG.

Evaluation of immunotherapeutic efficacy of MIP along with chemotherapy in animal model

of tuberculosis.

To evaluate Immunoprophylactic and Immunotherapeutic activity of MIP in mouse syngeneic

tumor model. Study of MIP as an adjunct to chemotherapy in combination with commercial

anti cancer drugs in tumor bearing mice and simultaneous study of mechanism of MIP

mediated host immune activation.

158

Work reported in 2013 -2014

A. Immunotherapeutic potential of MIP and the underlying mechanisms in mouse

tumor model

Ex-vivo studies with macrophages and dendritic cells showed that TLRs had major role in

MIP mediated activation of these cells. Further it was examined whether signaling through

these TLRs was responsible for MIP mediated protection against tumor. Tumors were

implanted in MyD88 knockout and wild type mice and MIP treatment was given by

peritumor injections. While in wild type mice treated with MIP about 40-50% mice had no

visible tumor or very small tumor, MIP treated MyD88 knockout mice had tumors

comparable to PBS treated mice. These results provided evidence of major role of TLRs in

MIP mediated protection against tumor. Further studies with TLR knockout mice strains

showed that TLR2 has important role in MIP mediated protection. TLR2 knockout mice

treated with MIP showed no reduction in tumor volume whereas MIP treated TLR4 knockout

mice had tumor volumes comparable to MIP treated wild-type mice. MIP treatment resulted

in increased IFN-γ expression in tumor milieu whereas MIP + cyclophosphamide treatment

induced type-I Interferons IFN-α and IFN-β. In the group treated with „only drug‟, expression

of type-I interferons was not observed. Results suggested that „drug + MIP‟ therapy

modulates the immune status of tumor microenvironment.

B. Protective efficacy of MIP against tuberculosis and mechanistic insights as compared

to BCG

Evaluation of immunostimulatory activity of different fractions of MIP

Differential immunostimulatory activity of cellular fractions of MIP was investigated. Cell

wall fraction showed highest immune stimulating activity. It was further fractionated into

aqueous soluble and lipid soluble parts and determined their immune stimulatory activity in

terms of macrophage and T cell activation. To analyse the TLR agonistic activity of MIP cell

wall fraction, immune response in the presence of specific pharmacological inhibitors of

TLRs was studied. About 20% reduction in secretion of major proinflammatory cytokine was

observed with TLR4 inhibitor, which further increased to almost 50% when a combined

TLR2 and TLR4 inhibitor was added to MIP cell wall treated macrophages. Experiments

were repeated with macrophages isolated from TLR2 and TLR4 knock out mice. About 75%

reduction in secretion of major proinflammatory cytokine was observed in TLR2 knock out

mice as compared to the wild type. Reduction in cytokine secretion was about 30% in TLR4

knock out mice. Data provided evidence of strong TLR2 agonistic activity and moderate

TLR4 stimulating property of MIP cell wall fraction.

Progress of work during the current reporting year (2014-2015)

A. Protective efficacy of MIP against tuberculosis and mechanistic insights as

compared to BCG

MIP induces dendritic cell activation, survival and Th1/Th17 polarization potential in

TLR-dependent manner

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Dendritic cells, by virtue of their unmatched antigen presentation potential, play a critical role

in activation of antitumor and antimycobacterial immune response. The effect of MIP on the

behavior of dendritic cells and the underlying mechanisms were investigated. MIP lead to

significant production of IL-6, IL-12p40, IL-10 and TNF-α by dendritic cells and expression

of costimulatory molecules CD40, CD80 and CD86 was upregulated.

Live MIP induced significantly higher response as compared to heat-killed MIP. Propidium

iodide and annexin V staining showed that MIP increases dendritic cell survival by inhibiting

apoptosis. Consistently, higher expression of anti-apoptotic proteins Bcl-2 and Bcl-xl was

observed in MIP-stimulated dendritic cells. Cytokines produced by naïve T cells co-cultured

with MIP-stimulated dendritic cells showed that MIP promotes Th1/Th17 polarization

potential in dendritic cells. Response to MIP was lost in MyD88-deficient dendritic cells

underscoring the critical role of TLRs in MIP-induced dendritic cell activation. Further

studies revealed that TLR2 and TLR9 are involved in dendritic cell activation by MIP-live,

whereas MIP-killed activates the DCs through TLR2. These findings define the behavior of

MIP-stimulated dendritic cells and highlight the role of TLRs in MIP-induced dendritic cell

activation.

MIP-live and MIP-killed have differential TLR agonistic activity

MIP in live and killed form was evaluated for macrophage activation and compared with

BCG. Using pharmacological inhibitors and knock out mice strains, major role of TLR2

was observed in the activation of macrophages by MIP and BCG. TLR2 signaling was

further delineated. With the help of HEK293 cells expressing TLR2 in homodimeric or

heterodimeric form, it was observed that live MIP had distinctly higher level of TLR2/2,

TLR2/1 and TLR2/6 agonist activity as compared to MIP-killed and BCG.

Decreased ability of MIP-killed to engage TLR2 could be due to denaturation or reduced

accessibility of TLR ligands because of heat treatment. Surprisingly, despite of showing

TLR2 engagement in macrophages, BCG didn‟t engage either of TLR2/2, TLR2/1 or

TLR2/6 in HEK 293 cells. We speculated that TLR2 agonists are not in exposed

form in the BCG which resulted in lack of response in HEK 293 cells. To address this,

we used MIP and BCG in sonicated form. Interestingly, when used in sonicated form,

both BCG and killed MIP induced substantial response from TLR2 expressing HEK 293

cells. Hiding of putative TLR ligands can be an important strategy of pathogenic

mycobacteria to evade immune-recognition. BCG also potentially inhibit phago-

lysosomal fusion which would further prevent degradation of bacterial components and

subsequent release of putative ligands. An important finding was that sonicated form of

MIP-killed induced significantly high response which would be further evaluated for its

protective efficacy.

B. Efficacy of MIP as a booster to BCG: Immunogenicity, protection and safety

study when given by aerosol route in animal models

BCG is effective against severe form of childhood tuberculosis however, effective protection

from TB in adults is still a challenge which indicates that there is a need to boost the

protective immune response against M.tb. However, paradoxically, multiple doses of BCG

had resulted in reduced protection and poor survival in the susceptible animal model. Initial

studies showed that vaccination with MIP gives protection against TB in both BCG responder

& non-responder strains of mice. In large scale human trial involving 28,948 people,

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protective efficacy of MIP against TB was observed. The vaccine/placebo was given to

healthy contacts of leprosy patients who had no evidence of tuberculosis. After 13 years, in

retrospective analysis significant difference was observed in the occurrence of TB cases in

the vaccinated and placebo groups. Further, higher protective efficacy of MIP was observed

in the group who had earlier received BCG in their childhood. Previous studies in our lab,

have shown higher protective efficacy of MIP as compared to BCG in animal models of

tuberculosis given by subcutaneous or aerosol route. Hence, it was proposed that a booster

with MIP may enhance the protective immunity in animals primed with BCG. The intranasal

route is non-invasive and proposed to induce both local lung and systemic immunity.

Two groups of mice were immunized with BCG by s.c. route and after a gap of 2 months,

they were given booster with MIP by aerosol/s.c. route. While one control group was

immunized with only BCG without any booster the other control group was immunized with

saline. M.tb specific memory recall response was studied at certain time points before booster

and after booster. Four week after BCG immunization M.tb specific immune response was

reduced to low level but it increased significantly after booster with MIP and was maintained

at significantly high level as compared to „only BCG‟ immunized group till experimental

observation period. To evaluate whether protective immune response also reflects in reducing

the bacterial load in lungs and spleen, M.tb challenge experiments were done in guinea pigs.

Significantly lower bacterial load was observed in the groups given booster with MIP as

compared to „only BCG‟ immunized group at 4 wk and 8 wk after challenge with M.tb. Lung

immune response was studied in the immunized and M.tb infected lungs at certain time points

after infection by analyzing mRNA expression of different cytokine genes. Expression of

IFN-γ, IL-12 and IL-2 was significantly high at 4 wk after infection in the groups given

booster with MIP as compared to „only BCG‟ immunized group. IL-10 level was also high in

the previous groups. Histopathological analysis of sections of infected lungs confirmed the

bacteriological findings. Significantly lower Gross pathological score was observed in the

groups given booster immunization. These results provide evidence of higher protective

efficacy in the groups given MIP booster after BCG priming. Further experiments are

underway, where dose of MIP aerosol is increased. Toxicological studies have been planned

to evaluate any untoward response in the lungs and other organs.

Future plans

Different cellular fractions of MIP will be evaluated for their immunoadjuvant property in

comparison to whole MIP. Mycobacterium cell wall consists of various molecules that play

important role in immune regulation and one of the unique molecules is Lipoarabinomannan.

Different types of lipoarabinomannans have been reported in slow growing/fast growing

mycobacterium species for example ManLAM, PILAM and ArabLAM and these exert either

immunomostimulatory or immunosuppressive activity. Hence, we will try to find out which

components or fractions of MIP are important for its immunostimulatory / immunoadjuvant

property and characterize the Lipoarabinomannan of MIP.

Efficacy of MIP immunotherpy would be evaluated in guinea pig model of tuberculosis

mimicking different clinical presentation of human TB e.g. in severe form of TB where

cavities and bacterial load is high. MIP immunotherapy will be given along with regular

chemotherapy. Lung immune response and bacterial load would be analyzed and compared

with the group where only anti TB chemotherapy is given.

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Whether immunization with MIP aerosol influences recruitment of T cells in airway lumen /

lung interstitial space would be studied. Specific memory T cells in these compartments of

lung have differential protective role.

M.tb can survive in the host for prolonged period of time as it employs different strategies to

evade the host immune response. Inhibition of autophagy and phago-lysosome fusion in the

infected macrophages is an important strategy which promotes its survival. We would like to

examine the role of autophagy in MIP mediated protection against M.tb infection. Effect of

MIP on autophagic process in macrophages and its role in phagosome maturation and phago-

lysosome fusion in M.tb infected macrophages will be studied. We will check whether MIP

induces/inhibits autophagy in murine macrophage cell line / primary macrophages. Level of

LC3II will be estimated as the turnover of LC3I to LC3II marks the increase in the

autophagosome formation, and thus the induction of autophagy. Formation of autophagosome

will also be analysed by ultrastructural analysis.The role of MIP mediated autophagy in

controlling the Mycobacterium tuberculosis infection will be examined.

Action taken on the RAP/SAC 2014 recommendations

The scientific and technical clarifications sought during the interactive session were provided.

Publications

Original peer-reviewed articles

1. Kumar P, John V, Marathe S, Das G, Bhaskar S (2015) Mycobacterium indicus pranii

induces dendritic cell activation, survival and Th1/Th17 polarization potential in a TLR-

dependent manner. J Leukoc Biol 97: 511-20.

2. Kumar P, Tyagi R, Das G, Bhaskar S (2014) Mycobacterium indicus pranii and

Mycobacterium bovis BCG lead to differential macrophage activation in Toll-like

receptor-dependent manner. Immunology 143: 258-268.

Reviews/Proceedings

1. Singh MS, Bhaskar S (2014) Nanocarrier-based immunotherapy in cancer management

and research. Immuno Targets Ther 3:121-134.

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Studies of Sertoli cells and spermatogonial stem cells of the testis and other

endocrinology related research

Principal Investigator Subeer S Majumdar

Research Associate Sayon Basu

Project Fellows Hironmoy Sarkar

Rajesh Sarkar

Bhola Shankar Pradhan

Ph. D. Students Satyapal Arya

Kamal Mandal

Souvik Sen Sharma

Collaborators U Rai, University of Delhi

S Bhattacharya, Visva-Bharati, Shantiniketan

MK Choudhury, Tezpur University, Tezpur

Theme of research

We use testis as an organ of multiple research interest 1) exploiting spermatogonial stem cells

for propagation of transgene; i.e. for generation of transgenic animals, 2) analyzing

differential gene expression by Sertoli cells (Sc) during active vs. inactive phase of

spermatogenesis to identify factors regulating germ cell division and differentiation with an

intent to divulge unknown (inborn or environmentally induced) non hormonal causes of

idiopathic male infertility and 3) undertaking germ cell transplantation studies to restore

fertility upon chemotherapy. In addition, we also participate in other endocrinological

research as collaborators.

Objectives

1. To exploit spermatogonial stem cells of testis for insertion and propagation of transgene

through several generations in an attempt to over express or knock down specific genes

2. To undertake gene expression studies of rat, mice and monkey Sertoli cells to identify

factors important for induction of spermatogonial stem cell division and differentiation in

the testis.

3. To study biology of spermatogonial stem cells and to use germ cell transplantation

technique for restoration of fertility following chemotherapy.

4. To study paracrine and endocrine modulation of signal transduction in target cells of the

endocrine system.

Work reported in 2013-2014

Functional genomic studies of genes selected from studies of differential genomics by

DNA microarray using mRNA from Rat Sertoli cells.

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In rats, in spite of sufficient level of circulating hormone, since birth, the division and

differentiation of spermatogonia do not occur until, 11-12 days of age. In view of this

hypothesis, we choose microarray technique to study the differential transcriptome related to

robust onset of spermatogenesis. Sc were cultured from infant (5 day old) as well as pubertal

(12 day old) rats and RNA from such Sc was further used for DNA microarray utilizing the

Agilent platform. DNA microarray reads were normalised and processed by Genespring Gx

software to generate comparative transcriptome profile of hormone treated infant and

pubertal Sc. We found that certain genes like Ell Associated Factor (eaf2), Ninjurin 2 (ninj2),

Neuramedin (nmu), Nuclear receptor 4A3 (Nr4a3) were up regulated in pubertal rat Sc as

compared to infant rat Sc. Sostdc1, Angiotensin type 2 receptor (Agtr 2), Extracellular

growth factor ligand 3 (Egfl3), Tetraspanin 8 (Tspn8) were found to be down regulated in

pubertal rat Sc as compared to infant rat Sc. Eaf2 is positive regulator of RNA Pol II, Ninj2 is

a cell- cell adhesion molecule, Nmu is involved in energy homeostasis, Nr4a3 is a orphan

nuclear receptor, Sostdc1 is a Wnt and BMP inhibitor, Agtr 2 is a receptor for apoptosis,

Tspn 8 is an inhibitor of differentiation whereas Egfl3 is a ligand for EGF pathway. From

functional genomics study of the differentially expressed genes obtained from rat microarray,

transgenic rat models were being generated

Endocrine signaling

Sertoli cell: We have demonstrated that unlike pubertal testicular Sertoli cells (Sc), infant Sc

fail to produce substantial amount of cAMP upon FSH treatment. In an effort to understand

why cAMP production is restricted in the infant Sc, we reported that the expression of GαS

and its activator, Ric8b are very low in infant Sc. This may be responsible for suboptimal

cAMP generation and insufficient expression of spermatogenically relevant genes by infant

Sc, in spite of sufficient expression of FSHR, circulating levels of FSH and binding of FSH to

FSHR on Sc. We also found that levels of Gαi increased upon FSH treatment in infant Sc

unlike pubertal Sc. A set of new information generated about some signal transduction

mediator molecules of primate Sc by us may provide basis for diagnosis and treatment of

certain forms of idiopathic male infertility.

Adipose tissue: We had shown earlier that Fetuin A is necessary for Free fatty acids (FFAs)

mediated augmentation of adipose tissue inflammation through the TLR4 pathway which

causes insulin resistance. Macrophage infiltration into adipose tissue during obesity and their

phenotypic conversion from anti-inflammatory M2 to proinflammatory M1 subtype has been

shown to significantly contribute towards developing a link between inflammation and

insulin resistance. Signaling molecule(s) for these events, however, remained poorly

understood. Further studies showed that lipid induced FetA from adipocytes is an efficient

chemokine for macrophage migration and polarization. These findings opened a new

dimension for understanding obesity induced inflammation.

Progress of work during the current reporting year (2014-2015)

Functional genomic studies of genes selected from studies of differential genomics by

DNA microarray using mRNA from Rat Sertoli cells

Testicular Sertoli cells (Sc) support the division and differentiation of germ cells (Gc) upon

their maturation during puberty. Recently, our lab has reported that in rats, despite sufficient

levels of FSH and T during infancy (upto 9 days of age), immature Sc fail to support the

robust division and differentiation of spermatogonial cells, which is only discernible during

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the onset of puberty, when the Sc start undergoing the functional maturation (from 12 days of

age onwards). Identification of factors produced by Sc upon their maturation and their mode

of actions in regulating germ cell differentiation are still missing Therefore, in order to

identify the factors associated with Sc maturation, a differential evaluation of hormone

responsive genes expressed by infant and pubertal rat Sertoli cell (Sc) was performed. From

the microarray analysis, Tetraspanin 8 (Tspan 8) was identified as one such factor which was

significantly up-regulated in 5-days-old infant Sc (immature) as compared to that of the 12-

days-old pubertal Sc (mature) in rat. Tspan 8 is a member of Tetraspanin superfamily having

four transmembrane domains.

The most distinctive feature of tetraspanin proteins is that they can form lateral associations

with multiple molecular partners and with each other, organize the surface membrane

proteins in dynamic microdomains, referred to as “tetraspanin microdomains” or the

“tetraspanin web.” This membrane organization, which is distinct from “lipid rafts,” can

regulate important cell functions, such as the immune response, cell adhesion, mammalian

fertilization, fungal invasion, cell fusion, trafficking and signal transduction. To delineate the

function of Tspan 8, we generated transgenic rat model expressing Tspan 8 in the pubertal Sc

( naturally, it is overexpressed in infant Sc). The full length ORF of Tspan8 without its stop

codon was amplified from the RNA isolated from embryo of rat during which flag tag

sequence (DYKDDDDK) was added to the c terminus of the Tspan8 ORF by PCR and the

sequencing data showed that there was no error in the coding sequence. The PCR product

was cloned into Rhox5-IRES-EGFP construct such that Tspn8 expression was governed by

Rhox5- promoter (this activates in Sc during puberty ) element. The resultant Rhox-5-Tspn8-

IRES-EGFP construct should putatively drive Tspn8 and EGFP expression only in Sc and

only from 14 days of post natal age - onwards. The 10 months old rats having Rhox5-Tspan8-

IRES-EGFP transgene were referred to as Tspan8 over expressing Tg rats and the age

matched WT rats were referred to as controls. F1 generation male rats from Tspan8 over

expression lines were analyzed for the defects in reproductive functions. Qualitative

histological evaluation showed abnormalities in the testes of the Tg rats at 10 months of age

in comparison to age matched controls. The testes of Tg rats showed sloughing of

spermatogenic cells in the tubules. Sperm were very less in the seminiferous epithelium

lumen. These tubular abnormalities were rarely found in testis of age matched controls. The

Tg rats showed significant (p<0.05) reduction in sperm counts as compared to their age

matched controls. Sperm counts were 148 ±0.37 × 106/ml/epididymis in control rats and 20

±0.05 × 106/ml/epididymis in Tg rats. Tspan8 over expressing F1 generation males (n= 5)

were found to be infertile when mated with healthy mature WT females (n= 10) for more than

three months (for each individual mating set).

Tspan 8 over expression in Sc upregulated matrix metaloproteinases (MMP7) which impaired

the tight junction molecules affecting maturation of Sc which ,in all probability led to

dysregulation in proliferation of Gc and caused their apoptosis leading to infertility in adult

rats. Therefore, identification of factors produced by Sc, one by one, by such functional

genomics approach may help to understand the molecular basis of male infertility.

Endocrine signaling

Sertoli cell: Signalling events are regulated by Wnt molecules in several tissues. In our

microarray studies, using infant amd pubertal monkey Sc, we found that along with several

other morphogens, expression of Wnt3 was upregulated many folds in pubertal monkey Sc as

compared to infant Sc. Wnt3 is known to activate Wnt/ β catenin signalling and affect crucial

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stages of development and differentiation. Importance of β catenin mediated signalling in

regulation of spermatogenesis has been reported previously . However, the identity and role

of Wnt family ligands, which act upstream of β catenin, remains to be understood with

reference to Sc mediated regulation of Gc differentiation. We generated transgenic mice with

Sc specific loss of Wnt3 at and after puberty for evaluating its role in spermatogenesis.

Transgenic mice were generated by us in which shRNA targeting Wnt3 was expressed under

control of Pem (Rox5) promoter thereby restricting resultant Wnt3 knockdown (KD) solely

to the postpubertal Sc. Expression of Wnt3 mRNA was significantly (p<0.05) low. On an

average, transgene positive male mice had ~85% reduction of testicular Wnt3 mRNA, in 10

weeks old adult Wnt3KD mice as compared to age matched LacZKD controls. Western blot

analysis of testicular proteins of 10 weeks old Wnt3KD and control mice also showed a

remarkable decrease in testicular Wnt3 levels in Wnt3KD mice.

Total β catenin levels were also lower in Wnt3KD mice. The ratio of phospho β catenin to β

catenin was significantly (p<0.05) higher in Wnt3KD mice which indicated increased β

catenin degradation in these mice. Immunohistochemical analysis revealed compromised

nuclear localization of β catenin in 10 weeks old Wnt3KD mice. In age matched, LacZKD

control mice, strong nuclear localization signal for β catenin was observed in advanced stages

of Gc. mRNA levels of β catenin were found to be significantly (p <0.05) low in Wnt3KD

mice as compared to LacZ KD mice. Testicular expression of Axin2 and Pitx2 genes, which

are transcriptional targets of nuclear β catenin, were also down regulated in Wnt3KD mice

as compared to the age matched LacZ KD control mice confirming limited nuclear

localization of β catenin in Tg mice.

Subfertility and oligozoospermia were noticed in such mice along with diminished levels of

Connexin-43, a gap-junctional molecule known to be essential for Gc development. We

report for the first time that Wnt3 governs Sc mediated regulation of spermatogenesis and

hence is crucial for fertility. A set of new information generated about some signal

transduction mediator molecules of primate Sc by us may provide basis for diagnosis and

treatment of certain forms of male infertility which are considered idiopathic, as of now.

Adipose tissue: Our studies suggested that Fetuin A (FetA ) is necessary for Free fatty acids

mediated augmentation of adipose tissue inflammation through the TLR4 pathway which

causes insulin resistance. Studies have also shown that lipid induced FetA from adipocytes is

an efficient chemokine for macrophage migration and polarization.

Insulin resistance set in obesity induced diabetes due to excess deposition of fat and as this

condition leads to disruption of demand and supply ratio of insulin. Elevated insulin

secretion by pancreas to compensate the loss of insulin sensitivity gradually fails due to

increased intensity of insulin resistance. There are several attempts to substitute insulin and

other small molecules that act as an insulin mimetic or PPARγ agonist or PPARγ receptor

agonist to deal with lipid induced insulin resistance. We, in collaboration with Prof.

