seminar final.key
DESCRIPTION
my seminar 1 slideTRANSCRIPT
![Page 1: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/1.jpg)
Function of NS2B for NS3 protease activation of
Japanese encephalitis virus
Mr.Chakard ChalayutAdvisor: Assist.Prof Gerd Katzenmeier
Laboratory of Molecular virologyInstitute of Molecular Biology & Genetics
![Page 2: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/2.jpg)
Japanese Encephalitis Virus(JEV)
Source: fehd.gov.hk Source: news.bbc.co.uk
Culex tritaeniorhynchus.
![Page 3: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/3.jpg)
Japanese Encephalitis Virus(JEV)
Source : vietnammedicalpractice.com