Bhattacharya have begun testing a small molecule made by Prof. Mihir K. Choudhury, on

diabetic high fat fed rodents and db/db mice , to determine its feasibility as an ideal treatment

for diabetes.

Spermatogonial stem cells: germ cell transplantation technique for restoration of

fertility following chemotherapy

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Techniques to culture mouse and monkey spermatogonial stem cells are being standardized.

Autologus germ cell transplantation has been done in mice to restore spermatogenesis in mice

treated with chemotherapeutic agents. Germ cells of one testis are now transfected with genes

before isolation and culture in vitro and transplanted in contralateral testis, post

chemotherapeutic exposure, to see colonization of such transplanted transgenic germ cells.

Action taken on RAP/SAC 2014 recommendations

No specific recommendation was made by the Committee.

Future plans

Since we are getting encouraging outcome, functional genomics of more differentially

expressed genes, obtained from microarray analysis of the testicular Sertoli cells (Sc) of rat

and monkeys will be undertaken. Transgenic mice will be generated by us in which shRNA

targeting CD30, Ninjurin and other differentially expressed genes will be expressed under

control of Pem promoter thereby restricting resultant knockdown (KD) solely to the

postpubertal Sc. Expression (decline) of mRNA in positive male mice will be determined in

10 weeks old adult KD mice as compared to age matched LacZKD controls. Western blot

analysis of such targeted proteins will be done. Fertility will be correlated with that. Such

studies will now be taken with mice as well as rat, both, as we have standardized technique of

rat transgenesis in our laboratory.

We have begun to undertake autologus germ cell transplantation after busulfan treatment of

mice. We will check success of autologus germ cell transplantation in mice post

chemotherapy and attempt doing similar kind of studies in monkeys also. For this, first, we

will establish monkey germinal stem cell cultures.

Collaborative research in the field of endocrinology /diabetes will be undertaken to divulge

basis of type 2 diabetes and its remedies with our collaborators using cell cultures, db/db

mice and high fat fed rodents.

Publications

Original peer-reviewed articles

1. Bhattacharya I, Basu S, Sarda K, Gautam M, Nagarajan P, Pradhan BS, Sarkar H,

Devi YS, Majumdar SS (2015) Low levels of Gs and Ric8b in testicular sertoli cells may

underlie restricted FSH action during infancy in primates Endocrinology 156: 1143-55.

2. Bhattacharya I, Gautam M, Majumdar SS (2015) The effect of IBMX and hormones on

gene expression by rat Sertoli cells, J Reprod Health Med 1:29-40

Reviews/proceedings

1. Sehgal L, Usmani A, Dalal SN, Majumdar SS (2014) Generation of transgenic mice by

exploiting spermatogonial stem cells in vivo. Methods Mol Biol 1194: 327-337.

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Production of transgenic and other animal models for biomedical research

Principal Investigator Subeer S Majumdar

Project Fellows Nirmalya Ganguli

Nilanjana Ghosh

Ph. D. Student Mansi Shukla

Collaborators S Goswami, JNU, Delhi

S Rath NII

V Bal, NII

SB Gokhale, BAIF, Pune

S Sengupta, NII

P Nagrajan, NII

A Mendez, IDIBELL, Spain

DP Sarkar, University of Delhi

D Modi, NIRRH, Mumbai

SN Dalal, ACTREC, Mumbai

S Chattopadhyay, NCCS, Pune

M Vaidya, ACTREC, Mumbai

R Ain, IICB, Kolkata

RS Gokhale, NII/IGIB, Delhi

Theme of research

Theme of the research is to generate transgenic animals for using them as a system for the

study of functional genomics and mammalian development and other animal models for use

in Biomedical research.

Objective

To develop new easier techniques for making transgenic animals. To develop transgenic

animal models using genes relevant to human health and diseases as well as to use this

technology for making large animals expressing therapeutic products in their milk for

increasing affordability of such therapeutics. The other objective is to extend collaborative

help in using specific animal models (transgenic or non transgenic) for Biomedical research.

Work Reported in 2013-2014

Generation of various transgenic mice for other investigators

All collaborative work for making various transgenic animals for other investigators were

undertaken as and when the constructs were given and fore founder animals were handed

over to P.I.‟s for generating transgenic lines to address their respective scientific goals.

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Attempts to generate transgenic farm animals expressing therapeutic protein in the

milk

We had isolated the β-casein genomic region (BuCSN2) which contains promoter region,

exon1, intron1 and exon2 (NCBI Accession No. KF612339) from the genome of Indian river

buffalo (Bubalus bubalis). We had reported the generation of transgenic mice in which milk

glands expressed EGFP when EGFP was cloned under this promoter (BuCSN2-EGFP) was

used to make the transgene.

There are difficulties and high cost of making transgenic large animals. In present day

scenario, transgenic animals may also be blamed to carry potential risk of germ line

transmission of introduced transgene to normal traits if not kept isolated, making them as

possible biohazard. Keeping all these in view, it is worth trying to transfect Mammary

Luminal Epithelial Cells, the cells which express and secrete milk proteins in the time of

lactation, in-vivo to have a possible bioreactor by somatic genomic modification. In spite of

several attempts using various gene delivery methods people have failed to generate a

efficient method for in-vivo gene delivery in mammary gland. It was shown before that

virosome mediated targeted delivery of transgene in liver cells is possible in vivo. Taking

clue from this, we used reconstituted Sendai Viral Envelope to generate a gene delivery

vehicle for easy, efficient and cost effective delivery of transgene in mammary gland in-vivo.

Generation of transgenic rat through testicular route

Several disorders can be efficiently and meaningfully studied only in rat models which are

10-12 times bigger in size than mice. Mouse cannot be bled frequently for determining

humoral changes and it cannot withstand major surgeries under anesthesia unlike, rat.

Structure – function relationship of various organs for toxicological evaluation is vastly

studied in rats and parameters to evaluate teratogenic effects are better established in rats,

mainly due to rat‟s extensive use in drug testing in the past. Hence, we established testicular

transgenesis for rats for overexpressing genes , modifying procedure reported for the mice, in

the past.

Generating a better model for post chemotherapeutic germ cell transplantation

A model for post chemotherapeutic infertility alleviation is made routinely by intraperitonial

injection of busulfan, which sometimes is lethal. We achieved germ cell (Gc) depletion

within 12-15 days by local injection of busulfan in the testis avoiding pain and suffering to

the animals.

Progress of work during current reporting year (2014-2015)

Generation of various transgenic mice for other investigators

This service is provided by NII to various laboratories of the nation. Task of making Various

transgenic animals for other investigators was undertaken as and when the constructs were

provided. Fore-founder animals were given to P.I.‟s for generating transgenic lines to

address their respective scientific goals. Work is being continued and major findings usually

lead to publications.

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Gene Knock down in rats

In the past, testicular transgenesis was successfully established for rats for overexpressing

genes. Following this new procedure, we have interrupted expression of specific genes in

vivo by shRNA in transgenic rats and determined physiological effects of such RNA-

interference. This will help in undertaking studies of functional genomics in rats.

Attempts to generate transgenic farm animals expressing therapeutic protein in the

milk

Although promoters of milk proteins from cow and goat have been isolated by Western

countries, buffalo which serves as the second largest species contributing to milk production,

worldwide, has not been exploited yet. Such promoters are required to facilitate generation of

therapeutic proteins in the milk for cost reduction and increased affordability. To this end, β-

casein genomic region (BuCSN2) which contains promoter region, exon1, intron1 and exon2

(NCBI Accession No. KF612339) was isolated by us from the genome of Indian river buffalo

(Bubalus bubalis). EGFP was cloned under the regulation of this promoter to examine the

efficacy and tissue specific activity of this promoter. Transgenic mice were generated using

this construct which showed mammary gland specific activity of the isolated promoter. To

check the ability of this promoter to drive human gene, expressing protein of therapeutic

importance, we cloned the cDNA of human Interferon-γ under the regulation of this

promoter and transfected MCF7 (human mammary adinocarcinoma cells) in vitro with this

construct bearing interferon-γ as a transgene . ELISA of cell lysates showed expression of

human interferon-γ by mammary cells.

Western blot of GFP ELISA of hIFNγ

Fig.1. Left Panel: Western Blot analysis of protein from various organs of BuCSN2-IRES2-

EGFP transgenic mice showing mammary gland specific expression of EGFP (transgene)

due to regulation of the gene by Buffalo beta casein promoter in mammary epithelial cells.

Right Panel: BuCSN2-hIFNγ-IRES2-EGFP transfected MCF7 (human breast carcinoma

cells) expressing hIFNγ, as detected by ELISA.

Concerning the difficulties and high cost in making transgenic farm animal, we were also

trying to transfect mammary epithelial cells directly in vivo. Mammary epithelial cells,

specifically mammary Luminal Epithelial cells (LEC) are responsible for expression of milk

protein at the time of lactation. These cells undergo repetitive cycle of regeneration and

apoptosis in each pregnancy/lactation cycle. A huge cell division event occurs starting from

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the early stage of pregnancy until advanced stage of pregnancy in the LEC. A possible

successful delivery of transgene in this time period in the LEC will create integration and

propagation of delivered transgene in this cell type which will eventually result in expression

of transgene at the time of lactation. We are presently working in the direction to determine

this crucial time period of mammalian development to target and convert mammary gland

into a possible bioreactor bypassing the difficulties of generating transgenic animals.

We are adopting various delivery methods and working with different transfecting agents to

successfully deliver transgene in the LEC. We have achieved limited success in transfecting

LEC. Transgenes delivered in this way will interact with cell and integrate in the genome at

somatic level causing genomic alteration of somatic cells and will not genetically alter the

crucial germ line of the species. If successful, this approach will help in generation of safe

Animal Bioreactor without potential environmental hazard posed by the Genetically

Modified Organisms (GMO), which stands as a major concern to many.

Generating a better model for post chemotherapeutic germ cell transplantation

We reported about development of a procedure where testis can be depleted of Gc by direct

injection of busulfan in the testis to create a model of infertility, post chemotherapy.

Intraperitoneal injections, used routinely, not only cause bone marrow depression but

sometimes are lethal.

The standardized dose resulted in reduction of the testis size and killing of most of the germ

cells of testis within 12-15 days post injection as opposed to about 70 days required for this

effect by traditionally used intraperitoneal injections. The animals were observed to be

healthy with no signs of health deterioration even after several months. This procedure helped

in preparing animal model with less pain and suffering to animal while objective of germ cell

depletion was achieved. For evaluating success of such procedure, Spermatogonial stem cells

(SSC) of one of the testis of pre-pubertal FVB/J mice was transfected with pCXEGFP

construct following in vivo testicular gene electroporation method. Electroporated testis was

removed surgically (hemicastrated) after 5 days of electroporation and GFP bearing SSC

were isolated, sorted through FACS using SSC specific antibodies and expanded in-

vitro using requisite growth factors to increase their number.

These cells were characterized for stemness using antibody against Oct-4. Testes of the

hemicastrated mice that were given testicular injections of busulfan became small after five

weeks, due to germ cell depletion. GFP expressing SSC cultured in- vitro were microinjected

into the seminiferous tubules of such germ cell depleted respective testes (autologus

transplantation) of recipient male through an efferent duct into rete testis. SSC colonized the

basement membrane of the tubules after 2 months as revealed by GFP expression by

donor SSC in recipient testis. The Gc transplanted testis was larger than the control testis of

other age matched animal, treated with busulfan but not transplanted with the Gc.

Future plan

1. We will continue to serve the scientific community for generation of transgenic mice

and other animal models for biomedical research.

2. We will work towards generating farm animals with an objective to produce important

171

rare therapeutic proteins in their milk.

3. Attempts will be made to generate animals bearing transgenes under control of drug

sensitive promoters.

4. To establish CRISPR-cas9 technology for genome editing.

Action taken on the RAP/SAC 2014 recommendations

No specific recommendation was given.

Publications

Original peer-reviewed articles

1. Ganguli N, Ganguli N, Usmani A, Majumdar SS (2015) Isolation and functional

characterization of buffalo (Bubalus bubalis) β-casein promoter for driving mammary

epithelial cell-specific gene expression. J Biotechnol 198:53-59

2. Chemmannur SV, Badhwar AJ, Mirlekar B, Malonia SK, Gupta M, Wadhwa N, Bopanna

R, Mabalirajan U, Majumdar S, Ghosh B, Chattopadhyay S (2015) Nuclear matrix binding

protein SMAR1 regulates T cell differentiation and allergic airway disease. Mucosal

Imunol doi:10.1038/mi.2015.11

172

The role of tumor suppressors in stress response

Principal Investigator Sanjeev Das

Research Associate Upasana Bedi (since Jan 2015)

Ph. D. Students Abhishek Bhardwaj

Rajni Kumari

Ruhi Deshmukh

Saishruti Kohli

Richa Kumari (since Dec 2014)

Theme of research

p73 is one of the tumor suppressors of the p53 family of nuclear transcription factors.

However the molecular mechanisms underlying p73 regulation remain unanswered. To

address the lacunae in the understanding of p73 stability and function, we carried out a

proteomics screen to identify p73 interacting proteins under normal and genotoxic stress

conditions. We have now identified TRIM28 and MED15 as potential p73 interacting

proteins.

The sirtuins are highly conserved enzymes that utilize NAD+ to modify other proteins. Of the

mammalian sirtuins, SIRT6 recapitulates many of the biological functions of founder member

of the sirtuin family, yeast Sir2. At the molecular level, SIRT6 regulates the expression of a

large number of stress-responsive and metabolism related genes, promotes genomic stability

and DNA repair. Since SIRT6-deficient mice exhibit profound metabolic defects which are

thought to be a primary contributor to their early lethality, understanding the role of SIRT6 in

metabolic homeostasis is an area of notable interest.

Objectives

A. Characterization of novel p73 interacting proteins involved in regulation of p73

stability and function

We have now identified an ubiquitin ligase TRIM28 and a transcriptional coactivator Med15

as potential p73 interacting proteins. Since TRIM28 is a RING-type E3 ligase, we plan to

investigate the role of TRIM28 in p73 homeostasis and its effect on p73 functions. Since

MED15 serves as a key p73 coactivator, we plan to investigate the role of MED15 in p73

tumor suppressor and anti-metastatic functions.

B. Understanding the function and regulation of SIRT6 in cancer metabolism

Our preliminary studies indicate that sirtuin 6 (SIRT6) protein levels are regulated in a

proteasome-dependent manner and our proteomics screen identified UBE3A as a novel

SIRT6 interacting protein. Hence we intend to examine the role of UBE3A in regulating

SIRT6 protein levels under different forms of metabolic stress. Since our proteomics screen

identified PKM2 as a SIRT6 interacting protein, we plan to investigate if SIRT6 can

deacetylate PKM2 and suppress its oncogenic functions. PKM2 acetylation is critical for its

nuclear localization and protein kinase activity and facilitates PKM2-mediated metabolic

173

reprogramming towards malignant phenotype. On the other hand SIRT6 functions to

maintain metabolic homeostasis.

Work reported in 2013-2014

A. Characterization of HDAC5 as a key modulator of p53-mediated transactivation

We performed a proteomics screen to identify novel p53 interacting proteins. We carried out

further studies to understand the function of one of the novel interacting proteins viz.

HDAC5. HDAC5 belongs to the class IIa HDAC (histone deacetylase) subfamily. Our

previous results indicate that HDAC5 is a deacetylase with specificity for K120 site of p53.

As previous studies report that K120 acetylation plays an important role in determining p53

target selection, we next determined the effect of HDAC5 on p53 functions. Our results

indicate that HDAC5 is required for the selective induction of p53 proarrest and antioxidant

target genes at early phase of genotoxic stress while at extended periods HDAC5 undergoes

nuclear export resulting in downregulation of p53 proarrest and antioxidant target genes and

induction of proapoptotic target genes. To corroborate the role of HDAC5 in genotoxic stress

response in vivo, we used RNAi to knockdown HDAC5 expression in mice which were then

subjected to genotoxic stress. Our results demonstrate that HDAC5 plays a key role in

modulating p53-mediated genotoxic stress response in vivo that augments prosurvival

functions of p53 over apoptosis. These findings provide novel insights into the role of

HDAC5 in regulating p53-mediated transactivation and have been published as a research

article in the journal Molecular Cell.

B. Characterization of novel p73 interacting proteins involved in regulation of p73

stability and function

TRIM28 encodes an 835-amino acid polypeptide containing a RING finger, B boxes, and a

PHD finger. TRIM28 has been shown to exhibit E3 ubiquitin ligase activity. Our results

indicate that coiled coil domain of TRIM28 binds to the N-terminal transactivation domain of

p73. We also examined this interaction under genotoxic stress conditions. Our results show

that TRIM28-p73 interaction is curtailed with increased duration of genotoxic stress and is

completely abolished at late time points. Previous reports suggest that tyrosine kinase c-abl

phosphorylates p73 at Tyrosine 99 upon genotoxic stress which is critical for p73

stabilization. Our results showed that there is no accumulation of p73 protein in absence of c-

abl but under such conditions if TRIM28 expression is abrogated there is significant

accumulation of p73. Thus we concluded that upon genotoxic stress p73 gets phosphorylated

in c-Abl-dependent manner leading to abrogation of its interaction with TRIM28.

Since MED15 serves as a key coactivator in various transcriptional complexes, we

investigated whether MED15 can serve as a p73 coactivator. We found out that upon

genotoxic stress the activation of p73 downstream targets is severely compromised in the

absence of MED15. These results establish that MED15 is an indispensable coactivator of

p73.

174

Progress of work during the current reporting year (2014-2015)

A. Characterization of novel p73 interacting proteins involved in regulation of p73

stability and function

Since TRIM28 has RING domain E3 ligase activity, we next determined whether it can

ubiquitylate p73. No ubiquitylated p73 was detected in HCT116p53-/-

TRIM28 knockdown

cells which confirms that it is the cognate E3 ligase responsible for p73 homeostasis in

unstressed cells. This was further corroborated by the fact that the RING deletion mutant of

TRIM28 was incapable of downregulating p73 levels. Furthermore, elevated levels of

polyubiquitylated p73 was observed in HCT116p53-/-

c-abl knockdown cells even upon

etoposide treatment. Additionally, p73 polyubiquitylation was found to be K48-linked which

is known to target proteins for proteasomal degradation. These results establish that Tyr99

phosphorylation by c-abl regulates TRIM28-mediated p73 polyubiquitylation and

consequently plays a critical role in p73 stabilization upon genotoxic stress. To assess the

functional consequences of TRIM28-p73 interaction, we investigated the effect of TRIM28

on key p73 functions viz. cell cycle arrest and apoptosis upon genotoxic stress. In

HCT116p53-/-

control cells genotoxic stress induced a pronounced G1 arrest at 24-36 hours,

after which there was a marked increase in the apoptotic population at 48 hours. In the case of

HCT116p53-/-

TRIM28 knockdown cells, significant population of apoptotic cells was

present from early time points (24-36 hours), which could be rescued with simultaneous

knockdown of p73. TUNEL staining also corroborated that abrogation of TRIM28 expression

augments p73-mediated genotoxic stress response. To strengthen our findings that the

increased sensitivity was p73-mediated, we checked the levels of different p73 target genes

including proarrest and proapoptotic genes. The proarrest target genes were rapidly induced

at early time points (12-36 hours) but upon prolonged stress (48 hours) their levels reduced

drastically. On the other hand, p73 proapoptotic target genes were induced only upon

prolonged stress (48 hours). In HCT116p53-/-

TRIM28/p73 double knockdown cells no

induction of the proarrest and proapoptotic target genes was observed at any of the time

points. Thus TRIM28 plays a key role in the regulation of p73-mediated genotoxic stress

response. These findings were also corroborated in vivo, wherein subcutaneous xenograft

tumors were generated in nude mice using HCT116p53-/-

control, TRIM28 knockdown and

TRIM28/p73 double knockdown cells and starting from fourth day post-implantation, mice

were administered etoposide every alternate day. Our results indicated that there was a more

pronounced decrease in the growth rate of tumors of HCT116p53-/-

TRIM28 knockdown cells

as compared to control cells. However this effect was abolished upon simultaneous

knockdown of p73.

Since our previous results indicated that MED15 could serve as p73 coactivator, we

examined the effect of MED15 on p73-mediated cell cycle arrest and apoptosis. In

HCT116p53-/-

control cells genotoxic stress induced a pronounced G1 arrest at 24-36 hours,

after which there was a marked increase in the apoptotic population. In the case of

HCT116p53-/-

MED15 knockdown cells there was no significant cell cycle arrest at any of

the time point and apoptosis was also significantly attenuated. TUNEL staining also

confirmed these observations. These results establish that MED15 is an integral component

of p73-mediated genotoxic stress response. To investigate the role of MED15 in p73-

mediated tumor suppression, subcutaneous xenograft tumors were generated in nude mice

using control, MED15 knockdown and p73 knockdown cells. Starting from fourth day post-

implantation, mice were administered etoposide every alternate day. Etoposide treatment led

to significantly reduced growth of tumors of control cells but tumors deficient in MED15 or

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p73 showed no significant response. The growth retardation observed was primarily due to

apoptosis as indicated by TUNEL staining on the tumor sections. Moreover the tumor

response was p73-MED15 interaction dependent as etoposide-mediated growth retardation in

HCT116p53-/-

p73 knockdown tumors was restored in presence of wild-type p73 but not

mutant p73 that does not interact with MED15. Since our results indicated that MED15

regulates p73-mediated transactivation of anti-metastatic genes, we evaluated the effect of

MED15 on p73 anti-metastatic functions in vivo. For this purpose, we orthotopically

transplanted HCT116Luc2 p53 knockdown control, HCT116Luc2 p53/MED15 double

knockdown or HCT116Luc2 p53/p73 double knockdown cells in caecum of nude mice.

Subsequently mice were given etoposide on every alternate day to induce genotoxic stress.

Significantly larger tumors were observed in mice bearing tumor with MED15kd cells as

compared to control cells. Moreover, the mice also developed ample number of spontaneous

metastatic nodules in lungs and liver. To confirm the metastasis from caecum to lungs and

liver, we performed ex-vivo imaging of these organs. We observed clear bioluminescent

signals from liver and lung. To confirm that the signals were due to metastatic tumors, we

further performed haematoxylin and eosin staining on those tissues, which revealed the

presence of metastatic foci. Moreover, the levels of anti-metastatic p73 target genes were

significantly lower in primary orthotopic tumors deficient in MED15. This was corroborated

by the fact that in the absence of MED15 the primary orthotopic tumors showed reduced

levels of epithelial marker E-cadherin and elevated levels of mesenchymal markers including

N-cadherin and Fibronectin which is indicative of increased propensity of these tumors to

metastasize. These results suggest that MED15 is essential for p73 anti-metastatic functions.

Thus our study reveals the pivotal role of Tyr99 phosphorylation in temporal regulation of

p73 levels as well as function.

B. Understanding the function and regulation of SIRT6 in cancer metabolism

Since SIRT6-deficient mice exhibit profound metabolic defects which are thought to be a

primary contributor to their early lethality, understanding the role of SIRT6 in metabolic

homeostasis is an area of notable interest. To gain mechanistic insights into SIRT6

regulation, we first tested the effect of different forms of metabolic stress including glucose

starvation, serum starvation and cold shock, on SIRT6 levels. We found that SIRT6 protein

levels were induced upon metabolic stress but there was no change in the transcript levels.

Since ubiquitin proteasome system plays a critical role in cellular protein homeostasis, we

used proteasome inhibitor MG132 to check if SIRT6 levels are regulated by ubiquitin

proteasome system. We found out that there was robust induction of the SIRT6 protein levels

in presence of MG132 while there was no change in the transcript levels. We then checked

the SIRT6 protein for polyubiquitylation and found that SIRT6 protein was indeed

undergoing K48-linked polyubiquitylation under normal conditions, which was abrogated

upon starvation. These results suggest that SIRT6 levels are regulated by ubiquitin

proteasome system.

To understand the complexity of SIRT6 interactome, we also carried out a LC-MS/MS based

proteomics screen to identify SIRT6 interacting proteins. The result of LC-MS/MS screen

identified several novel interacting proteins including Ubiquitin-protein ligase E3A (UBE3A)

and Pyruvate Kinase M2. UBE3A functions as an E3 ligase in the ubiquitin proteasome

pathway. UBE3A was originally discovered to ubiquitylate and promote degradation of

tumor suppressor p53, through which it plays a pathogenic role in human papillomavirus-

induced cervical epithelium neoplasia. Since SIRT6 is regulated by ubiquitin proteasome

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system, UBE3A might be the cognate E3 ligase regulating SIRT6 protein levels. This is also

supported by our preliminary studies which indicate that there is a decline in UBE3A

transcript and protein levels concomitant to increase in SIRT6 protein levels upon metabolic

stress.

The M2 splice isoform of pyruvate kinase (PKM2), is an enzyme that catalyzes the final step

of glycolysis- the conversion of phosphoenolpyruvate to pyruvate. PKM2 levels are increased

in human cancer tissues. It is believed that high expression of PKM2 leads to anabolic

metabolism of glucose for macromolecular biosynthesis, thereby promoting cancer cell

proliferation and tumor growth. In addition to the glycolytic functions, non-glycolytic

functions of PKM2 in cancer cells are of particular interest. PKM2 can undergo nuclear

translocation by virtue of its nuclear localization signal in the C-terminal domain. In the

nucleus, PKM2 activates transcription of various genes by interacting with and

phosphorylating specific nuclear proteins, endowing cancer cells with a survival and growth

advantage. These biochemical activities are controlled by allosteric regulators and post-

translational modifications of PKM2 including acetylation. Thus it would be interesting to

study the regulation of PKM2 by SIRT6 which exhibits deacetylase activity.

Future plans

Our proteomics screen clearly demonstrates that SIRT6 interacts with UBE3A but upon DNA

damage this interaction is abolished. This suggests that UBE3A could be regulating SIRT6

protein levels in unstressed cells. We propose to look for physical interaction between SIRT6

and UBE3A. We also plan to map the domains of SIRT6 and UBE3A responsible for the

interaction. We also plan to investigate whether UBE3A can ubiquitinated SIRT6 leading to

its degradation. We also plan to investigate how UBE3A itself is regulated under metabolic

stress conditions.

PKM2 acetylation is critical for its nuclear localization and protein kinase activity and

facilitates PKM2-mediated metabolic reprogramming towards malignant phenotype. On the

other hand SIRT6 functions to maintain metabolic homeostasis. Since our proteomics screen

identified PKM2 as a SIRT6 interacting protein, we plan to investigate if SIRT6 can

deacetylate PKM2. In addition to the glycolytic functions, non-glycolytic functions of PKM2

in cancer cells are of particular interest. PKM2 can undergo nuclear translocation by virtue of

its nuclear localization signal in the C-terminal domain. In the nucleus, PKM2 activates

transcription of various genes by interacting with and phosphorylating specific nuclear

proteins, endowing cancer cells with a survival and growth advantage. Thus we plan to

investigate whether SIRT6 suppresses PKM2 oncogenic functions.

Action taken on the RAP/SAC 2014 recommendations

The scientific and technical clarifications sought during the interactive session were provided.

No specific recommendations were made.

177

Publications

Original peer-reviewed articles

1. Satija YK, Das S (2015) Tyr99 phosphorylation determines the regulatory milieu of

tumor suppressor p73. Oncogene (in press).

Reviews/Proceedings

1. Kumari R, Sen N, Das S (2014) Tumour suppressor p53: understanding the molecular

mechanisms inherent to cancer. Curr Sci 107: 786-794.

2. Kumari R, Kohli S, Das S (2014) p53 regulation upon genotoxic stress: Intricacies and

Complexities. Mol Cell Oncol doi: 10.4161/23723548.2014.969653.

178

Elucidating the molecular mechanisms of aging and innate immunity using

Caenorhabditis elegans as a model system

Principal Investigator Arnab Mukhopadhyay

Project Associates Awadhesh Pandit (SSII-Project)

Neeraj Kumar

Ph. D. Students Anupama Singh

Syed Shamsh Tabrez

Sonia Verma

Anita Goyala

Amit Garg

Collaborators S Sengupta, NII

MJ Kulkarni, CSIR-NCL, Pune

K Chakraborty, CSIR-IGIB, Delhi

S Srinivasan, IBAB, Bangalore

SG Sampathkumar, NII

Theme of research

We use Caenorhabditis elegans as a model system to understand the molecular basis of

aging. Our laboratory uses a combination of genetics, molecular biology and genomics to

decipher signalling events that culminate in alterations in gene expression during aging. We

are trying to understand the complex interplay of transcription factors and co-regulators

downstream of the Insulin-IGF-1-like signalling (IIS) pathway in regulating longevity,

metabolism, stress- and pathogen resistance. Since Dietary Restriction (DR) is the only

intervention that increases life span and delays age-onset diseases, we are trying to decipher

the molecular events that follow initiation of DR. Using chemical genetics, we are trying to

find novel longevity extending compounds and are studying their mechanisms of action.

Objectives

A. Deciphering the coordinate regulation of genes downstream of the IIS pathway

B. Involvement of miRNA-Transcription factor networks in dietary restriction

C. Involvement of novel kinases in dietary restriction

D. Role of the Endoplasmic reticulum (ER) in DR-mediated longevity

Work reported in 2013-2014

A. Deciphering the coordinate regulation of genes downstream of the IIS pathway

We performed DAF-16/FOXO chip-sequencing and analyzed the data. We reported the

dynamic distribution of DAF-16 binding sites on the genome and compared the data with

published expression data. We also performed motif discovery within the DAF-16 binding

peaks to identify additional transcription factor binding sites. These transcription factors may

co-regulate DAF-16 target genes.

Fig

ure

2.

Fig

ure

2.

179

B. Involvement of miRNA-Transcription factor networks in dietary restriction

We performed miRNA sequencing of wild-type and a genetic mimic of DR [eat-2(ad1116)]

in worms that revealed 81 miRNA which were upregulated during day 1 of DR; none was

significantly downregulated. We found that the FOXA transcription factor PHA-4 binds to

the promoters of most of the upregulated miRNAs. This was interesting as DR is known to

require PHA-4/FOXA to extend life span. We also validated the expression of the 10 miRNA

and found them to be dependent on PHA-4.

C. Involvement of novel kinases in dietary restriction

We have identified and characterized a novel kinase IDR-1 (now called DRL-1) that when

knocked down initiates a process of DR without compromising food intake. We performed

microarray analysis to determine gene expression changes associated with DR and found that

those involved in xenobiotic detoxification are upregulated in a PHA-4/FOXA-dependent

manner. We also showed that the low levels of Reactive Oxygen Species (ROS) generated

during DR are due to the reprogramming of metabolism towards fatty acid oxidation and not

due to an active ROS detoxification system. Further, we started a detailed characterization of

another gene that is homologous to drl-1. We call this gene idr-2 (or drl-2). Knocking down

the gene either by mutation or by RNAi leads to depletion of fat storage and increased life

span. We showed that the life span is dependent on PHA-4/FOXA, hinting to the fact that drl-

2 knockdown may be bringing about a DR-like state similar to drl-1. But interestingly, this

mutant increases life span only when grown on a particular strain of bacteria (HT115, a K12

strain) and not on another (OP50, a B strain).

D. Drugs that can extend C. elegans life span

We identified Rifampicin as a glycation inhibitor in vivo that dramatically increases life span

in DAF-16/FOXO as well as JNK-dependent manner. Rifaximin did not have significant

activity in vitro or in vivo and failed to increase life span. Using LC-MSE, we identified that

Rifampicin reduces glycation on essential metabolic proteins. This research has been

published and so this objective will not be pursued further in the current form.

Progress of work during the current reporting year (2014-2015)

A. Deciphering the coordinate regulation of genes downstream of the IIS pathway

We performed next generation sequencing-based transcriptomic analysis using low insulin

signaling mutant daf-2(e1370) and compared it to daf-16(mgdf50);daf-2(e1370) [where DAF-

16 is deleted]. This analysis revealed genes that are dependent on daf-16 when insulin

signaling is low, a condition that dramatically increases life span. We compared this data to

our DAF-16 ChIP-seq data and found that not all DAF-16 binding on the chromatin has

transcriptional consequences. We compared our ChIP-seq and transcriptomics data to all

published data on DAF-16 gene expression that revealed a set of 43 direct targets of high

confidence. Using RNAi, we systemically knocked down all the 43 genes and studied their

effects on daf-2-dependent phenotypes, including dauer diapause, stress resistance and

longevity that are dependent on DAF-16. We find that this set of 43 genes is required

differentially for all the phenotypes of daf-2(e1370).

Fig

ure

2.

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We bioinformatically searched the DAF-16 binding peaks for additional transcription factor

binding sites. We found that the binding motif for an orphan nuclear hormone receptor

(oNHR) is enriched in the DAF-16 peaks. We performed transcriptomics analysis to compare

the dependence of genes upregulated in daf-2(e1370) (in a DAF-16-dependent manner) on

the presence of the oNHR. We found that a large contingent of DAF-16 indirect targets and

33 direct targets are also transcriptionally regulated by the oNHR. This shows that DAF-16

and oNHR may collaborate to coordinately regulate gene expression downstream of the IIS.

We are now studying the contributions of these 33 targets on IIS phenotypes.

We have also started a systematic analysis of a zinc finger protein gene (zfp-1) that DAF-

16/FOXO directly regulates. ZFP-1 is a chromatin modulator and interacts with another

chromatin-associated protein GFL-1. Even gfl-1 is a direct transcriptional target of DAF-16.

Preliminary data suggests that ZFP-1 is regulated by multiple isoforms of DAF-16 and

knocking it down affects multiple phenotypes associated with the IIS pathway.

B. Involvement of miRNA-Transcription factor networks in dietary restriction (DR)

After mapping our small RNA sequencing data [from wild-type and eat-2(ad1116)] with the

miRNA database, we determined that a large number of reads do not match any annotated

miRNA. We performed novel miRNA discovery using the miRdeep2 package. We identified

novel miRNA both in wild-type as well as in eat-2(ad1116) and validated three from the

latter.

Next, we performed transcriptomics analysis to compare the gene expression changes in eat-

2(ad1116) as compared to wild-type. We found that a large contingent of genes is

upregulated on DR. Since PHA-4 is required for DR-mediated longevity, we asked whether

PHA-4 regulates these genes. Interestingly, we found that PHA-4 directly binds to a

significant number of the upregulated genes. Thus during DR, PHA-4 transcriptionally

upregulates the expression of both mRNA and miRNA.

MiRNAs target mRNA to affect translational arrest or degradation. We predicted the targets

of the miRNAs upregulated during DR and found that they overlapped significantly with the

mRNA that were upregulated. Thus, following DR the PHA-4-controlled miRNA and mRNA

form a large network that is enriched for feed-forward motifs. This may be an efficient

strategy for reducing fluctuations in gene expression during times of energy crisis.

Interestingly, such networks were not found downstream of the IIS pathway, showing that

these two nutrient sensing pathways function differently to control longevity.

C. Involvement of novel kinases in dietary restriction

We have earlier shown that following initiation of DR, there is a shift in metabolism towards

fatty acid oxidation. Also, there is an increase in expression of xenobiotic genes in a PHA-4-

dependent manner. Next we asked whether the increased expression of xenobiotic genes was

the result of increased fatty acid oxidation or was an unlinked event. For this, we initiated DR

in a fatty acid oxidation defective strain nhr-49(nr2041) where the transcription factor

required for the expression of beta oxidation genes is mutated. We found that DR failed to

increase the expression of the xenobiotic genes under this condition. This proved that

xenobiotic detoxification is required to detoxify putative lipotoxins generated due to fatty

acid oxidation. We found that apart from PHA-4, other transcription factors (NHR-8 and

181

AHR-1) that are required for transcription of xenobiotic genes are also required for DR-

mediated life span extension.

In order to further understand how metabolic reprogramming can signal to upregulate the

expression of the xenobiotic detoxification genes, we performed a targeted genetic screen.

We used genetic mutants of the important components of signal transduction pathways and

asked whether they can suppress life span extension of a long-lived DR worm. Preliminary

data suggest that p38 MAPK pathway but not JNK pathway may be important for this

purpose.

The drl-2 gene was also characterized in details and was found to possess most of the

phenotypes associated with drl-1. However, unlike drl-1, knocking down drl-2 specifically in

the intestine is sufficient to extend life span and decrease fat storage. We also found that the

drl-2 mutants have increased osmotolerance and we are trying to link it to the life span.

D. Role of the Endoplasmic reticulum in DR-mediated longevity

We have recently observed that in two independent genetic models of DR, a transient

upregulation of an ER stress marker (ER chaperon HSP-4) occurs. This upregulation is

dependent on the ER sensor IRE-1 and the ER stress-responsive transcription factor XBP-1.

This response was specific to ER as we did not witness upregulation of mitochondrial stress

marker HSP-6 or cytosolic stress marker HSP-16.2. We are now studying the functional

consequences of this phenomenon on life span.

Future plans

1. Functional role of oNHR and DAF-16 common targets on life span, stress resistance,

development and dauer diapause, phenotypes controlled by the IIS pathway

2. Study the role of ZFP-1 and GFL-1 in IIS pathway-regulated phenotypes and determine

the mechanism by which these proteins interact with the upstream components

3. Decipher the role of p38 MAPK signaling during DR

4. Linking the osmotic stress tolerance to longevity in case of drl-2

5. Further studying the implications of early upregulation of ER stress markers during DR

Action taken on the RAP/SAC 2014 recommendations

No specific recommendation was received.

Publications

Original peer-reviewed articles

1. Pandit A, Jain V, Kumar N, Mukhopadhyay A (2014) PHA-4/FOXA-regulated

microRNA feed forward loops during Caenorhabditis elegans dietary restriction. Aging

6: 835-55.

2. Golegaonkar S, Tabrez SS, Pandit A, Shalini S, JagadeeshaPrasad MG, Bansode S,

Sampathkumar SG, Kulkarni MJ, Mukhopadhyay A (2015) Rifampicin reduces

Advanced Glycation End products and activates DAF-16 to increase life span in

Caenorhabditis elegans. Aging Cell doi:10.1111/acel.12327.

182

3. *Chamoli M, Singh A, Malik Y, Mukhopadhyay A (2014) A novel kinase regulates

dietary restriction-mediated longevity in C. elegans. Aging Cell 13: 641-55.

*in press last year, since published

183

Structural studies on proteins, dynamics and ligand interactions using

NMR

Principal Investigator Monica Sundd

Ph. D. Students Rohit Singh Dangi

Usha Yadav

Shalini Verma

Vinod Kumar Meena

Theme of research

The theme of our research is to understand the structure, backbone dynamics and interactions

of proteins using NMR, and other biophysical techniques to understand their function. We are

working on some of the key proteins of fatty acid metabolism in Leishmania viz. the acyl

carrier protein, acyl CoA binding proteins, and enzymes of type II fatty acid biosynthesis

pathway. Comparative studies with similar proteins from P. falciparum, M. tuberculosis, E.

coli and human are also being conducted to understand their similarity or differences with

Leishmania proteins.

Objectives

The objective of the study is to structurally and functionally characterize important proteins

of fatty acid biosynthesis as well as dispersal in Leishmania major using NMR and other

biophysical techniques in order to understand their structure, function and interactions with

naturally occurring partners. We are pursuing two major projects that are interlinked:

A. Understanding the sructure and function of the type II fatty acid biosynthesis pathway of

Leishmania major

B. Structural characterization of the proteins involved in fatty acid dispersal in Leishmania

Work reported in 2013-2014

A. Understanding the sructure and function of the type II fatty acid biosynthesis

pathway of Leishmania major.

Last year, we reported the structure of the acyl carrier protein of Leishmania major and its

acyl intermediates. From our studies with the acyl-intermediates, we concluded that the

longer acyl chain intermediates in Leishmania are extremely unstable and hence the final

product of the pathway could possibly be C8- or C10-ACP. C8-ACP is a precursor of lipoic

acid synthesis, necessary for the functioning of several enzyme complexes in the

mitochondria. Based on these results, we speculated that this pathway has probably been

designed by nature to fulfil the lipoic acid requirements of the mitochondria as it cannot

synthesize long acyl chains.

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B. Structural characterization of the proteins involved in fatty acid metabolism in

Leishmania

In Leishmania, three ACBPs (acyl CoA binding proteins) play an important role in the

dispersal of fatty acids. The three proteins display good dispersion in an NMR spectrum and

the chemical shifts of these proteins were assigned using NMR. The binding affinity of these

ACBPs towards standard acyl-CoAs like C4-, C8-, C10-, C12-, C14- C16-, C18- and C20-CoAs

was also analyzed using ITC. The three ACBPs displayed very different affinities towards

various acyl-CoAs, underscoring their importance in the survival of Leishmania.

Progress of work during the current reporting year (2014-2015)

In the past year, our studies were focused primarily on the fatty acid biosynthesis project.

Understanding the sructure and function of the type II fatty acid biosynthesis pathway

of Leishmania major

Heterologous expression of Leishmania ACP in E. coli results in the expression of only one

form of the protein, apo-ACP, despite the presence of a type I 4'-phosphopantetheinyl

transferase AcpS (Acyl carrier protein synthase) as a part of type II fatty acid machinery in E.

coli. Other type II ACPs express in two or more forms, apo-, holo- and acyl-intermediates.

These results gave the first hint that the acyl carrier protein of Leishmania doesn't act as a

good substrate for AcpS. With Sfp (a type II 4'-phosphopantetheinyl transferase, from B.

subtilis), which is highly promiscuous in its activity, it shows several folds lower activity

compared to E. coli, P. falciparum and M. tuberculosis ACP. Careful analysis of the NMR

structure of the acyl carrier protein suggested a remarkably different 4'-phosphopantetheinyl

transferase (PPT) binding interface of Leishmania ACP. Type II ACPs have a conserved

'DSL' motif present at the beginning of helix II containing a serine which undergoes

posttranslational modification by the attachment of a phosphopantetheine arm from CoA, the

reaction catalyzed by PPTs. In Leishmania ACP, this DSL motif is replaced with an 'NSL'

motif. Likewise, a few other residues present at the PPT binding interface are also different.

For instance, the conserved Met 44 is replaced with a Phe in Leishmania ACP and Glu 48 is

replaced with a Gln. The latter two residues are present in helix II of ACPs and form the

protein-protein interaction interface.

Leishmania ACP does not interact with E. coli AcpS.

Wild type Leishmania ACP fails to convert into holo-ACP when expressed in E. coli.

Mutagenesis studies were carried out in Leishmania ACP at the three positions (present in the

PPT interaction interface), singly as well as in combination as shown in Fig. 1. Asn 36 lies

close to the active site of Sfp in the structure of Sfp-PCP complex (PDB ID 4MRT).

However, the other two residues are far away from the catalytic site. The single mutants

N36D, and F45M expressed in E. coli in a single form. However, the double mutant

N36D+F45M expressed in two forms, 78% apo and 22% holo-ACP suggesting that E. coli

AcpS has a stringent requirement for the residues at the abovementioned positions.

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Enzymatic conversion of Leishmania ACP with B. subtilis Sfp

Figure 1. A helix wheel projection of the helix II of E. coli and Leishmania ACP displaying

the sites that show disparity in sequence. These three sites were mutated to the residues in E.

coli ACP singly as well as in combination. The Sfp binding surface is also shown.

Enzyme assays were also carried out on wild type and the mutants using Sfp, a type II PPT

from B. subtilis. Km and Kcat values were obtained by varying the CoA concentration from 0-

100M, keeping the PPT and ACP concentrations constant in all enzyme assays at 2M and

40M, respectively. Kinetic constants were obtained by performing Michaelis-Menten fits to

the raw data using Graphpad Prism version 6.00 for Windows (GraphPad Software, La Jolla

California USA, www.graphpad.com). As illustrated in the Fig 2, lowest Vmax was observed

for the mutant F45M, displaying a Kcat/KM ratio (catalytic efficiency) 5 fold lower than wild

type LmACP. A mutant F45A was also generated that failed to convert to holo-ACP even

after 4 hrs or longer incubations suggesting that a hydrophobic anchor residue at position 45

in ACPs is necessary to interact with Sfp. The Kcat values were in the order; F45M< Wild<

N36D+Q48E>N36D+F45M< N36D< P. falciparum ACP< E. coli ACP< M. tuberculosis

ACP as illustrated in Fig. The wild type Leishmania ACP has a Vmax 7 folds lower than E.

coli ACP and the single mutant N36D displays a Vmax, ~2 fold lower than E. coli ACP. In the

double mutant N36D+Q49E, the Kcat of Sfp was higher than wild type Leishmania ACP but

lower than N36D mutant. A mutant M44F of E. coli displays much higher Km of Sfp for CoA

compared to wild type protein, as shown in the Fig 2, but the Vmax remained unchanged. All

this data points towards some very interesting facts; the same residue, at the same site in two

different ACPs contributes differently to catalysis. Any mutation at the protein-protein

interaction interface (position 45 or 49) results in a decrease in Vmax. Notably, all the

mutations have been introduced in helix II, at sites remote (>10Å) from the catalytic residues,

and they cast a very different effect on enzymatic activity. In addition to the quantitative

information furnished by our kinetic experiments, our studies allowed us to precisely identify

the reaction steps influenced upon mutagenesis. The low Vmax (E*k2) value of Sfp for CoA

using wild type LmACP and its mutants compared to E. coli, P. falciparum and M.

tuberculosis ACPs, suggests that the catalytic step involving the conversion of Enzyme-

Substrate complex into product (k2) is altered in the equation shown below:

In case of the E. coli mutant M45F, Vmax remains unaltered but Km increases. As the reaction

rate k1 is diffusion controlled 108-10

9 M

-1s

-1, the rate of dissociation of the enzyme-substrate

complex (k-1) possibly increases in this mutant.

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Figure 2. Enzyme kinetics curves for Sfp (B. subtilis) using various ACPs as substrates viz.

E. coli ACP, P. falciparum ACP, M. tuberculosis ACP, Leishmania ACP and its various

mutants.

Chemical shift perturbation studies suggest weak binding between Leishmania ACP and

Sfp (B. subtilis).

Figure 3. Average chemical shift changes in the backbone amides of Leishmania ACP A)

Wild type upon binding to Sfp in the absence of Mg2+

and CoA B) Wild type upon binding to

Sfp (Bacillus subtilis) in the presence of Mg2+

and CoA C) double mutant N36D+F45M upon

binding to Sfp in the presence of Mg2+

and CoA and d) N36D upon binding to Sfp in the

presence of Mg2+

and CoA. A discontinuous line in the figure marks one standard deviation.

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The interaction of Leishmania ACP and its mutants with Sfp was also investigated at the

atomic level using NMR. All the mutant ACPs appeared well folded in the 1H

15N HSQC

spectra. 1H

15N labeled samples (0.3mM) of wild type Leishmania ACP, N36D mutant and the

double mutant N36D+F45M were titrated with increasing concentration of unlabeled Sfp, to

a final molar ratio of 1:2 in 50mM Tris, pH 8.0. Figure 3 illustrates the average backbone

amide chemical shift changes of Leishmania ACP wild type and the mutants upon addition of

Sfp. The overall small magnitude of the amide chemical shift changes in LmACP residues

upon Sfp binding offers a window into the fairly weak, and transient nature of the ACP-Sfp

interaction, which is probably necessary for the timely release of the substrate and end

products. Relatively large amplitude chemical shift change were observed at two sites, a)

residues surrounding Glu 16 and b) residues surrounding Ile 55 HN. The balance appeared to

tip in favour of either of these two sites between wild type and the mutants. In wild type

ACP, the chemical shift change was much larger for Glu 16, while in the single mutant N36D

and the double mutant N36D+F45M, Ile 55HN displayed much larger change. The two

aforementioned sites interact with two different domains of the pseudo-dimer of Sfp, that is

connected by a hinge region. Thus, the data points towards the rearrangement of the two

domains of Sfp with respect to ACP, highlighting the flexibility of the domains, allowing

them to attain a preferred conformation with respect to the ACP molecule, that in turn

determines the activity of Sfp towards CoA. The fact that the mutant N36D+F45A of

Leishmania ACP fails to show any activity with Sfp suggests that position 45 of ACP is

extremely important for its interactions with Sfp.

Future plans

Based on our results with AcpS and Sfp, we speculate that the interaction interface of

Leishmania ACP might have evolved convergently with its cognate PPT, and could serve as a

better substrate for the latter enzyme, a group II 4'-phosphopantetheinyl transferase, 272

amino acids in length. We plan to clone and purify the cognate PPT of Leishmania and

characterize it structurally as well as functionally. As the enzyme is slightly larger in size for

NMR, we are planning to express perdeuterated protein (Leishmania PPT) in order to

understand the interactions of the enzyme with its own ACP molecule, as well as other type II

ACPs. Chemical shift assignments of the PPT molecule will be carried out on a 700 MHz

NMR spectrometer that will be used to follow the conformational changes. Biochemical

studies will also be carried out on this enzyme and its mutants and compared with Sfp and

Human PPT.

We are also initiating studies on a few other enzymes involved in lipoic acid synthesis, i.e.

lipoate protein ligase (Lip B), Lip A etc that also interact with the ACP molecule and are a

part of fatty acid biosynthesis pathway of Leishmania.

Action taken on the RAP/SAC 2014 recommendations

No specific recommendations were made.

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Genetic and functional analyses of host and HIV-1 genes that affect

progression of HIV-1 and development of nucleic acid based antiviral

approaches

Principal Investigator Akhil C Banerjea

Project Associates Sanket S Ponia

Amjad Ali

Sakshi Arora

Ph. D. Students Richa Kapoor

Rameez Raja

Binod Kumar

Shubhendu Trivedi

Project Fellows Jyotsna Singh

Larance Ronsard

Shaista Ahmed

Collaborators V Ramachandran, UCMS/GTB Hospital, Delhi

S Das, UCMS/GTB Hospital, Delhi

V Bhattacharya, BHU, Varanasi

T Dhole, SGPGI, Lucknow

A Vyakarnam, IISc, Bangalore

A Shet, St John‟s Medical College, Bangalore

V Tandon, JNU, Delhi

Theme of research

Host mounts its own anti-viral responses and the viruses have evolved over the years to

systematically overcome or evade several cellular restriction factors including innate

immunity to support their persistence and replication. How HIV-1 exploits the catabolic and

anabolic pathways for its own advantage is poorly understood and needs to be studied in

detail. An understanding of the mechanisms governing host-virus interaction is critically

important. Viruses exploit the host cell machinery in a manner that confers greater viral

fitness. Various kinds of RNAs are now known to influence the disease process and our broad

objectives are to explore the functional implications of small RNAs with respect to HIV-1

replication besides studying the role of both regulatory and accessory proteins of HIV-1in

pathogenesis.

Objectives

HIV-1 displays great genetic heterogeneity and this helps the virus to evade the immunity.

We are therefore interested to study the genetic variations in the HIV-1 genes among

circulating strains of HIV-1 from North India and delineate the functional implications that

govern overall pathogenesis of HIV. The various accessory proteins like Nef, Vpu, Vif and

Vpr are known to play a major role in pathogenesis. We, therefore, wish to understand the

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role of accessory genes in modulating cellular functions important for causing pathogenesis.

The two major regulatory proteins, Tat and Rev, play an important role in HIV-1 gene

expression and replication besides influencing the RNAi machinery. It is therefore important

to study (a) how key regulatory and accessory proteins of HIV-1 are modulated inside the

mammalian cells (b) how they directly or indirectly modulate the key regulators of cell

cycle/apoptosis etc. Finally it is important to determine how micro-RNAs (viral & cellular)

influence these functions and contribute to pathogenesis and study in detail what strategies

are adopted by HIV to suppress the RNAi machinery and thereby subvert host defences.

Work reported in 2013 - 2014

Our preliminary findings suggested the role of HIV-1 Vpr in causing inhibition of whole cell

ubiquitination. Using selected genes in a transient transfection system it was established that

Vpr alone was sufficient for this remarkable function. This was observed by transient as well

as by HIV-1 infection in a relevant cell line. The possible role of miRNA34a in HIV-1

replication was reported in human T-cells. We also reported the possible role of AKT in virus

infection. We reported how HIV-1 modulates metabolic sensors like mTOR during HIV

infection.

Progress of the work during the current reporting year (2014 -2015)

HIV-1 exploits the cellular ubiquitination machinery

To determine if the whole cell ubiquitination (Ubn) is affected by HIV-1 infection , infectious

clone, pNL4-3, was used. HEK-293 cells were cotransfected with HIs-UB encoding plasmid

along with pNL4-3(HIV-1 prototype subtype B) for 48 hrs and ubiquitinated proteins were

pulled down by Ni-NTA beads. Immunoblot analysis was carried out using anti-His antibody.

It was clear that in the presence of HIV-1, a significant reduction in the total cellular

ubiquitination was observed. This was further confirmed in a physiologically relevant setting

by infecting HIV-1 into T-cell line-Jurkat E6.1. Here also, it was evident that upon HIV-1

infection the whole cell ubiquitination was reduced. Infection to the extent of 80% was

confirmed by FACS analysis using HIV-1 gag p24 antiserum. Next we wanted to find out

which gene was responsible for this function. All the accessory/regulatory genes (Nef, Vpr,

Vpu, Vif, Tat & Rev) were cloned in Myc-tagged vector and independently co-transfected

with His-ubiquitination encoding plasmid for 48 hrs and checked for the cellular

ubiquitinated proteins. It was only the Vpr that caused very significant reduction. We

additionally confirmed that this observation was Vpr-dose dependent. A time course analysis

of Vpr expression suggested that 12 hrs post transfection Vpr could be detected which

correlated with reduction in ubiquitination. This observation was further confirmed by doing

infection with pNL4-3 (WT) and Delta-Vpr-PNL4-3 in Jurkat cells also. Earlier structural

studies with Vpr showed that it consisted of 3 short helical domains. We constructed deletion

mutants in all the three helical regions (17 to 33; 38 to 48 and 53 to 77 aa) to find out the

determinants of this activity. Results suggested that all three helices are important. We tested

Vpr C (derived from an Indian isolate 93IN905) along with natural variants of Vpr

(especially L64P mutant that we earlier reported from India – J Gen Virol. 90, 2768-2776,

2009). Vpr C was just as efficient as Vpr B in causing reduction in whole cell Ubiquitination

and the natural L64 P mutant lost this property. A series of experiments suggested that

although whole cell ubiquitination is reduced by Vpr, but quite remarkably ubiquitination of

antiretroviral restriction factors is retained and in specific instances (like APOBEC3G) it is

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increased even further, suggesting redirection of cellular ubiquitination machinery which is

beneficial for the virus.

Role of MicroRNA34a in HIV-1 replication

miR34a is one of the cellular microRNAs which is shown to be upregulated during HIV-1

infection in T cells. However, the role of miR34a in HIV-1 replication is not known. We

determined the importance of miR34a during HIV-1 infection in T cells and show that there

exists a positive feedback loop between microRNA34a and HIV-1 replication. It is known

that miR34a targets a cellular protein PNUTS (Phosphatase 1 Nuclear Targeting Subunit).

PNUTS is known to interact with the P-TEFb complex which is a component of

transcriptional elongation complex. Experimentally we found out that PNUTS negatively

regulates HIV-1 transcription by inhibiting the assembly of core components of P-TEFb i.e.

CyclinT1 and CDK9. Also, PNUTS expression in T cells decreases in a time-dependent

manner post HIV-1 infection. Hence, we conclude that HIV-1 increases miR34a expression

to overcome the negative effects of PNUTS on HIV-1 transcription.

Role of AKT in HIV-1 pathogenesis

A number of knockdown based studies have reported cell survival pathways to be important

for HIV-1 infection.PKB/AKT is one of the crucial factors needed by cells for their survival.

The exact role of this kinase which lies downstream of P1,3 Kinase pathway has not been

properly elucidated in HIV-1 infection.Our preliminary results suggest that HIV-1 Tat and

Vif are regulated by AKT/PKB which needs to be explored further. Further, our data shows

that MDM2 (a target substrate for AKT) is post- translationally modified in presence of HIV-

1 Tat. We are currently investigating the regulation of MDM2 in HIV-1 infection and its

functional consequences.

Modulation of mTORC by HIV-1 to meet its biosynthetic demand

HIV-1 has to depend on the host factors to meet its biosynthetic demands. The cellular

catabolism is controlled by AMPK while anabolism is controlled by Akt. The delicate

balance between anabolism and catabolism is controlled by mTORC, hence mTORC acts as a

homeostatic regulator of metabolism. In this study, we show that HIV-1 modulates

intracellular levels of mTORC1 in a time-dependent manner; it is up regulated up to 24 hour

post infection which decreases drastically 36 hours post infection. HIV-1 proviral single

mutant constructs (Delta pNLs) indicated the involvement of more than one viral gene for

mTORC1 modulation. The viral determinants were validated by overexpression based

studies. We also observed that Deptor, an endogenous inhibitor of mTORC1, is also

modulated upon HIV-1 infection. The results indicated that Deptor is also involved in

mTORC1 modulation. Finally we observed that CAD, an enzyme which is necessary for

pyrimidine biogenesis, is also modulated in a time-dependent manner during infection.

Overexpression of CAD increases p24 levels (indicator of HIV-1 replication). Overall these

results highlight that HIV-1 modulates in time dependent manner to derive its biosynthetic

demand.

Future plans

Mechanistic details of how HIV-1 affects cellular metabolism, role of AKT in HIV-1

pathogenesis; potential role of miRNA34a in HIV-1 infection, will be explored in detail.

191

Viral protein-protein (accessory and regulatory) interaction and its implications in HIV-1

pathogenesis will be explored.

Action taken on the RAP/SAC 2014 recommendation

None suggested.

Publications

Original peer reviewed articles

1. Arora S, Verma S, Banerjea AC (2014) HIV-1 Vpr redirects host ubiquitination pathway.

J Virol 88: 9141-9152.

192

Therapeutic Interventions in Chronic Diseases

Principal Investigator Sarika Gupta

SERB Young Scientist Viji Vijayan

Project Associates Tandrika Chattopadhyay

Sakshi Gupta

Ph. D. Students Nuzhat Ahsan

Kapil Manglani

Madhuraka Pal

Ibrar Ahmed Siddique

Shikha Salhotra

Research Fellow Mayuri Khandelwal

Collaborators A Surolia, IISc, Bangalore

B Kundu, IIT, Delhi

Theme of research

My group is a multi-disciplinary group adapting an integrated approach in drug discovery

that combines medicinal chemistry, basic biology and biochemistry principles for efficient

drug design process. Interests of the group lie in identifying underlying principles in a disease

pathogenesis to discover new targets, designing molecular intervention strategies and

confirming the biological/therapeutic activities of the designed compounds. The small

molecule regulators contribute to both drug development and understanding biological

systems in human body.

Objectives

To design and synthesize stable curcumin analogs and study their effect on aggregation and

cytotoxicity of Wild type and Mutant α-Synuclein.

Work reported in 2013-2014

1. Simvastatin induced neurite outgrowth unveils role of cell surface cholesterol and

acetyl CoA carboxylase in SH-SY5Y cells

Statins are known to modulate cell surface cholesterol (CSC) and AMP-activated protein

kinase (AMPK) in non-neural cells; however no study demonstrates whether CSC and

AMPK may regulate simvastatin induced neuritogenesis (SIN). We found that simvastatin

(SIM) maintains CSC. Modulation of CSC revealed that SIN is critically dependent on this

CSC. Simultaneously, phospho array for MAP kinases revealed PI3K / Akt as intracellular

pathway which modulates lipid pathway by inhibiting AMPK activation. Though, SIM led to

a transient increase in AMPK phosphorylation followed by a sudden decline; the effect was

independent of PI3K. Strikingly, AMPK phosphorylation was regulated by protein

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phosphatase 2A (PP2A) activity which was enhanced upon SIM treatment. Moreover, it was

observed that addition of AMP analogue and PP2A inhibitor inhibited SIN. Bio-composition

of neurites shows that lipids form a major part of neurites and AMPK is known to regulate

lipid metabolism majorly through acetyl CoA carboxylase (ACC). AMPK activity is negative

regulator of ACC activity and we found that phosphorylation of ACC started to decrease after

6 hrs which becomes more pronounced at 12 hrs. Addition of ACC inhibitor showed that

SIN is dependent on ACC activity. Simultaneously, addition of Fatty acid synthase (FAS)

inhibitor confirmed that endogenous lipid pathway is important for SIN. This study highlights

a distinct role of CSC and ACC in SIN which might have implication in process of neuronal

differentiation induced by other agents. In conclusion, this study unravels a co-ordinated

action of CSC and ACC in SIN. We highlight two major events involved during SIN: 1)

retention of CSC which acts as stabilizer to orchestrate signaling events necessary to promote

neuritogenesis; 2) initiation of fatty acid biosynthesis by ACC activation through PP2A

phosphatase dependent de-phosphorylation of AMPK.

2. Methionine down-regulates TLR4/MyD88/NF-κB signalling in osteoclast precursors

to reduce bone loss during osteoporosis

In this study, methionine, a nutritionally essential amino acid has been employed as a tool to

evaluate whether an anti-osteoporotic pharmacological agent can perform similarly at varying

lengths of time. Administration of methionine to a rat osteoporotic system prevented

pathophysiological bone resorption. Methionine inhibited trans-differentiation of blood

mononuclear cells to functional osteoclasts and this was due to its ability to down-regulate

TLR-4/MyD88/NF-ĸB cascade in developing osteoclasts, a signaling mechanism that was

originally thought to be involved only in pathogen recognition. A combination therapy of

Alendronate (bisphosphonate)+methionine significantly improved bone physiology of

osteoporotic rats (even at a significantly lower dose of Alendronate) highlighting methionine

as a pharmacological drug for anti-resorptive therapy.

Progress of work during 2014-2015

Curcumin Pyrazole and its derivative (N-(3-Nitrophenylpyrazole) Curcumin inhibits

aggregation, disrupt fibrils and modulate toxicity of Wild type and Mutant α-Synuclein.

The aberrant self assembly and deposition of misfolded proteins is the leading cause for

several conformational disorders like Alzheimer‟s disease, multiple system atrophy and

Parkinson‟s disease. The term “α-synucleinopathy” refers to a specific group of such

neurodegenerative diseases that exhibit abnormal aggregation and accumulation of α-

synuclein, a pre-synaptic protein. Physiologically α-synuclein is a natively unfolded protein

monomer regulated via lysosomal and proteosome degradation pathways. Nonetheless

genetic and environmental stress factors disrupt normal physiological state of α-synuclein

causing it to assemble and form toxic amyloid aggregates or highly ordered fibrils.

Substitutional mutations (A30P, E46K and A53T) in α-synuclein and overexpression of the

wild type α-synuclein (WtAS) due to gene multiplication are some of the identified genetic

causes for this aberrant aggregation of α-synuclein.

Parkinson‟s disease (PD), a well-known α-synucleinopathy and most prevalent movement

disorder in humans is characterized by the presence of α-synuclein aggregates as Lewy body

inclusions in specific regions of the brain such as substantia nigra, thalamus and neocortex.

Though numerous clinical and experimental studies have shown that soluble oligomeric and

194

protofibrillar forms of α-synuclein are potentially neurotoxic, there are also reports stating

neurodegenerative potency of fibrils via cell membrane permeabilization (Melki and Pieri,

2012). Molecules that inhibit α-synuclein fibrillization and stabilize it in a non-toxic state can

therefore serve as therapeutic molecules for both prevention of accumulation of aggregated α-

synuclein and maintenance of normal physiological concentrations of α-synuclein.

Accumulating evidences suggest that deposition of neurotoxic α-synuclein aggregates in the

brain during the development of neurodegenerative diseases like Parkinson‟s disease can be

curbed by anti-aggregation strategies that either disrupt or eliminate toxic aggregates.

Curcumin, a dietary polyphenol exhibits anti-amyloid activity but the use of this polyphenol

is limited owing to its instability. As chemical modifications in curcumin confiscate this

limitation, such efforts are intensively performed to discover molecules with similar but

enhanced stability and superior properties. Keeping in view the enormous beneficial effects

of curcumin and its minimal toxicity, we have modified the chemical space around curcumin

scaffold to synthesize stable curcumin analogs. This study focuses on the inhibitory effect of

two stable analogs of curcumin viz. curcumin pyrazole and curcumin isoxazole and their

derivatives against α-synuclein aggregation, fibrillization and toxicity. Curcumin pyrazole

has shown better potency than curcumin in inhibiting synuclien aggregation. Hence, 15 more

derivatives of curcumin pyrazole have been synthesized. Employing biochemical, biophysical

and cell based assays we discovered that curcumin pyrazole (3) and its derivative N-(3-

Nitrophenylpyrazole) curcumin (15) exhibit remarkable potency in not only arresting

fibrillization and disrupting preformed fibrils but also preventing formation of A11

conformation in the protein that imparts toxic effects (Fig1). Compounds 3 and 15 also

decreased neurotoxicity associated with fast aggregating A53T mutant form of α-synuclein.

These two analogues of curcumin described here may therefore be useful therapeutic

inhibitors for the treatment of α-synuclein amyloidosis and toxicity in Parkinson‟s disease

and other synucleinopathies.

Our data also suggests that compounds like compound 6 (N-(3-Fluoro phenylpyrazole)

Curcumin which seem therapeutic via conventional biophysical and imaging techniques do

not impart any beneficial effects in reducing cytotoxicity. The results indicate that

conventional dye binding assays like ThT and Congo Red along with microscopic imaging

should be done in conjunction with cell-based assays to determine the nature of the

compound under study which again underscores the importance of our work. Any strategy to

discover new anti-amyloidogenic molecules should take into account the toxicity of the

resulting oligomers. The future prospects of the study include investigating the modulatory

role of not only compounds 3 and 15 but also compound 6 in disease progression in α-

synuclein overexpressing transgenic mice models. The use of compounds 6 and 3 in

mechanistic studies may enable a better understanding of the pathogenesis of α-synuclein in

the progression of PD and other synucleinopathies.

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Figure 1. (a) Percentage inhibition of aggregation of WtAS by compound 1-19 as monitored

by ThT assay. WtAS (210 µM) was co-incubated with equimolar concentration of

compounds 1-19 for 30 days. (b) Comparative bar diagram showing the effect of compounds

1, 3, 6 and 15 on 15 days (c) and 30 days aggregated WtAS by ThT assay. WtAS (210 µM)

samples were aggregated alone for 45 days and 60 days or equimolar concentrations of

compounds 1, 3, 6 and 15 were added at day 15 and 30 days and further co-incubated for 30

days. Percentage increase in aggregation was calculated by measuring corrected ThT

fluorescence intensity. (d) Representative image of A11 dot blot showing effect of

compounds 1, 3, 6 and 15 on WtAS A11 epitope formation. (a) WtAS (210 µM) aggregated

alone or co-incubated with equimolar concentration of compound 1, 3, 6 and 15 for 30 days

(b) 60 days. The compounds were added at day 15 or 30 and further co-incubated for 30

days.(e) Comparative bar diagram showing MTT reduction by oligomers of WtAS(210 µM)

alone or co-incubated with compounds 1, 3, 6 and 15 in neuronal cell cultures. Results are

the mean of three different experiments done in duplicates and error bars show standard

deviation. *, #, $ denote p < 0.05, p < .005 and p < 0.0001, respectively.

Future plans

1. Screening of small molecular inhibitors of amyloid beta, alpha synuclein and

transthyretin aggregation and their preventive and therapeutic efficacy in animal models.

2. Understanding the mechanism of cytotoxicity of transthyretin oligomers.

3. Role of Bone Morphogenetic Proteins and testosterone in Glucose Homeostasis and

Diabetes.

4. To study the interaction of ERAP with -synuclein and role in Parkinson‟s disease.

5. Investigating the role of gelsolin as a common cellular player for modulating amyloid

load and neurodegeneration.

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Action taken on the RAP/SAC 2014 recommendations

All suggestions by the RAP/SAC members were addressed and incorporated in the research

program.

Publications

Original peer-reviewed articles

1. Pasi S, Kant R, Gupta S, Surolia A (2015) Novel multimeric IL-1 receptor antagonist for

the treatment of rheumatoid arthritis. Biomaterials 42:121-33.

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Molecular mechanism of enzymatic reactions and enzyme-ligand

interactions

Principal Investigator Apurba Kumar Sau

Project Fellow Ginto George

Ph. D. Students Sudeepa Rajan

Nikunj H. Raninga

Vineet Sadarangani

Ankita Dutta

Theme of research

The aim of this project is to understand molecular mechanism of different classes of GTPases

induced by immunomodulatory cytokine interferon- (IFN-) and to compare the mechanistic

similarities and differences with other GTPases within the same as well as different classes.

The study has been currently focused on human guanylate binding protein-1 (hGBP-1) and

other proteins in the same family. The mechanism along with the structural data may provide

an insight to design drug candidates on novel GTPases and their effectors involved in the

disease.

Objectives

A. IFN-γ induced GTP-binding proteins and their role in the regulation of GTP

hydrolysis

To study the regulation of GTP hydrolysis in IFN- induced guanylate binding proteins p67

(hGBP-1 and hGBP-2) and to understand their similarities and differences within the same

family as well as within the same and different classes.

B. Understanding the function of arginine metabolic enzymes in Helicobacter pylori

The aim is to investigate a detailed molecular mechanism of two arginine metabolic enzymes

arginase and ADC in H. pylori. The mechanism along with structural data from other

organisms may provide a novel strategy to develop new inhibitors with greater efficiency

against H. pylori infection.

Work reported in 2013 – 2014

A. IFN-γ induced GTP-binding proteins and their role in the regulation of GTP

hydrolysis

To investigate whether the stability of hGBP1 upon binding with the substrate is related to

the product formation, thermodynamic and kinetic assays of the wild type and mutant

proteins were performed. The data suggest that the difference in ΔGD between the analogue

free and bound proteins plays an important role in the product formation; larger difference

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(higher stability) favors GMP formation, but smaller (lower stability) favors dissociation of

GDP-bound enzyme dimer resulting in more GDP formation.

B. Understanding the function of arginine metabolic enzymes in Helicobacter pylori

To examine the role of a sequence motif in H. pylori arginase, individual mutational analysis

as well as deletion studies were performed. The data demonstrate that the motif is extremely

critical for the function of the protein. MD simulations suggest that the sequence motif is

located near the active-site with a loop-cum-helix structure. The motif is also critical for

retaining the bimetallic center of the protein, which is crucial for the function.

Progress during the current reporting year (2014-2015)

Circular dichroism study to understand the role of the sequence motif in secondary

structure

To examine the role of the sequence motif in the secondary structure of the protein, CD

measurements were performed in the far UV range. The experiments were carried out for the

wild type and mutant proteins (apo and holo). For the apo proteins, the CD value was found

to change for certain mutations. Trp159Ala and Glu155Ala showed significant decrease in

the CD value compared to wild type, suggesting that these mutations lead to the loss of

secondary structure of the protein. The deconvolution of the CD spectra yielded an average

-helix content of ~ 6.3 and 18% for Trp159Ala and Glu155Ala, respectively as compared to

29% in wild type. To understand whether Trp159 is important for the secondary structure of

the protein, similar studies were performed with Trp159Phe. In contrast to Trp159Ala,

Trp159Phe regained the secondary structure. These data demonstrate that both Trp159 and

Glu155 of the motif are individually important for maintaining the structure of the protein.

But Glu153Ala, Ser154Ala, Glu156Ala and Asp126Ala exhibit similar secondary structures

to wild type. On the other hand, Gln160Ala, Lys161Ala, Leu162Ala, Cys163Ala and

Ser164Ala showed an increase in the CD value compared to wild type. The increase in the

helicity of these mutants may be due to the presence of Ala, which is known to have higher

propensity to form helical structure.

To understand whether the loss of activity in Glu155Ala is due to the decrease in the helical

content, CD measurement of Glu155Ala was carried out in the presence of 5-10 % TFE (tri-

fluoro ethanol), a known -helical structure inducing agent in protein. The secondary

structure of the mutant was found to increase with TFE. Glu155Ala with 10% TFE exhibited

CD value similar to wild type, indicating that TFE increases the secondary structure of the

mutant protein. To examine whether the structural gain in Glu155Ala with TFE makes the

protein catalytically functional, the activity assay was carried out in the presence of 10%

TFE. The mutant showed negligible activity (~ 2% of wild type), suggesting that Glu155 has

also a catalytic role.

To understand whether the metal ions have role in the secondary structure of the mutant

proteins, similar CD measurements were carried out. In the presence of the metal ions, the

CD values of Glu153Ala, Ser154Ala, Lys157Ala, Trp159Ala, Trp159Phe and Lys161Ala are

found to be higher than their apos, indicating that the metal ions increase the secondary

structure of these mutant proteins. However, for other mutants the increase in the CD values

is marginal in the presence of the metal ions.

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Heat-induced denaturation studies for the role of the sequence in stability

To determine whether the sequence motif is important for the stability of the protein, heat-

induced unfolding studies were carried out using CD measurements. The change in the

secondary structure was measured with increase in the temperature from 30 to 90 0C. The CD

values at 220 nm were fitted to a two-state unfolding model to determine the Tm (midpoint

transition temperature). All mutants showed sigmoidal transitions suggesting that temperature

induced unfolding of the proteins occurred in a cooperative manner. Comparison of the Tm

values between the wild type and mutants showed that in the absence of the metal ions the

mutant proteins are significantly less stable than the wild type, as reflected in the reduction of

the Tm by ~ 5-17 0. Interestingly, only Glu155Ala and Trp159Ala showed the notable

decrease in the Tm (~14-17°) compared to wild type, suggesting that both electrostatic (by

Glu155) and hydrophobic (by Trp159) interactions are very critical in providing stability to

the apo-proteins. But Trp159 has a slightly larger role in the stability than Glu155, as

observed from the difference in the Tm values compared to wild type (17 vs 140). To see the

involvement of Trp159 in these interactions, the stability of Trp159Phe was examined. The

data showed an increase in the Tm of about 9 degree as compared to Trp159Ala (60 vs 51 0C),

suggesting the role of Trp159 in the hydrophobic and/or π-π interactions for the stability of

the protein.

To see the effect of the metal ions in the stability, similar measurements were done on the

mutant proteins in the presence of the metal ions and their Tm values were also evaluated. As

observed, the interaction of the metal ions with the residues at the active-site compensates the

loss of stability in the mutant proteins. Unlike other mutants, the metal ions in Glu155Ala did

not compensate the loss of stability significantly compared to their apos, suggesting that the

interaction of Glu155 with its surrounding residues is vital for the stability of the protein. But

for Trp159Phe, the loss of one metal as well as absence of the Trp159-Asp126 interaction is

likely to be the main reasons for not regaining the stability. Thus, the heat-induced unfolding

studies clearly show that the sequence motif is a critical component for the stability of H.

pylori arginase.

Higher GMP formation is regulated through tetramerization of hGBPs

Although dimerization has been reported to be crucial for GMP production, truncated

hGBP1307

, which completely dimerizes with the substrate analogue GppNHp, showed a

marked decrease in GMP to GDP formation (~ 0.6:1vs ~ 5:1) compared with the wild type

hGBP1. To understand whether the enhanced GMP formation in wt-hGBP1 is regulated

through a higher order assembly, analytical gel-filtration assay was carried out with the wild

type, mutant and truncated proteins in the absence and the presence of the transition state

analogue, GDP.AlF4. With GDP.AlF4, wt-hGBP1 eluted as a tetramer. The assay was also

performed with S157A mutant, which shows activity similar to the wild type. Like wild type,

it also eluted as a tetramer. We performed similar assays with a series of the mutants, D108A,

D103L.D108L, T75A and R48P, where these residues are known to be primarily critical for

GMP formation. Interestingly, with GDP.AlF4 D108A mutant, which forms significantly

reduced GMP compared with the wild type, eluted as a mixture of monomer and tetramer. In

contrast, D103L.D108L mutant, which was completely impaired in GMP formation, eluted

only as a monomer, indicating that the double mutant did not tetramerize with GDP.AlF4.

R48P is known to have a negligible GTP binding or hydrolysis, whereas T75A hydrolyzes

GTP to mainly GDP. With GDP.AlF4, R48P and T75A eluted as a monomer and dimer,

respectively, indicating that none of the two mutants tetramerize with GDP.AlF4.

Importantly, hGBP1307

, which is known to produce lower GMP, eluted only as a dimer with

200

GDP.AlF4, suggesting an important role of the helical domain in tetramer formation. Taken

together, these data suggest that the enhanced GMP formation is associated with

tetramerization of the protein.

To examine whether the lower GMP production in the wild type hGBP2 is correlated with

lower tetramer formation, similar studies with GDP.AlF4 were performed. Unlike wt-hGBP1,

wt-hGBP2 eluted as a mixture of monomer (~10%), dimer (~40%) and tetramer (~50%),

implying that the protein did not form a complete tetramer after the first phosphate cleavage.

However, it exists as a complete dimer with GppNHp. The lower amount of GMP in wt-

hGBP2 is, however, not consistent with the proportion of tetramer formed, suggesting that

tetramer of wt-hGBP2 is catalytically less efficient than that of wt-hGBP1 and might be

associated with a different conformation.

Future plans

Although, dimerization was found to be crucial for GMP production, truncated hGBP1307

that

lacks the helical domain, shows a marked decrease in GMP to GDP production compared

with the full-length hGBP1(~ 0.6:1vs ~ 5:1). The full-length hGBP1 and truncated hGBP1307

have been found to completely dimerize with GppNHp. hGBP2 also completely dimerizes

with GppNHp, but shows a significantly lower GMP formation (~10% of hGBP1) than

hGBP1. These observations suggest that dimerization cannot completely explain the

stimulated GMP formation in the full-length hGBP1. The analytical gel-filtrations assays on a

series of the mutant proteins of hGBP1 in the presence of the transition state analogue,

GDP.AlF4 indicate that tetramerization seems to be responsible for higher GMP formation.

This will be examined by the chimeric proteins where the catalytic domain will be kept intact,

but the other domains will be systematically swapped with that of the homologous protein.

We will examine whether tetramerization is associated with the structural change of the

protein and also whether the tetramer in hGBP1 and hGBP2 exhibits conformational

difference.

Action taken on the RAP/SAC 2014 recommendations

The scientific and technical clarifications sought during the presentation were provided.

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Deciphering the role of cell signalling in M. tuberculosis biology

Principal Investigator Vinay Kumar Nandicoori

Fellow Gagan Jhingan (Wellcome-DBT)

Research Associates Savita Lochab (NII Young Investigator)

Sandeep Upadhyay

Ph. D. Students Yogesh Chawla

Vijay Soni

Divya Arora

Prabhjot Kaur

Preeti Jain

Basanti Malakar

Mehak Jahoor Khan

Project Fellow Saba Naz

Collaborators D Sriram, BITS, Hyderabad

B Prakash, IIT, Kanpur

Y Singh, IGIB, Delhi

S Khosla, CDFD, Hyderabad

D Mohanty, NII

Theme of research

Protein phosphorylation events are especially known for their influence on the regulation of a

number of cellular processes including gene regulation, cell growth and division. Analysis of

the Mtb genome sequence suggested the presence of 11 putative eukaryotic-like STPKs and 3

protein phosphatases. The M. tuberculosis STPKs affect key mycobacterial processes -

including determining cell shape, cell division, morphology, virulence, cell division, growth

rate and response to hypoxia. Our aim is to decipher the signaling pathways in M.

tuberculosis and investigate their role in modulating the host signaling network and the

survival of pathogen in the host.

Objectives

A. To identify novel downstream targets and determine the role of kinase mediated

phosphorylations of these targets.

B. Generate gene replacement mutants of STPKs and investigate their role in the growth and

survival of pathogen in the host.

C. Investigate modulation of host signalling pathways upon infection with M. tuberculosis

H37Rv wild type or kinase gene replacement mutants.

Work reported in 2013-2014

A. Protein Kinase B (PknB) of Mtb is essential for both in vitro growth and in vivo

survival of pathogen in the host

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The Mtb protein kinase B (PknB) comprises an intracellular kinase domain, connected

through a transmembrane domain to an extracellular region that contains four PASTA

domains. We found stringent regulation of PknB expression necessary for cell survival, with

depletion or overexpression of PknB leading to cell death. While PknB-mediated kinase

activity was essential for cell survival, active kinase lacking the transmembrane or

extracellular domain faied to complementconditional mutants not expressing PknB. By

creating chimeric kinases, we found that the intracellular kinase domain has unique functions

in the virulent strain, which cannot be substituted by other kinases. Although the presence of

the carboxy-terminal PASTA domain is dispensable in the avirulent M. smegmatis (Msmeg),

all four PASTA domains are essential in Mtb. Mouse infection studies were performed to

determine the role of PknB in mediating pathogen survival in the host demonstrate that PknB

was not only critical for growth of the pathogen in vitro, but was also essential for the

survival of the pathogen in the host.

B. Phosphorylation of nucleoporin Tpr governs its differential localization and is

required for mitotic progression

A major constituent of the nuclear basket region of the NPC, nucleoporin Tpr plays a multi-

dimensional role in the cell. Studies have implicated a role for Tpr in regulating important

processes including chromosome segregation, chromatin organization and unspliced RNA

export. We have previously established that Tpr is phosphorylated in both, MAP kinase

dependent and independent manner, and found that Tpr acts as both a substrate and as a

scaffold for MAP kinase ERK2. Last year we reported the identification of S2059 and S2094

as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and

S2034 as the minor ERK independent phosphorylation sites found in the Tpr protein in vivo.

Our results suggest that protein kinase A phosphorylates the S2094 residue, and the site was

hyperphosphorylated during mitosis. Further, we found that Tpr phosphorylated at the S2059

residue distinctly localizes with chromatin during the telophase. Abrogation of S2059

phosphorylation abolished its interaction with Mad1, thus compromising nuclear distribution

of Mad2, and resulted in cell cycle defects. The identification of novel phosphorylation sites

on Tpr and the observations presented in this study open fresh avenues for the better

understanding of Tpr functions.

Progress of work during the current reporting year (2014-2015)

Protein kinase A (PknA) of Mycobacterium tuberculosis is independently activated and

is critical for growth in vitro and survival of the pathogen in the host

Although ser /thr/tyrosine kinases are widely prevalent in higher eukaryotes, in bacterial

systems cellular processes are largely modulated by two-component signaling cascades and

bacterial tyrosine kinases (BY kinases). The analyses of the whole genome sequences of

several pathogens including mycobacterial species, Yersinia, Streptococcus etc., however, has

revealed the presence of ser/thr protein kinases in them. PknA and PknB are known to have

profound effects on processes involved in determining cell shape and morphology, and

possibly cell division. PknA has been shown to regulate morphological changes associated

with cell division, and its overexpression gives rise to elongated and branched structures.

PknB overexpression has been reported to result in widened and bulging cells.

Overexpression in both cases led to decreased growth rate of the bacilli. PknA and PknB

consist of an ~270 aa intracellular kinase domain, an ~60-70 aa juxtamembrane domain, and

203

an ~20 aa transmembrane domain connected to an extracellular domain. While the

extracellular region of PknA is relatively short (~70 aa), the extracytoplasmic domain of

PknB contains iterative PASTA (penicillin binding protein and serine/threonine kinase

associated) domains. Based on transposon mutagenesis both PknA and PknB have been

thought to be essential. Though PknB has been demonstrated to be essential both for in vitro

growth and in vivo survival, to date there are no reports addressing the question of whether

PknA is essential for growth or survival.

PknA and B phosphorylate a number of proteins required for mycolic acid synthesis, cell

division and peptidoglycan synthesis. Despite the fact that many of their substrates were

initially identified as substrates for one kinase or the other, most are actually phosphorylated

by multiple kinases. PknB and PknH have recently been proposed to be the master regulators

that are capable of phosphorylating seven STPKs in their activation loops in vitro, thus

controlling their activation status. Based on the results from in vitro kinase assays, PknB has

been suggested to activate four kinases (including PknA), which in turn phosphorylate their

target substrates. It is conceivable that PknB, which participates in similar functions as PknA

may regulate activation and functioning of PknA in vivo, akin to the cross-talk seen in most

eukaryotic signaling pathways. The mode of PknA activation in mycobacteria and its

dependence on PknB have not been examined thus far.

Thus we investigated the functional importance of PknA in vivo. We observed aberrant cell

shape and severe growth defects when PknA was depleted. Using the mouse infection model

we observed that PknA was essential for survival of the pathogen in the host.

Complementation studies affirmed the importance of the kinase, juxtamembrane and

transmembrane domains of PknA. Surprisingly, the extracytoplasmic domain was

dispensable for cell growth and survival in vitro. We found that phosphorylation of the

activation loop at T172 of PknA was critical for bacterial growth. Using phospho-specific

PknA antibodies and conditional pknB mutant we found that PknA autophosphorylated its

activation loop independent of PknB. Fluorescently tagged PknA and PknB show distinctive

distribution patterns within the cell, suggesting that while both kinases are known to

modulate cell shape and division, their modes of action are likely to be different. This was

supported by our findings that expression of kinase-dead PknA versus kinase-dead PknB in

mycobacterial cells led to different cellular phenotypes. Data indicated that though PknA and

PknB are expressed as part of the same operon, they appear to be regulating cellular

processes through divergent signalling pathways.

Comparative proteomic analysis of avirulent, virulent and clinical strains of Mtb

identifies strain specific expression profiles.

The ability of the organism to survive under harsh conditions in the host is connected to its

ability to modulate host cellular processes to its advantage. Advent of multiple-drug resistant

(MDR) and extensive drug resistant (XDR) Mtb strains are a major global heath concern,

compromising the existing therapy. In recent years systems biology approaches, which

studies the complex interactions between genes, proteins and other elements have been

successfully applied to generate predictive models of the networks and dynamic interactions

between host-pathogen. Various genomic approaches dealing with comparison between drug

sensitive and resistant mycobacterium phenotypes have identified multiple SNPs related to

DNA repair, replication and recombination genes, thereby providing insights into genetic

basis of drug resistance. Transcriptomics studies dealing with drug treatments and analysis of

MDR strains have highlighted the role of altered gene expression of type II fatty acid

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synthases, efflux genes, central metabolic pathway members, ABC transporters and genes

related to stress response.

To understand the adaptation of mycobacterium to different stress conditions, it is necessary

to quantify the protein expression profiles. 2-DE based approaches were also used to identify

strain specific differences among virulent and avirulent strains of Mtb. An elegant proteome

comparison between H37Rv and BCG strains combining both 2-DE and ICAT has provided

complementary information among these strains. Quantitative proteomics studies have

highlighted differential expression of proteins among Mtb strain H37Rv and M.bovis BCG

especially related to lipid biosynthesis pathways as well as during different growth phases

and nutrient starvation. Recently utilizing a combination of discovery and targeted

approaches a SRM based Mtb proteome library was generated to accurately quantitate most

of the proteins of Mtb and related clinical strains. Despite many reports dealing with Mtb

proteome very few detailed studies have been performed on clinical strains which are directly

related to drug resistance.

In order to determine the protein expression profile differences between laboratory and

clinical strains, we performed systematic analysis of the whole cell proteome analysis of

laboratory avirulent strain H37Ra, laboratory strain H37Rv and single-drug resistant (BND-

433) and multi-drug resistant (JAL-2287) clinical isolates. Liquid chromatography and mass

spectrometry analysis of lysates from four biological replicates of the four strains identified a

total of 2161 protein groups covering ~54% of the predicted Mtb proteome. Label free

quantification analysis of the data revealed presence of 257 differentially expressed proteins

that showed prominent up and down- regulation with respect to JAL strain. The differentially

expressed proteins could be classified in to seven K-means cluster bins, which broadly

delineated strain specific variations. Expression pattern analysis of the transcription factors

and the downstream targets demonstrated substantial differential modulation in JAL,

suggesting a complex regulatory mechanism. While abrogating higher levels of ESAT6 in

JAL decreased its virulence, it had marginal impact on the other strains. Lower levels of

mce1 operon protein expression observed in BND, significantly compromised its ability to

utilize cholesterol as the sole carbon source. Taken together our study reveals that the strain

specific variations have meaningful impact on the biology of the pathogen.

Future Plans

Deciphering the role of Mtb Serine/Threonine protein phosphatase PstP

1. Generation of conditional gene replacement mutant of M. tuberculosis serine/threonine

phosphatase PstP.

2. Deciphering the function of PstP using ΔpstP mutant and investigating its impact on

phosphorylation status of protein kinases PknA, PknB and some of their substrates.

3. Mouse infection studies to determine the role of PstP in mediating pathogen survival in

the host.

Investigating the role of phosphorylation in modulating the cell division and cell wall

synthesis in Mtb

1. Identification of novel targets involved in cell division and peptidoglycan synthesis.

2. Identification of phosphorylation site(s) and characterization.

3. Generation of gene replacement mutants of novel targets identified.

205

Action taken on the RAP/SAC 2014 recommendations

Suggestions given by the RAPSAC members during the interaction were incorporated into

the research program.

Publications

Original peer-reviewed articles

1. Nagarajan SN, Upadhyay S, Chawla Y, Khan S, Naz S, Subramanian J, Gandotra S,

Nandicoori, VK (2015) Protein kinase A (PknA) of Mycobacterium tuberculosis is

independently activated and is critical for growth in vitro and survival of the pathogen in

the host. J Biol Chem doi:10.1074/jbc.M114.611822.

2. Rajanala K, Sarkar A, Jhingan GD, Priyadarshini R, Jalan M, Sengupta S, Nandicoori VK

(2014) Phosphorylation of nucleoporin Tpr governs its differential localization and is

required for its mitotic function. J Cell Sci 127: 3505-3520.

3. Chawla Y, Upadhyay S, Khan S, Nagarajan SN, Forti F, Nandicoori VK (2014) Protein

Kinase B (PknB) of Mycobacterium tuberculosis is essential for growth of the pathogen in

vitro as well as for survival within the host. J Biol Chem 289: 13858 – 13875.

206

Molecular biology of infectious diseases

Principal Investigator Lalit C Garg

Ph. D. Students Vineet Kumar Gupta (up to June 2014)

Bharti Bhatia (up to June 2014)

Amit Kumar Solanki

Suresh Kumar

Theme of research

In global surveys, infectious diseases rank among the leading causes of death of both humans

and animals. Vaccination against infectious agents continues to be one of the most effective

methods of limiting the cost of management of many infectious diseases. The goal of this

study is to clone and express genes of biomedical importance with an emphasis on the

development of vaccines against pathogens and to unravel the molecular mechanisms of

infectious diseases to explore new drug targets.

Objectives

(A) Development of recombinant and DNA based vaccine against Clostridium

perfringens

Gram positive Clostridium perfringens is a major cause of human and veterinary enteric

diseases largely because this bacterium can produce several toxins when present inside the

gastrointestinal tract. Epsilon toxin (Etx), produced by C. perfringens types B and D, is the

key antigen implicated in the Enterotoxaemia and Pulpy kidney disease of domestic animals.

C. perfringens type B and C produce beta toxin (btx) which is responsible for necrotizing

enteritis and enterocolitis, Being the most common causes of cattle mortality, these bacteria are

of great economic importance. The project aims to develop recombinant and DNA vaccine

against the toxins produced by C. perfringens. We also aim to assess the role of various

residues within the toxin for its toxicity, oligomerization, binding to host cell and

immunogenicity.

(B) Studies on functional characterization of PE_PGRS and PE_PPE proteins of

Mycobacterium tuberculosis H37Rv

Sequence analysis of the Mtb H37Rv genome resulted in identification of novel multigene

families- the PE (proline-glutamic acid) and the PPE (proline-proline-glutamic acid). These

families account for much of the genomic difference between the pathogenic and

nonpathogenic mycobacterial genomes. Therefore, they may play role in Mtb's virulence and

host specificity. However, their exact role in Mtb biology is not clearly understood. We aim

to explore the possible role of the PPE and PE-PGRS proteins in the biology of Mtb.

Work reported in 2013-2014

(A) Development of recombinant and DNA based vaccine against Clostridium

perfringens

207

Codon biased beta toxin gene of Clostridium perfringens was cloned and expressed in E. coli.

Recombinant beta toxin produced as inclusion bodies was purified and refolded. Biological

activity of the refolded protein was evaluated in HL-60 cells and BALB/c mice. Antibodies

generated against the recombinant beta toxin were able to neutralize the toxicity of beta toxin

in vitro and in vivo.

(B) Studies on functional characterization of PE_PGRS and PE_PPE proteins of

Mycobacterium tuberculosis H37Rv

We assessed the role of PE_PGRS30 in modulation of host immune response. Infection of

PMA-differentiated human THP-1 macrophages with M. smegmatis expressing PE_PGRS30

resulted in reduced production of pro- inflammatory cytokines compared to those infected

with vector control M. smegmatis. PE_PGRS30 did not have any effect on survival of M.

smegmatis in THP-1 macrophages. Down-regulation of pro-inflammatory cytokines by

PE_PGRS30 was not mediated by IL-10. Infection of THP-1 macrophages with M.

smegmatis expressing deletion mutants of the protein showed that only PE + PGRS mutant

was able to reduce the levels of pro-inflammatory cytokines.

PPE14, a mycobacterial secretory protein, stimulated the production of pro-inflammatory

cytokines by PMA-differentiated THP-1 macrophages, which was TLR2 and MyD88-

dependent.

Progress of work during the current reporting year (2014-2015)

(A) Development of recombinant and DNA based vaccine against Clostridium

perfringens

Earlier, we have reported the immunogenic and vaccine potential of recombinant beta toxin

of C. perfringens. In order to develop sub-unit vaccines, immunodominant epitopes of the

beta toxin were identified using bioinformatics tools. Four putative identified regions (25-40

aa, 75-87 aa, 140-156 aa and 185-200 aa) were cloned in translational fusion with B-subunit

of heat labile enterotoxin (LTB) of Escherichia coli and were expressed in Vibrio cholerae

secretory expression system. The four recombinant fusion proteins obtained from the culture

supernatants of transformed V. cholera cells were analyzed for pentamer formation and

ability to bind to the GM1 receptor. LTB in the fusion protein retained its ability to bind to

GM1 receptor.

Immunization of BALB/c mice with the fusion proteins generated a very good immune

response with antisera of high titers. The antisera generated against all the fusion proteins

were analysed by Western blotting for the presence of toxin specific antibodies.

In vitro antibody neutralization assays indicated that the antiserum generated against the

fusion protein LTB-Epi140-156 significantly neutralized the toxin in an in vitro assay using

HL60 cells. The toxin-antisera formulations reduced cell death when compared to serum

from the PBS immunized mice.

LTB-Epi140-156 fusion protein immunized mice demonstrated nearly 40 % protection against

btx toxicity when challenged with minimal lethal dose of beta toxin, whereas no protection

was observed in the control mice immunized with PBS alone within 12 h of challenge.

208

For the development of candidate DNA-based vaccine against beta toxin of C. perfringens,

the btx gene was cloned in pDisplay vector for secretory expression of the recombinant

protein. To investigate the immunogenic potential of this construct, C57 BL/6 mice were

immunized through intramuscular route. Immunization with the pDisplay vector alone was

included as a negative control. Booster doses were given 2 weeks after primary immunization

and sera were collected weekly for monitoring the immune response. Two booster strategies

i.e. homologous prime boost (DNA followed by DNA) and heterologous prime-boost (DNA

followed by heat inactivated recombinant btx) were used. Heterologous boosted mice

mounted stronger anti-rbtx antibody response when compared to that obtained with

homologous boosted mice.

Antisera generated by immunization with pDisplay vector expressing secretory beta toxin

was tested for its neutralization ability using THP-1 and HL60 cells, and in vivo by

recombinant beta toxin challenge. Serum obtained from C57BL/6 mice immunized with the

DNA construct using heterologous prime boosting offered better neutralization potential than

that obtained from homologous boosting.

Intranasal immunization with the recombinant DNA construct was also evaluated using both

homologous and heterologous prime boosting strategies. Heterologous prime boosting

generated stronger immune response when compared to homologous prime boosting. It was

observed that immunization with the DNA vaccine construct of beta toxin resulted in the

generation of protective immune response as the antisera exhibited good neutralization ability

against the toxic effects of the recombinant beta toxin. The heterologous prime-boosted mice

showed 100% protection in vivo against the beta toxin challenge.

Structure of beta pore forming toxins have been divided into three domains – the cap, the

stem and the rim. In order to generate a non-toxic mutants for safer vaccine, and to

understand the role of different residues in biological properties of the beta toxin such as

binding to the host cell receptor, oligomerization and pore formation, a large number of

amino acid residues were targeted in different regions of the toxin. A number of mutants

(both point and deletion) were generated by PCR and site-directed mutagenesis and the

introduction of mutations was confirmed by DNA sequencing. The mutated genes were then

cloned in E. coli expression vector.

(B) Studies on the functional characterization of PE_PGRS and PE_PPE proteins of

Mycobacterium tuberculosis H37Rv

We have earlier reported characterization and function of PE_PGRS30 of M. tuberculosis by

expressing the respective proteins in M. smegmatis. M. smegmatis cells expressing

PE_PGRS30 were used as this mycobacterial species naturally lacks PE_PGRS family of

genes. During the reporting period , we have examined the function of another protein of

PE_PGRS family of M. tuberculosis i.e. PE_PGRS35. Function of PE_PGRS35 of M.

tuberculosis in its pathogenesis and immune evasion was examined by recombinant

expression of the protein in Mycobacterium smegmatis. Expression of PE_PGRS35 in M.

smegmatis altered the growth profile of the bacterium and the recombinant protein localized

in the mycobacterial cell wall. Infection of PMA-differentiated human THP-1 macrophages

with pVV1983

M. smegmatis expressing PE_PGRS35 led to diminished production of pro-

inflammatory cytokines including IL-12, TNF-α and IL-6, as compared to those infected

with pVV16 M. smegmatis (vector control). No change in intra-cellular bacterial survival and

macrophage viability were observed upon infection with the two recombinants.

209

Future plans

Deletion/substitution mutant clones of beta toxin will be subjected to expression analysis and

the recombinant mutant proteins will be evaluated for for their toxicity, binding to the host

cell and oligomerization. Non-toxic mutants will be evaluated for their vaccine potential.

Detailed functional characterization of PE_PGRS and PE_PPE proteins of mycobacterium

will be carried out.

Action taken on the RAP/SAC 2014 recommendations

No specific recommendations were made.

Publications

Original peer-reviewed articles

1. Karan S, Kaushik H, Saini N, Sahoo PK, Dixit A, Garg LC (2014) Genomic cloning and

sequence analysis of Interleukin-10 from Labeo rohita. Bioinformation 10: 623-629.

2. Dash P, Sahoo PK, Gupta PK, Garg LC, Dixit A (2014) Immune responses and

protective efficacy of recombinant outer membrane protein R (rOmpR)-based vaccine of

Aeromonas hydrophila with a modified adjuvant formulation in rohu (Labeo rohita).

Fish Shellfish Immunol 39: 512-523.

3. Bhatia B, Ponia SK, Solanki AK, Dixit A, Garg LC (2014) Identification of glutamate

ABC-Transporter component in Clostridium perfringens as a putative drug target.

Bioinformation 10: 401-405.

4. Bhatia B, Solanki AK, Kaushik H, Dixit A, Garg LC (2014) B-cell epitope of beta toxin

of Clostridium perfringens genetically conjugated to a carrier protein: Expression,

purification and characterization of the chimeric protein. Prot Expr Purif 102: 38-44.

210

Understanding the role of interferon regulatory factors in dendritic cell

development and innate immunity

Principal Investigator Prafullakumar Tailor

Project Fellow Monika Kaushik

Ph. D. Students Hemant Jaiswal

Rohit Verma

Irene Saha

Kuldeep Singh Chauhan

Bhupendra Singh Rawat

Theme of research

Dendritic cells (DCs) are composed of multiple subsets that collectively provide early innate

immunity, leading to subsequent adaptive immunity. Plasmacytoid dendritic cells (pDC),

CD4+ DC, CD8α

+ DC and CD4

-CD8

- DC are four major subtypes in the murine spleen.

These subtypes of DCs express different sets of genes and assume distinct functions. We are

interested in understanding the mechanisms of development of DC subsets and their

functions. Members of Interferon regulatory factors (IRFs) play important role in DC subset

development and their respective functions. Main area of research of the laboratory is to

understand the significance of signaling pathways and contribution of IRFs and other critical

transcription factors in DC subset development and functions.

Objectives

The principal aim of the project is to understand the role of IRF family members in the DC

development and functions. Interferon regulatory factor 4 (Irf4) and Interferon regulatory

factor 8 (Irf8) plays pivotal role in generation of diverse DC subtypes. The development of

CD8α+

DC and pDC requires Irf8, whereas CD4

+ DC subset is dependent

on Irf4. Studies

from knock out mice models led to identification of critical role of Inhibitor of DNA binding

2 (Id2), Basic leucine zipper transcription factor 3 (Batf3) and Irf2, Relb in development of

CD8α+ DC and CD4

+ DC respectively. Major areas of focus this year are 1] Understanding

the cross talk between Irfs and other transcription factors in defining DC subtype

development and 2] understanding contribution of TGF-β signaling in Irf8 directed DC

development.

Work reported in 2013-2014

Understanding the interactions between transcription factors in defining DC subtype

development

We had shown that expression of Irf8 in DC9 (Irf8-/-

) cells led to growth inhibition and

guided cells towards pDC and CD8α+ DC differentiation. Irf8 expression in DC9 cells also

showed an increase in Id2 and Batf3 transcript levels, transcription factors critical to

development of CD8α+ DCs. We further confirmed that Id2 and Batf3 expression without Irf8

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was not sufficient; while Batf3 and Id2 when co-expressed with Irf8 resulted in a synergistic

effect on classical CD8α+

DC development. We demonstrated that Irf8 is upstream of Batf3

and Id2 in the classical CD8α+ DC developmental program and had defined the hierarchical

relationship of transcription factors important for classical CD8α+ DC development (J.

Immunol. 2013, 191:5993-6001).

Understanding the role of TGF-β signaling in Irf8 regulated DC development

Transforming growth factor-β (TGF-β) signaling is shown to have important roles in DC

development. In DCs, TGF-β signaling induces Id2 expression, a member of helix-loop-helix

(HLH) group of transcription factors which are antagonists of activator class of HLH

members (Nat. Immunol. 2003, 4:380-386). Id2 expression is essential for CD8α+ DCs and

Langerhans cells (Nat. Immunology 2003, 4:380-386). A recent study identified Irf8 as a

direct target of TGF-β signaling in DCs (Eur. J. Immunol. 2007, 37: 1174–1183); and as per

our previous studies, Irf8 expression leads to induction of Id2 gene. Thus, suggesting that

TGF-β signaling may regulate CD8α+ DC development by Irf8 directed Id2 gene expression.

To check our hypothesis, we conducted preliminary experiments by blocking TGF-β

signaling using chemical inhibitors (SB431542 or LY364945) or by over-expressing

signaling blocking proteins (Smad7 or dnTgfbr2); neither affected Irf8 or Irf4 transcript levels

nor did it affect different DC subtype development. Thus our observations imply that TGF-β

signaling may be redundant in Irf8 directed pDC or CD8α+ DC development and further

studies were needed to confirm our observations.

Progress of work during the current reporting year (2014-2015)

Understanding the interactions between transcription factors in defining DC subtype

development

Irf8, Batf3 and Id2 play an essential role in development of CD8α+ DC. Our recently

concluded study defined the hierarchical relationship of transcription factors important for

classical CD8α+ DC development, where we demonstrated that Irf8 is upstream of Batf3 and

Id2 in the classical CD8α+ DC developmental program. Also we had shown that Batf3 and

Id2 when co-expressed with Irf8 resulted in synergistic increase in CD8α+ DC development.

Though, interactions (if any) between Irf8, Batf3 and Id2 during CD8α+ DC development

remained unanswered. Hence, to better understand the interaction between transcription

factors we initiated a study to examine in vivo interactions by two independent approaches. In

first approach, we used checkmate mammalian two hybrid system (Promega, USA), whereby

Irf8 and its mutants (R289E mutant deficient in protein interactions and R79E mutant

deficient in DNA binding) were cloned as a protein fused with VP16 activation domain. Id2

and Batf3 were expressed as a protein fused with yeast GAL4 DNA binding domain in a

vector expressing the Renilla reniformis luciferase (to normalize the transfection

efficiency)VP16 activation domain. Batf3 was expressed as a fusion protein with VP16

activation domain and Id2 was expressed as a fusion protein with GAL4 DNA binding

domain to study interaction between Id2 and Batf3. Interaction between the two test proteins,

as GAL4 and VP16 fusion constructs, results in an increase in firefly luciferase expression

under promoter containing five GAL4 binding sites. Alternatively, we are performing bi-

florescence complementation assay where the N-terminal and C-terminal split of Yellow

fluorescent protein (YFP) are expressed as a fusion of different proteins. N-terminal half of

YFP was expressed as a fusion protein ahead of Id2 and Batf3 and later half of YFP was

cloned as fusion protein at C-terminus of Irf8 and its mutants. Only when the two proteins

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interact, it brings two halves of YFP protein together to generate the fluorescence signal of

YFP. These experiments would enable us to understand physical interaction (if any) between

transcription factors that regulate the specific DC subtype development. PU.1, a know partner

of Irf8 was expressed as fusion protein N-terminal half of YFP and used a positive control.

Our preliminary results which needs further confirmation; suggests that Id2 and Batf3 do not

interact with each other and that Id2 and Batf3 might have independent roles in directing

CD8α+ DC development. We are analysing our results by conducting further experiments

checking more controls. To understand how Irf8 controls gene expression patterns to direct

DC differentiation, we conducted a gene expression profiling by affymetrix chip microarray

analysis. Our global gene expression analysis by expressing Irf8 in DC9 cells (Irf8-/-

background) showed 537 genes were up regulated and 295 genes were down regulated by

Irf8. Further experiment by Chromatin Immunoprecipitation (ChIP) assay would confirm the

genes that are directly under control of Irf8 and their significance in DC development would

be studied.

Understanding the role of TGF-β signaling in Irf8 regulated DC development

Our previous studies suggested that TGF-β signalling may be redundant in Irf8 directed DC

development. Though, our studies were performed on the DCs or ongoing BMDC cultures

and hence we performed experiments by using chemical inhibitors of TGF-β signalling from

the start of DC cultures from bone marrow cells. We confirmed that TGF-β signalling was

inhibited by SB431542 till day 9, as SMAD-2 phosphorylation was efficiently inhibited and

different DC subtype development was not affected. To check our observations in vivo, we

procured and analyzed the DC development in transgenic mice model expressing dominant

negative form of TGFBR2 under CD11c promoter (CD11c-dnTGFBR2 represented as TG

mice henceforth). Representations from pDC and CD8α+ DC populations from in vitro

BMDC cultures were comparable between control mice (WT) and TG mice. CD8α+ DC

specific Irf8, Batf3, Id2 transcripts along with pDC specific Tcf4 and SpiB and CD4+ DC

specific Irf4 and Irf2 transcripts were also expressed at comparable levels in WT and TG

BMDCs. Our data from the TG mice are in agreement with the recently reported phenotype

from CD11c specific deletion of TGFβRII, wherein multi organ autoimmune inflammation

and spontaneous activation of T and B cells led to death at 3-4 months (J. Immunol. 2012,

189: 3878-93). Our study shows that in adult TG mice in absence of inflammatory condition,

homeostatic development of major DC subtypes were unaffected (Nat. Immunol. 2005,

6:600-607). Our results differ from the reported study with human cord blood based culture

system in terms of dependency of Irf8 on the TGF-β signaling during dendritic cell

development (Eur. J. Immunol. 2007, 37:1174-1183) and it may be due to basal differences

in the mouse adult bone marrow precursors vis a vis human cord blood precursors or reported

study from cord blood cells were cultured using two step method with cocktail of cytokines

whereas our study was based on the FL-DC cultures system a bona-fide representative of

diverse DC subtypes from the mouse spleen (J. Immunol. 2005, 174:6592-6597). Our

observations are supported by study from adult CD11c-dnTGFΒR2 mice and also from

CD11c targeted TGFΒR2 deletion in CD11c mice. Thus we conclusively demonstrate that

TGF-β signaling may be redundant in controlling Irf8 dependent DC development in mouse

system. Recent studies suggested that IRF8 directly controls Itgb8 gene in APCs and triggers

development of Treg and Th17 cells by activating latent TGF-β (Immunity 2014, 40:187-198).

In light of this report we are currently revisiting our studies by understanding the role of Irf8

in regulating TGF-β signaling leading to the DC subtype specific gene transcription. From

gene expression array, we have identified genes from TGF-β superfamily pathway that are

213

specifically induced by Irf8 and we are currently analysing their role in Irf8-directed DC

development.

Future plans

We have performed the global gene expression analysis by microarray experiment and have

identified genes that are differentially regulated by Irf8 during DC development. We have

standardized ChIP assay in the lab and we will be performing ChIP-sequencing experiment to

identify the direct targets of Irf8. Earlier, we demonstrated that Irf8 is upstream of Batf3 and

Id2 in the classical CD8α+ DC developmental program and had defined the hierarchical

relationship of transcription factors important for classical CD8α+ DC development.

Similarly, Irf4, Relb and Irf2 are essential to development of CD4+ DC subtype, we have

initiated the study to analyse role of individual transcription factors and to study interaction

between these factors during CD4+ DC development. Together, Irf8 and Irf4 forms the axis

DC diversity development and in future studies we would also like to address how, Irf4 and

Irf8 differentially regulate development of different DC subtypes.

Action taken on the RAP/SAC 2014 recommendations

The scientific and technical clarifications sought by the SAC were provided during the

discussions. No specific recommendations were made.

Publications

Original peer-reviewed articles

1. Bradfute SB, Anthony SM, Stuthman KS, Ayithan N, Tailor P, Shaia CI, Bray M, Ozato

K, Bavari S (2015) Mechanisms of immunity in post-infection vaccination against Ebola

virus infection. PLoS One (in press)

214

Structural and functional biology of Mycobacterial proteins

Principal Investigator Bichitra K Biswal

Project Associate Avishek Anant (up to Sep 2014)

Ph. D. Students Nazia Nasir (up to June 2014)

Mohd. Saeed Ahangar

Deepak Chandra Saroj

Khundrakpam Herojit Singh

Abhisek Dwivedi

Bhavya Jha

Deepak Kumar

Theme of research

We have been pursuing two major research projects to understand structural and functional

aspects of proteins from Mycobacterium tuberculosis (Mtb), the organism that causes

tuberculosis (TB) in humans. In the first project, we aim at elucidating the 3D structures and

biochemical properties of enzymes of histidine (His) biosynthesis pathway of Mtb to unravel

the molecular mechanisms underlying their actions and to design enzyme specific anti-TB

inhibitors through a structure-based approach. The second project deals a similar line of study

on membrane associated proteases (MAPs).

Objectives

(i) Over-express, purify to homogeneity, perform biochemical assay of and crystallize the

target enzymes.

(ii) Determine 3D structures of their native and ligand-bound forms mainly using X-ray

crystallography.

(iii) Unravel the mechanisms underlying their action at the molecular level.

(iv) Design inhibitors against these targets through a structure-based-inhibitor design

approach and examine in vitro and in vivo efficacies of these inhibitors.

(v) Elucidate the 3D structures of enzyme/inhibitor complexes, analyse the interactions

between these, and use this information to design more potent inhibitors

(vi) Identify the plausible physiological substrates of MAPs through a proteomics approach.

(vii) Examine the role MAPs in Mtb pathogenesis through in vivo studies.

Work reported in 2013-2014

In the previous year, reported data were largely pertaining to the structural and biochemical

studies of HisC, a PLP-dependent histidinol-phosphate aminotransferase involved in the

catalysis of seventh step, conversion of imidazole acetol phosphate to L-histidinol phosphate.

We have shown that biological functional unit of HisC consists of a symmetric dimer. Like

HisC2, each protomer of HisC consists of two domains, a larger PLP-binding domain having

an α/β/α topology and a smaller domain comprising of a three-helix bundle resting over a

small β-sheet. The PLP-binding domain contains a seven-stranded β-sheet sandwiched

215

between four α-helices. Analysis of the ligand (2-(N-morpholino) ethanesulfonic acid)

[MES] -bound and free forms suggests that the N-terminal arm consisting about 40 amino

acids undergoes a large conformational change upon ligand binding. Briefly the loop adapts

a closed conformation in the ligand bound form and is open in the native structure.

Importantly, the ligand bound structure of the enzyme has led to the mapping of its active site

pocket. The active site is situated in the dimeric interface and residues that line the active site

region include Tyr25A, Asn37A, Thr38A, Asn39A, Gly101A, Ser102A, Asn103A, Tyr127A,

Met129A, His130A, Ala172A, Asn176A, Asp201A, Ala203A, Tyr204A, Thr229A, Ser231A,

Lys232A, Arg240A, Leu241A, Gly242A, Arg337A, Arg346A, Tyr67B, Pro260B, and

Tyr261B. It was established through biochemical study that Rv1600 is a PLP-dependent

histidinol-phosphate aminotransferase (HspAT). In the membrane protein project, we have

reported that a truncated version of Rv2224c was crystallized and native and anomalous X-

ray diffraction from the crystals data were collected.

Progress of work during the current reporting year 2014-2015

A. His pathway enzymes

During the past one year, we have determined high resolution structures of the native and

liganded forms of HisC (Rv1600) and HisC2 (Rv3772) and through extensive structural

analyses we have shown that HisC is an HspAT and Rv3772 in contrast is an aromatic amino

acids aminotransferase. Also MES was shown to be a competitive inhibitor of Rv1600. That

humans do not make histidine de novo, MES scaffold inhibitors lay a solid platform for the

design of more potent inhibitors against the Mycobacterial enzyme Rv1600.

Rv1600 and Rv3772 exhibit topology similar to subfamily Iβ ATs

The trend for catalytic efficiency (kcat/KM) of Rv1600 for its substrates was observed as

Hsp>Tyr>Phe while that for Rv3772 was Phe>Tyr>Trp. After determining the substrate

specificities of Rv1600 and Rv3772, we crystallized both enzymes and elucidated their 3D

(three-dimensional) structures to determine the structural similarities and differences between

Rv1600 and Rv3772. Both Rv1600 and Rv3772 monomers fold in a similar manner,

resembling the tertiary structure of members of the PLP-dependent subfamily Iβ ATs. The

spatial arrangement of the monomeric polypeptide chain of both the enzymes can be

described as analogous to a curved left hand with three distinct clustering of structural motifs

as thumb, palm and fingers (Figure 1). The PLP-binding domain in the palm position

encompasses major portion of the entire polypeptide chain consisting of a seven-stranded β-

sheet sandwiched between six α- helices. The thumb domain, which is comprised of a bundle

of three helices, serves as a nodal point. Protruding from this domain is a 40-residue long

loop which constitutes the N-terminal arm and passes over the PLP-binding domain (Figure

1). The C-terminal domain resembling the fingers forms a three-helix bundle resting over a

small β-sheet.

Typically, Iβ ATs are biologically active as a homodimer with the monomers related by

molecular 2-fold pseudo symmetry. Both Rv1600 and Rv3772 also adapt the same functional

unit, where the protomers of the dimer are non-covalently linked to each other via H-bonds

and van der Waals interactions. The dimer contains two identical active sites, with each

monomer contributing critical residues to both the active sites.

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Figure1. Representative 3D structures of Rv1600 and Rv3772. The sub-domain structures

of Rv3772 monomer. The N-terminal arm, palm, thumb and fingers domains are colored in

pink, lime, magenta and blue respectively.

Active site of Rv3772 can accommodate a dicarboxylate and an aromatic amino acid

To dissect the molecular basis underpinning biological activity of Rv3772, we determined

crystal structures of cofactor-Rv3772 complex bound to its preferential substrate, Phe and a

dicarboxylate, succinate to a resolution limit of 1.97 Å and 1.98 Å, respectively. While the

carboxylate group of free-form of Phe is held in place through interactions with Asn157, the

bound-form makes an additional interaction with Arg330. Further, superimposition of the two

active sites of the monomers from a homodimer highlights a structural deviation of a part of

the N-terminal arm (Leu9 to Ala24), leading to an inward movement of Tyr15 towards the

PLP-Phe complex in the substrate binding pocket. Thus, the N-terminal arm plays an

important role in regulating the binding and exit of ligand in this AT as well. PMP-Rv3772

complex was also captured with a dicarboxylate, succinate, bound in the active site. Succinate

exists as a non-covalently linked moiety, forming a bidentate H-bond/ion pair with Arg322

and Arg330 of the C-terminal domain.

Active site of Rv1600 is polar

A structural alignment of the ligand bound form of Rv1600 with E. coli HspAT bound to PLP

and Hsp -shows that MES of Rv1600 binds in a position identical to that of Hsp in E. coli

HspAT. In order to delineate the molecular interactions involved in Hsp binding in the active

site of Rv1600, Hsp was docked into the PLP-Rv1600 complex. Analysis of the Rv1600-

MES and Rv1600-Hsp structures suggests that the interactions of the two respective ligands

in the active site pocket are nearly identical. In particular, three active site residues, Tyr25,

Asn103 and Tyr127, are involved in H-bonding with the imidazole ring and amino group of

Hsp. Also, Met129 and Pro260 provide van der Waals interactions stabilizing the molecule in

the binding pocket. The phosphate moiety of Hsp is held in place by Arg337, Arg346 and

Asn176. And Tyr25, Asn103, Tyr127, Met129, Tyr67* and Tyr261* (* indicates residue

protruding from the adjacent protomer) forms a polar pocket which carves a favourable

environment for the binding of Hsp (Figure 2).

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Rv3772 harbors a hydrophobic binding pocket

In the Phe-Rv3772 complex, the residues Val86, Phe110, Leu112 and Phe246* form a

hydrophobic environment in the substrate binding pocket. This hydrophobicity of the

binding pocket aids in the specific binding of neutral or less polar substrates such as Phe and

Tyr/Trp in Rv3772. To determine the molecular basis of substrate specificities of Rv1600 and

Rv3772, we compared their 3D structures. No significant differences were observed in their

overall structures but their active site residues and their spatial disposition show distinct

features (Figure 2). In Rv1600 dimer, four polar residues, Asn103, Tyr127, Met129, and

Try261* form a bowl-shaped depression providing a favorable binding platform for polar

head groups such as the morpholine-ring of MES and imidazole ring of Hsp. On the other

hand, in Rv3772 non-polar residues present at the equivalent positions provide a conducive

setting for the binding of non-polar head group such as the phenyl-ring of Phe. Thus, Rv1600

and Rv3772 have varied substrate specificities owing to the marked differences in the degree

of polarity of their substrate binding pockets.

Figure 2. Difference in polarity of Rv1600 and Rv3772 active sites. A stereoview of the

superimposition of the active sites of MES-Rv1600 and Phe-Rv3772 complexes shows a

clear replacement of the polar residues of Rv1600 (Asn103, Tyr127, Met129 and Tyr261 *)

with non-polar residues in Rv3772 (Val86, Phe110, Leu112 and Phe246*).

B. Membrane proteases

The membrane protein (encoded by Rv2223c, Rv2224c and Rv2672) were over-expressed in

Mycobacterium smegmatis over-expression system and were purified using affinity and gel-

filtration chromatography. Diffraction quality crystals of 30-residue truncated version of

Rv2224c have been grown. X-ray diffraction data have been collected. Efforts to determine

the structure are underway.

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Future plans

(i) Standardize over-expression and purification protocols for the preparation of milligram

quantities of homogeneous samples of the remaining enzymes of His pathway. And

perform appropriate biochemical assays to establish their functions.

(ii) To grow diffraction quality crystals of these enzymes, medium- to high-throughput

crystallization experiments, using commercially-available and custom-made

crystallization reagents, will be carried out. Potential lead conditions will be optimized

to grow diffraction quality crystals. Subsequently, 3D structures of these enzymes will

be elucidated employing appropriate phasing method(s).

(iii) Determine the substrate-bound complex structure of each enzyme, comprehensibly

characterize its active site pocket, and elucidate underlying mechanism of its action.

(iv) Perform mutational studies particularly of those variants of the enzyme involving active

site mutation to pinpoint catalytic residues.

(v) Utilize this structural information to design small molecule inhibitors and examine their

in vitro and in vivo efficacies.

(vi) As mentioned in the previous year report, a triazole scaffold inhibitor (3-Amino-1,2,4-

triazole) was shown to inhibits HisB competitively. New derivative compounds of this

scaffold are being made and their complex structures with the enzymes will be

determined and in vitro activity will be measured. Moreover, their potency on Mtb

growth at cell culture level will also be examined. Compounds that show good activity

and appropriate pharmacokinetic property will be chosen for next level study. As

imidazole glycerol phosphate is the natural substrate of HisB, we are planning to screen

a library of imidazole-based compounds against HisB to identify new inhibitors for

HisB.

(vii) Over the last few years, we have successfully over-expressed and purified a few

membrane proteases (such as Rv2223, Rv2224 and Rv2672) in sub-milligram quantities

and importantly have carried out kinetic studies. Ongoing efforts to grow diffraction

quality crystals of these enzymes in the presence of various detergents and solve their 3D

structures will continue. To gain insights into their physiological substrates proteomics

approach will be used.

Action taken on the RAP/SAC 2014 recommendations

The recommendations made by the RAP/SAC committee were implemented to their

satisfactions.

Publications

Original peer-reviewed article

1. Saroj DC, Singh KH, Anant A, Biswal BK (2014) Overexpression, purification,

crystallization and structure determination of AspB, a putative aspartate aminotransferase

from Mycobacterium tuberculosis. Acta Cryst F70: 928-932.

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Plasmodium proteins involved in virulence and host modulation: Host-

parasite interactions in Plasmodium liver stages

Principal Investigator Agam Prasad Singh

Project Associate Rajesh Anand

Ph. D. Students Surabhi Dixit

Parul Sahu

Afshana Quadri

Collaborators S Kar, KIIT University, Bhubaneswar

Theme of research

Plasmodium species introduce effector molecules into hepatocyte cytosol to manipulate host

metabolic and /or signaling pathways for its own benefit. Those could prove good targets for

drug development. Parasite kinases, phosphatases etc. targeted to hepatocytes are likely

candidates. The host processes affected by them could also be target for intervention. Basic

theme is to identify, new parasite molecules that affect the host cellular processes, and

possible intervention strategies. We use reverse genetics tool to characterize the parasite

proteins with regard to its function.

Objectives

Objective of this study is to identify new parasite derived proteins that are involved in host

modulation, and required for parasites to grow and complete their life cycle. Currently,

Primaquine is the only drug available for malaria liver stages (LS) treatment, but it can‟t be

administered to pregnant women and people with G6PD deficiency as it causes toxicity.

Alternative drugs are the need of hour. Drugs can be targeted against parasite or host

processes. Using genetic, cell biology and biochemical methods, we identified that

Plasmodium circumsporozoite protein (CSP) is introduced in the hepatocyte cytoplasm and

hepatocyte nucleus, and ectopic expression of CSP alter thousands of host transcripts. The

overall effect is improved liver schizont growth. The current project aims to identify other

parasite proteins, like CSP, that play role in liver schizont development. The newly identified

proteins detail interaction with host cell will provide opportunity for developing new

interventions.

Work reported in 2013-2014

Host parasite interactions: In this section following were reported.

1. Characterization of a P. berghei FIKK Kinase (PBANKA_122500): Pb122500 protein

contains conserved S/T kinase domain in its C-terminal region therefore we tested the kinase

activity. We used full-length Pb122500 protein obtained through immuno-precipitation (IP)

from mid gut of infected mosquitoes using anti-Pb122500 antibody or c terminus of protein

expressed in E.coli. Result shows that full-length Pb122500 and c-terminus protein both had

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kinase activity. Using Protein kinase-A inhibitor, in an in vitro experiment with purified

kinase protein, we were able to completely inhibit the kinase activity of Pb122500.

2. Transcriptome analysis of hepatocytes infected with Pb-FIKK Kinase-KO parasites: We

did mRNA sequencing of host cells (using NGS), infected with Pb122500-KO or WT-

PBANKA parasites. In Pb122500- KO infected host cell, when compared with WT infected

cells, 288 [219 upregulated, 69 down regulated] changed more than two folds. Pie chart

analysis showed that major pathways affected were associated with signaling, metabolism,

immunity, focal adhesion and cell cycle pathways of host cells. NGS data was validated by

qRT-PCR and qPCR results coincide with NGS data.

3. Transcriptome analysis of hepatocytes infected with SLTRiP-KO parasites: In order to

know the function of exported SLTRIP in host modulation we performed whole

transcriptome analyses of host transcript at 22 hours post sporozoite infection of HepG2

cells.. When compared with WT infected cells, 2840 transcripts changed more than two folds.

Pie chart shows the major pathways affected were RNA transport/processing, metabolism,

immunity and ribosome structure /function. To validate NGS data, we selected two or more

genes from various pathways and a total of 23 genes were tested by qPCR on independent

biological repeats of the experiment. Quantitative-PCR results coincide with NGS data.

Novel drug targets: Curcumin has been shown to inhibit blood stages of malaria parasite.

Bioavailability of Curcumin is a matter of concern. Nano form of Curcumin has been shown

to be more effective against malaria blood stage parasites due its better bioavailability. We

tested both Curcumin and nano-Curcumin for their potency against liver stage malaria, in in

vivo condition (mouse). We did not find any in-vivo activity against liver stage parasite at the

highest dose tested [10 mg/mouse/day x3).

Progress of work during the current reporting year (2014-2015)

Project 1: Host parasite interactions during Malaria liver stage parasite

A) SLTRiP (Pb-871) Immunization provides protection against sporozoite challenge and is

T cell dependent: Mice immunized with purified SLTRiP protein yielded a very high

antibody titer of 5x106 after second booster immunization (Figure 1A). The specificity of

mouse anti-SLTRiP antibody was determined by western blot (Figure 1B). Mouse anti-

SLTRiP antibody specifically recognizes SLTRiP in P.berghei wild type sporozoites lysate

but not in SLTRiP-KO sporozoites and P.berghei (wild type) blood stage parasite lysate

(Figure 1B) showing a very high degree of specificity. Absence of SLTRiP antibody cross-

reactivity, with blood stage parasite highlights that SLTRiP is different from other tryptophan

rich proteins expressed during blood stages. To test the vaccine potential of SLTRiP protein,

BALB/c mice were immunized with purified SLTRiP protein and challenged with 1x104 wild

type sporozoites. In control and SLTRiP immunized group, blood stage parasites were

detected on 3rd

and 7th

days respectively (Figure 1D), highlighting that SLTRiP gives strong

but partial protection against sporozoite challenge. The above result was further verified by

checking parasite burden in the livers of immunized and control mice 48 hrs post challenge

with sporozoites. Results show that the parasite load in liver of SLTRiP immunized mice was

four logs (10,000 times) less as compared to the control group of mice (Figure 1E). To

ascertain the contribution of cell mediated immunity, we immunized RAG-1null

mice with

SLTRiP and measured the loss of protection compared to genotype matched controls

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(C57BL/6). RAG-1null

mice which lacks both B and T cells, showed a 100-fold loss in

protection [out of 1000 fold protection in matched wild type control] indicating T cells play

major role in protection since antibodies give minimal protection (Figure 1F). SLTRiP

immunization generated very high antibody titer and one would naturally expect these

antibodies to play role in protection against sporozoite challenge. Using sporozoite antibody

neutralization assay [SNA] we tested the contribution of anti-SLTRiP antibodies in

protection. To our surprise, at 1:5 dilution we observed only two-fold less infection compared

to control (Figure 1F), indicating minimal role of antibodies in protection.

Figure 1. A) Antibody titer of mouse anti-SLTRiP polyclonal sera. B) anti-SLTRiP antibodies specifically

recognizes SLTRiP in sporozoite lysate. Lane 1- purified recombinant GST-SLTRIP (72.5 kDa, E.coli

expressed), Lane 2- SLTRiP-KO sporozoite lysate, Lane-3 WT blood stage lysate, Lane-4 WT sporozoite lysate

and Lane-5 Protein molecular weight markers. β-actin was used as loading control. C) BLOT in 2B was probed

with anti CS antibodies and shows equal loading. D) SLTRiP immunized mice show a delay in first emergence

of blood stage infection as compared to control group. E) The parasite load in liver of SLTRiP immunized mice

is four log‟s (10,000 times) lower as compared to the control group of mice. F) Sporozoite neutralization assay

(SNA) with SLTRiP-Ab compared with the control sera.

B) New drug screening against liver-stage parasites

Andrographolide a diterpene lactone purified from methanolic fraction of the plant

Andrographis paniculata has been shown to have antimalarial activity against the blood stage

of Plasmodium. Andrographolide also exhibits a broad range of biological activities, such as

anti-inflammatory, immunomodulatory, antibacterial, antitumor, antidiabetic and

hepatoprotective. We tested Andrographolide against the liverstage of Plasmodium. We

found that after drug treatment [per oral] at 5mg/mouse x3 days followed by sporozoite

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challenge, the Andrographolide treated mice led to a 3 day delay in the pre patent period thus

causing a 1000 fold reduction in initial parasite load with respect to the control.

Project 2: Aspergillus flavus disease pathology.

This is a new project initiated during the current reporting period. The objective and progress

of the work are described below.

Objective

Aspergillus flavus is one of the leading Aspergillus spp. resulting in invasive aspergillosis of

central nervous system (CNS) in human beings. Immunological status in aspergillosis of

central nervous system remains elusive in case of both immunocompetent and

immunocompromised patients. An intravenous model of A. flavus infection was utilized to

determine the pathogenicity of infection and cytokine profile in the brain of male BALB/c

mice.

Progress of the work

Host responses to A. flavus CNS infection: Mice responded to the systemic infection marked

by defined perturbations in the physical, histological and immunological parameters. In order

to gain insights to the induction of A. flavus mediated pathological conditions inside the brain

of infected mice, we next observed the histological sections microscopically. Histological

sections of brain showed activated microglial cells, leukocytes particularly neutrophils and

few monocyte or lymphocytes in the brain tissues from the mice sacrificed at 48 hPI (Fig. 2a

and b). Growing fungal hyphae surrounded by leukocytes were observed in the brain sections

taken on second day (Fig. 2c and d). The brain sections sampled on second day demonstrated

a number of inflammatory granulomatous foci formed by the microglia and permeated

leukocytes specially neutrophils and few monocytes (Fig. 2e and f).

Inflammation and neuropathogenesis. During the experiment two kinds of pathology was

observed in infected mice. Initially during the first two days major pathology in the form of

ischemic events in neurons characterized by the eosinophilic cytoplasm and necrosis were

observed (Fig. 3a and b). Additionally to a lesser extent necrosis by forming pyknotic nuclei

were also observed in the sections taken at 48 hPI (Fig. 3a and b). Compared to control there

was a significant increase in ischemic necrotic neurons and pyknotic neurons at 48 hPI and at

96 hPI (Fig. 3c and d). Sections also demonstrated significant increase in ischemic necrotic

neurons and pyknotic neurons in the 96 h infected brain sections compared to 48 h infected

samples. CNS structures like dentate gyrus, cerebellum etc., where neurons are densely

packed, necrotic degeneration of neurons mostly manifested pyknosis from the mice about to

die on fourth day (Fig. 3c,). However, in a few neuron rich area major pathology observed

was ischemic necrotic neurons (Fig. 3d). Microscopic examinations of sections from fourth

day control mice demonstrated very few ischemic events and negligible number of neurons

showing pyknosis (Fig. 3e and f). The number of inflammatory granulomata was higher at

second day compared to that of fourth day post infection suggesting active machinery that

resolved the inflammatory granulomatous foci once the fungal components were killed. This

work was published in journal cytokine (2015). Cytokine 72 (2): 166-172

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Figure 2. Brain tissue sections, displaying the leukocyte infiltrations, granuloma and hyphae. BALB/c mice

were infected intravenously with 3.5 x105 conidia of A. flavus. The microphotographs displaying activated

microglia (a) and the leukocyte infiltrations (b) in the sections from the mice sacrificed at 48 hPI. Hyphae were

also visible in the sections of mice on second day after infection (c and d). Further a number of granulomatous

lesions/foci were also observed in the mice at 48 hPI (e and f).

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Figure 3. Sections of brain tissue from A. flavus infected BALB/c mice infected intravenously with 3.5x 105

conidia of A. flavus. Ischemic and pyknotic neurons as observed in the sections taken at 48 hPI (a and b). The

pyknotic neurons displayed in the sections from the mice that was about to die on fourth day (c). CNS structures

where neurons are densely packed, necrotic degeneration of neurons mostly manifested pyknosis from the mice

about to die on fourth day (c). At few places in neuron dense area sections sampled at 96 hPI demonstrated

ischemic neurons as a major event (d). Histological examination of brain from control mice sampled at 96 hPI

suggested a few ischemic events and a negligible pyknosis of the nucleus of neurons (e and f).

Future plans

1. Phenotypic characterization of PB823-KO parasite line. We identified that

immunization with PB823 protein provides protection [1000 fold] against sporozoite

challenge. We want to understand the PB823 protein role in parasite. We have created a

knockout parasite line and in future would characterize this mutant with regards to parasite

growth and development at various life cycle stages.

2. Screening of new chemical scaffolds against liver stage parasites. This is an ongoing

effort to screen new chemical entities for their inhibition potential against malaria liver stage

parasite. We collaborate with multiple researchers to source the new chemical entities.

3. Biochemical and functional characterization of Pb-FIKK kinase domain. Previously

we identified this protein and its role in parasite, now we want to identify its substrate and

phosphorylation site in its substrate. We would also test a kinase inhibitor library (from GSK)

to identify a potent inhibitor against this kinase.

Action taken on the RAP/SAC 2014 recommendations

Committee suggested for reduction in the transcriptome work, which we have followed and

rather focused more on biology and biochemical work.

225

Publications

Original peer-reviewed articles

1. Patel TK, Anand R, Singh AP, Shankar J, Tiwary BN (2014) Evaluation of Aflatoxin B1

biosynthesis in A. flavus isolates from central India and identification of atoxigenic

isolates. Biotech Bioproc Engn 19:1105-13.

2. Kaiser K, Camargo N, Kappe SHI, Singh AP (2014) Plasmodium axenically developed

exo-erythrocytic forms immunization confer strong protection against infectious

sporozoite challenge. Austin J Vaccines Immunother 1: 6.

3. Anand R, Shanka, J, Tiwary BN, Singh AP (2015) Aspergillus flavus induces

granulomatous cerebral aspergillosis in mice with display of distinct cytokine profile.

Cytokine 72: 166-172.

Reviews/Proceedings

1. Jaijyan, DK, Singh H, Singh, AP (2014) Malaria liver stage parasites strategies for

immune evasion and host modulation: Implication for vaccine design. Austin J Vaccines

Immunother 1: 5.

2. Dalai SK, Yadav N, Patidar M, Patel H, Singh AP (2015) Liver-stage specific response

among endemic populations: diet and immunity. Front Immunol doi:

10.3389/fimmu.2015.00125

226

Biophysical and biochemical characterization of Leishmania

phosphoglycerate kinase: an enzyme in the glycolytic pathway of parasitic

protozoa

Principal Investigator Vidya Raghunathan

Collaborator H Attreya, IISc, Bangalore

Theme of research

It is known that Leishmania sp. unlike mammalian counterparts uses multiple isoforms for

many enzymes of the energy pathway, one of which is phosphoglycerate kinse or PGK.

Leishmania PGK isoforms has some distinct structural features, as PGKB and PGKC differ in

a handful of internal residues and in the presence of a long extension at the C-terminus of

PGKC. Drug development efforts can be targeted, either at the glycosome itself or at the

enzymes present within them for which, targeting unique structural features is critical. We are

interested to use nuclear magnetic resonance spectroscopy to study the enzymology as well as

structure of PGK isoforms. We also want map the metabolic profile of Leishmania spp

cultures and correlate this to the enzymological studies with purified proteins.

Objectives

1. Expression, purification and determination of specific activities of PGKB-Lmex and

PGKC-Lmex and steady state kinetics study.

2. Comparison between PGKB-Lmex and PGKC-Lmex of, pH optimum of activity and

enzyme inhibition by salt and suramin.

3. 31P NMR studies using substrate / enzyme (PGKB-Lmex or PGKC-Lmex) mixtures, with,

either no metal, MgCl2, CaCl2, MnCl2 or CoCl2 to determine the change in the

dissociation constant of substrate with metal ions. Comparison with data from similar

experiments in literature with yeast PGK using Mg-ADP and Mg-ATP.

4. Peptide based studies of glycosomal membrane association of PGKC-Lmex. The peptides

used in these studies will be evaluated as useful models to understand the structural basis

of the biochemical differences between PGKC-Lmex and PGKB-Lmex.

5. Conformational studies by NMR using site non-radioactive isotope labeling.

6. Using promastigote and amasitoge cultures of Leishmania spp for metabolome mapping.

The concentration of specific metabolites in the cell at a particular time can be monitored

at the micromillimolar level by 31

P, 1H and

13C NMR spectroscopy. The metabolites that

can be detected are alanine, lactate, acetate, pyruvate, succinate, glycerol, urea, CO2,

oxalate, valine, glutamine and arginine.

Work reported in 2013 – 2014

Looking at the previous reports we took the glycolysis reaction for testing the activities of

PGKB and PGKC from Leishmania mexicana.

227

PGK NADH

ATP + 3-PG ADP+ 1,3-PG 3-PGA + Pi + NAD+

GAPDH

The recombinant clones of L. mexicana PGK (PGKB and PGKC) in E.coli available to us

have been checked by isolating the plasmid and sequencing to confirm the presence of the

PGK genes in the correct reading frame. The enzymes were characterized in terms of kinetic

parameters.

Phosphoglycerate Kinase Assay for ADP binding followed a protocol using pyruvate

Kinase/PEP for ATP generation.

ADP ATP + 3-PG ADP+ 2,3-PG

PGK in leishmania cultures: The PGK activity was measured using the standard assay, in the

amastigote cultures. Suramin dependent inhibition of activity was also observed as expected

from a known inhibitor of PGK.

The specific activity of PGKB-Lmex and PGKC-Lmex was found to be different, PGKB-

Lmex being more active than PGKC-Lmex. The ATP binding of PGKC-Lmex is stronger as

compared to PGKB-Lmex whereas 3-PG binding is stronger in the case of the latter. ADP

binding is stronger in the case of PGKB-Lmex. When compared with data published by

others on yeast PGK we find the ATP affinity is higher with the Leishmania enzymes

whereas yeast has a higher affinity for the other ligands 3-PG and ADP. When trying to

understand the basis of protein function one is inevitably led to the structure of the protein (if

it is known) or biochemical studies based on structure or sequence of the protein. No

structure of Leishmania PGK is available so far and only two sequences published, that of L.

major and L.mexicana. Our goal is also to determine the structure of these enzymes by NMR

and in conjuction do dynamic conformational studies.

Based on the results of computational analysis 3 synthetic peptides derived from the C-

terminal sequence of L. mexicana PGKC, were complexed with lipids or micelles and studied

by circular dichroism spectra and NMR. Proton NMR spectra of the peptide complexes

reconstituted in SDS micelles were recorded. Structure of the 26-mer peptide derived from C-

terminus of PGKC-Lmex was determined in MeOH and deuterated SDS. This revealed that

the peptide adopted a complete helical structure in the membrane-like environment provided

by these solvents (see publication). In plain aqueous media the peptide was insoluble. Further

structural studies outlined below will help us understand the structural biochemistry of the 62

residue c-terminal domain of PGKC-Lmex.

Progress of work during the current reporting year (2014-2015)

In lieu of the structure of the peptide as determined by NMR in solution [S. Kaushik, et al.,

Mol. Biochem. Parasit. 185 (2012) 27-35], we have launched into looking at the structure of

the entire C-terminal domain extension of PGKC-Lmex by cloning in E. coli. The 62 residue

domain of the C-terminus of PGKC has been cloned in E. coli BL21 (RP) strain. The protein

expression is good and western blotting shows the presence of a band after purification by

metal affinity chromatography at the molecular weight expected for the peptide of about 7

PEP

Pyruvate kinase

PGK NADH

GAPD

H

2 -PGA+ Pi+ NAD+

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kDa. Optimization of the purification for the purpose of making samples suitable for NMR

studies is underway. We have also cloned PGKC-Lmex in E.coli.

Future Plans

Using homology based modeling to have a base structure for PGKC-Lmex and PGKB-Lmex.

We will generate PGKB [13

C, 15

N-Trp] and [13

C, 15

N-Trp] PGKC for experimental studies by

high resolution NMR. Position of L. mexicana tryptophan in PGKB is W334 and in PGKC

are W334 and W447. NMR with these labeled proteins will be used to determine the

conformational change in the protein upon binding of substrate. All other structural studies

listed in objectives will become possible too.

Complete all structural studies.

Action taken on RAP/SAC 2014 recommendations

No specific suggestions were made. The advise of the committee members was taken on the

approach to determine structure of the stated proteins and the challenges faced therein.

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Chemical Glycobiology: Glycoform modulation, carbohydrate-based drug

design, and glycomics

Principal Investigator Srinivasa-Gopalan Sampathkumar

Ph. D. Students Syed Meheboob Ahmed

Surbhi Goswami

Vandita Dwivedi

Pratima Saini

Sandeep Kalal

Collaborators S Neelamegham, SUNY, Buffalo, USA

Arnab Mukhopadhyay, NII

Theme of research

Our laboratory of chemical glycobiology strives to develop carbohydrate-based small

molecules as probes, tools, and inhibitors to shed light on the importance of glycosylation in

biological processes. Understanding the functional roles of glycocalyx – which covers the

mammalian cells consisting of glycoproteins, glycolipids, glycosaminoglycans, and

glycosylphosphatidyl inositol anchors – has been challenging due to inherent complexity and

non-template driven biosynthesis. We design and synthesize novel molecules that intercept

glycan biosynthesis and enable bio-orthogonal functionalization. We investigate effects of

these molecules in vitro in mammalian cells, characterize changes to glycosylation using

biochemical, cell biological, and glycoproteomic methodologies, and in vivo in living

animals.

Objectives

Our main objective is to harness the power of carbohydrate-based synthetic small molecules

to unravel the importance of glycosylation in biological systems, as follows: (A)

Development of non-natural monosaccharide analogues for interception and metabolic glycan

engineering (MGE), wherein the promiscuity of enzymes of glycan biosynthesis is exploited

for processing of non-natural analogues. Small molecules that alter glycosylation of cell

surface antigens could potentially provide valuable inhibitors of glycan biosynthetic

pathways and disease modulators for auto-immune diseases. (B) Development of novel

carbohydrate-neuroactive (CH-NA) hybrid molecules in order to achieve MGE of the central

nervous system (CNS) across the blood-brain barrier (BBB). Although MGE has been

successfully shown in peripheral organs of living animals, no expression of non-natural

glycans were found in organs of the CNS. Our approach which enables MGE in brain

provides a vital access key to study importance of glycosylation in CNS development,

diseases, and disorders. (C) Development of glycopeptidomimetics (GPM) – based small

molecules carrying a zinc binding group (ZBG) on the carbohydrate moiety as novel

inhibitors of matrix metalloproteinases (MMP), which are known to trigger cancer metastasis.

(D) Development of inhibitors of human neuraminidase (sialidase) – NEU3 which is

expressed on the cell surface and might regulate ganglioside mediated processes.

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Work reported in 2013-2014

A. Inhibition of mucin-type O-glycosylation (MTOG) of cell surface molecules using

non-natural monosaccharide analogs via metabolic glycan engineering (MGE)

Biosynthesis of mucin-type O-glycans is initiated by the addition of N-acetyl-D-

galactosamine (GalNAc) to Ser/Thr (S/T) on the polypeptide backbone. It is known that

mucin-type O-glycans (MTOG) could be engineered to express non-natural functional groups

by exploiting the promiscuity of the GalNAc salvage pathway. Efficient thiol-dependent

inhibition of MTOG by peracetyl N-thioglycolyl-D-galactosamine (Ac5GalNTGc, 1) in

human lymphoid and myeloid cell lines was reported. Peracetyl N-glycolyl-D-galactosamine

(Ac5GalNGc, 2) and N-acetyl-D-galactosamine (Ac4GalNAc, 3) were employed as controls.

Our studies using flow cytometry, western blotting, microscopy, and mass spectrometry

showed that 1 abrogated sialylation and elaboration. Structure-activity relationship studies

confirmed that both the sulfhydryl and C-4 axial hydroxyl moieties are essential for MTOG

inhibition. Investigations of glycoforms of CD43 revealed drastic hypo-sialylation and hypo-

glycosylation in Jurkat (human T-lymphoma) cells. Incubation of Jurkat cells stably

expressing CD43-myc/FLAG with 1 followed by immunoprecipitation and thiol-selective

biotinylation confirmed the direct metabolic incorporation of N-thioglycolyl-D-galactosamine

(GalNTGc) (Ref: Agarwal, K., et al. J. Am. Chem. Soc. 14189-14197 (2013)).

B. Development of carbohydrate – neuroactive (CH-NA) hybrid molecules for

metabolic glycan engineering (MGE) across the blood-brain barrier (BBB)

We reported the synthesis and characterization of a panel of ManNAc analogues conjugated

to neuroactive molecules, their evaluation in vitro in cell lines and in vivo in mice. Previous

studies by Reutter and Bertozzi have shown metabolic engineering of sialic acids in tissues

such as heart, kidney, and liver but not in brain. Strikingly the CH-NA hybrids carrying N-

azidoacetyl-D-mannosamine (ManNAz) moiety, but not the corresponding non-hybrid

compounds, showed significant expression in brain tissue. Modulation of polysialic acid –

neural cell adhesion molecule (PSA-NCAM) expression in vivo using appropriate N-alkyl

variants of CH-NA hybrids was reported.

C. Design, synthesis, and development of carbohydrate-based small molecules for

inhibition of matrix metalloproteinases (MMP)

Design, synthesis, and characterization of a panel of monosaccharide derivatives carrying a

zinc binding group (ZBG) at the C-6 position were reported. Synthesis of selectively

protected glycosyl donors for utilization in glycosidation reactions towards synthesis of

glycopeptidomimetics (GPM) was reported.

D. Development of nanotools for glycobiology: Design and development of inhibitors

for human sialidases

Towards our investigations on human sialidases, evaluation of mRNA expression levels of

NEU 1-4 in mammalian cells (SH-SY5Y and CaCO-2), by RT-PCR were reported.

Expression and characterization of FLAG-tagged NEU3 (ganglioside sialidase) in Cos-1

(African green monkey kidney) cells was reported.

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Progress of work during the current reporting year (2014-2015)

A. Inhibition of mucin-type O-glycosylation (MTOG) of cell surface molecules using

non-natural monosaccharide analogs via metabolic glycan engineering (MGE)

Our goal is to develop carbohydrate-based small molecule inhibitors of biosynthesis of

mucin-type O-glycans by application of the metabolic glycan engineering (MGE)

methodology. Such small molecules would act as a first step in manipulating mucin-type O-

glycans and may serve as valuable tools in probing the dynamic structure-function

relationships of mucins in biological processes. Most of CD antigens carry both N-linked and

O-linked glycans which are built post-translationally by an army of glycosyl transferases in

endoplasmic reticulum (ER) and Golgi. MTOG expressed on blood group antigens and sialyl-

Lewis-X are known to play key roles in immune recognition, homing, and extravasation.

In continuation of our efforts on the development of carbohydrate-based small molecule

inhibitors for MTOG, we investigated K562 (human erythroleukemia) and U937 (human

histiocytic leukemia) cells. Additionally, investigations in animal cell lines EL-4 (mouse T-

lymphoma) and RAW264.7 (mouse macrophage) have been carried out with a goal to test

effects in primary cells and in vivo in mice.

Incubation of K562 cells with Ac5GalNTGc (1) (100 M, 48 h) showed reduction in the

neuraminidase-sensitive CD43-L60 epitopes of CD43 in a thiol-dependent manner, by

western blotting; while, control experiments incubated with Ac5GalNGc (2), Ac4GalNAc (3),

and vehicle (DMSO) did not show any change. However, lectin blots using Maackia

amurensis - II (MAL-II) lectin (binds to NeuAc2→3Gal moieties) showed only minor

changes at the global level. In contrast, Jurkat cells had shown drastic abrogation of MAL-II

epitopes, particularly, in the molecular weight range 100 – 150 kDa. Differential effects

between Jurkat and K562 cells, although similar in direction, could be attributed to the

expression and activity of enzymes of the glycosylation pathway. For example, Jurkat cells

are known to be defective in C1GalT (core1 galactosyl transferase), arising due to a mutation

in the chaperone Cosmc, thus resulting in low levels of elaboration of Tn-antigen (GalNAc-

S/T). Whereas, C1GalT is active in K562 and thus results in extensive core1 elaboration.

Cell surface glycans, owing to their location on the periphery, are invariably involved in

many biological and immunological processes, including cell-cell, cell-pathogen, and cell-

matrix interactions. In order to study the role of MTOG in immune cell differentiation and

phagocytosis, we chose U937 cells as an in vitro model. U937 cells were incubated with

GalNAc analogues (at various doses and time points) and studied by western blotting using

various anti-CD43 antibodies, namely, neuraminidase-sensitive (clones L60 and 1G10),

neuraminidase-resistant (clone L10), and glycan-independent (C-terminal). Again, drastic

abrogation of anti-CD43-1G10 and anti-CD43-L60 was observed with 1, but not with 2 and

3, confirming the thiol-dependent effects. Further lectin blots using MAL-II showed

consistent overall hypo-sialylation. It was also observed by microscopy that treatment with 1,

but not 2 or 3, induced strong homotypic cell clumping which might be a consequence of loss

of sialic acids on the cell surface.

Studies on mouse cell lines EL4 and RAW264.7 showed similar thiol-dependent effects.

Interestingly, EL4 and RAW264.7 cells are known to express CD43, respectively at 115 kDa

and 130 kDa, due to inherent variations in their glycosylation machinery. Probing of overall

glycosylation using MAL-II showed a thiol-dependent hypo-sialylation in EL-4 cells treated

232

with 1 but not in controls. Conversely, blots using peanut agglutinin (PNA) (binds to

Gal1→3GalNAc, also known as T-antigen or Thomson-Friedenreich antigen (TF-antigen))

showed increase in PNA epitopes due to loss of sialic acids on the glycans. Additionally,

lectin blots using Vicia villosa agglutinin (VVA) showed increased intensity suggesting

exposure of non-elaborated GalNAc residues on glycoproteins. EL-4 and RAW264.7 cells

were incubated with GalNAc analogues and probed by western blotting using various anti-

CD43 antibodies, namely, neuraminidase-sensitive (clone S7 and 1B11, which bind,

respectively to 115 kDa and 130 kDa glycoforms of CD43) and glycan-independent (C-

terminal, M19). Incubation of EL4 cells with 1, but not controls, specifically abrogated

CD43-S7 epitopes as well as showed bands at lower apparent molecular weight in CD43-

M19 blots. These results are found to be consistent with those observed in Jurkat cells. In

RAW264.7 cells, the CD43-1B11 epitopes were decreased upon incubation with 1 consistent

with those observed for THP-1 cells. Results obtained by flow cytometry for the CD43

glycoforms at the intact cell surface were found to be in agreement with western blotting

results.

B. Development of carbohydrate – neuroactive (CH-NA) hybrid molecules for

metabolic glycan engineering (MGE) across the blood-brain barrier (BBB)

Our goal is to design and develop CH-NA hybrid molecules for achieving MGE across the

blood-brain barrier (BBB). The ability to modulate glycan structures in brain across BBB

would provide key access to study the importance of glycoconjugates in development and

disorders of the central nervous system (CNS) in living animals. Sialoglycoconjugates, such

as polysialic acid – neural cell adhesion molecule (PSA-NCAM) are abundantly present in

CNS and are critically important for development, learning, memory, and nerve regeneration.

Ability to manipulate sialoglycoconjugates in living animals might shed light on the changes

to glycosylation under normal and disease conditions.

Having confirmed the unique ability of CH-NA hybrids, but not the parent non-hybrids, to

access CNS tissues including brain across the BBB, we extended our studies using additional

analogues and neuroactive carrier molecules. A panel of conjugates of N-acetyl-D-

mannosamine (ManNAc) analogues with neuroactive molecules was synthesized through

multi-step synthesis in multi-gram scale and characterized using NMR (1H and

13C), HPLC,

and mass spectrometry. Expression of N-azidoacetyl-D-neuraminic acid (NeuAz) carrying

sialoglycoproteins were characterized via Cu (I) assisted azide-alkyne cycloaddition (Cu-

AAC) click chemistry using a novel water soluble propiolamide carrying biotin reagent, in

the presence of ascorbic acid and Cu (I) stabilizing ligands. Optimization of dosage and

concentrations were performed to identify maximal engineering of brain using heart tissues as

controls. First the NeuAz was used as an abiotic bio-orthogonal handle to indentify best

carrier molecules. Subsequently, using the best carriers, N-alkyl-ManNAc derivatives were

used for modulation of PSA-NCAM levels in brain of living mice. Western blotting of PSA-

NCAM using anti-PSA and anti-NCAM antibodies showed significant reduction of PSA

levels on NCAM upon intravenous administration of CH-NA hybrid molecules.

C. Design, synthesis, and development of carbohydrate-based small molecules for

inhibition of matrix metalloproteinases (MMP)

Our goal is to design and develop carbohydrate-based small molecules as potential inhibitors

of MMPs and as anti-metastatic agents. MMPs, on the one hand, are critically important in

normal development and tissue remodeling. On the other hand, MMPs play a detrimental role

233

by aiding cancer cells to break-away from primary site and enhance metastasis. Development

of MMP inhibitors (MMPi) as anti-metastatic agents has been a focus of intense research for

more than three decades.

Towards glycopeptidomimetics (GPM) based MMPi, several selectively protected glycosyl

donors have been synthesized. Under Königs-Knorr and Schmidt glycosidation conditions,

the products were obtained only in moderate to poor yields. Preliminary cytotoxicity assays

in vitro using monosaccharide with a pendant ZBG revealed minimal toxicity.

D. Development of nanotools for glycobiology: Design and development of inhibitors

for human sialidases

Our goal is to develop small molecules for inhibition of human sialidases, particularly NEU3.

Mammalian glycocalyx is modified in a dynamic manner during development and

homeostasis. Particularly, sialic acid present at the termini of glycoproteins and glycolipids

on cell surface are removed by human sialidase in response to changes in environment.

Towards development of NEU3 inhibitors we have established activity assays using bacterial

sialidases using NeuAc-MU (4-methylumbelliferyl -D-neuraminic acid sodium salt) as a

substrate using fluorimetry. Additionally, FLAG-tagged NEU3 was over-expressed in

HEK293T cells and purified by immunoprecipitation using anti-FLAG (M2) beads as

confirmed by silver staining and western blotting.

Future plans

Carbohydrate-based small molecule inhibitors serve as invaluable tools to probe the

functional significance of cell surface glycans. Biochemical characterization of the

mechanism of transfer of GalNTGc and other GalNAc analogues on Ser/Thr of mucin-type

O-glycoproteins is currently underway. Based on a screening study of ppGalNAcT isoforms

(T1-T14) by real-time PCR in Jurkat and K562 cells, we have identified ppGalNAcT14 and

ppGalNAcT2 as candidates. Expression and biochemical characterization of FLAG-tagged

ppGalNAc T14 and T2 (expression plasmids obtained as a kind gift from Prof. H. Narimatsu,

AIST, Tsukuba, Japan) are currently underway. Activity assays will be performed using

UDP-GalNAc, the natural donor. Peptide fragments, designed based on CD43 sequence, will

be synthesized. In parallel, chemical synthesis of the non-natural donor UDP-GalNTGc will

be carried out.

Towards characterization of site occupancy and micro-heterogeneity of CD43 using

glycoproteomics, we have established Jurkat cells lentivirally transduced to express soluble

CD43-Fc-His (in collaboration with Prof. Sriram Neelamegham, SUNY, Buffalo, USA).

Studies on the effects of GalNAc analogues on glycoform modulation and nano-LC/MS

based characterization are currently underway.

The biological consequences of thiol-dependent MTOG inhibition will be studied using in

vitro models of T-cell activation. Studies to investigate effects of GalNAc analogues in

primary lymphocytes cultures ex vivo are currently underway. These will be extended to

studies on homing in vivo after pre-treatment with analogues ex vivo and via direct

administration.

234

In our endeavour to develop carbohydrate-based small molecules as tools that could provide

invaluable access to glycobiology of CNS tissues, we will expand the current panel of CH-

NA hybrids. CH-NA hybrids will be developed both for the engineering of glycans as well as

modulation / inhibition of glycosylation in brain across BBB. Characterization of azidosialo-

glycoconjugates will be pursued using Cu-free strain promoted cycloaddition reactions. In

order to identify glycoproteins that have undergone metabolic engineering covalent capturing

of azidosialo-glycoproteins on alkyne-functionalized agarose beads under Cu-AAC

conditions will be carried out. Identity and N-glycan site occupancy of covalently captured

glycoproteins will be investigated using trypsin and PNGase-F, respectively.

Towards development of GPM as MMPi and as anti-metastatic agents, we will intensify our

efforts in synthesis of carbohydrate analogues and glycosidation for glycopeptide synthesis,

purification, and characterization. The panel of molecules will be first screened using model

MMPs (e.g. thermolysin), toxicity screening in cancer cell lines, and standard in vitro

metastasis model assays.

In our studies on the development of inhibitors for human sialidases, we will pursue

expression, characterization, and activity assays of NEU3 and NEU1. NEU3 is a membrane

sialidase and is known to be localized in membrane rafts. Studies to identify the partners in

the membrane complex of NEU3 using proteomics-based approaches using FLAG-tagged

NEU3 and co-immunoprecipitation are currently underway.

Action taken on the RAP/SAC 2014 recommendations

Suggestions received from RAP-SAC members have been duly incorporated.

Publications

Original peer-reviewed articles

1. Golegaonkar S, Tabrez SS, Pandit A, Sethurathinam S, Jagadeeshaprasad MG, Bansode S,

Sampathkumar SG, Kulkarni MJ, Mukhopadhyay A (2015) Rifampicin reduced advanced

glycation end products and activates DAF-16 to increase lifespan in Caenorhabditis

elegans. Aging Cell doi: 10.1111/acel.12327.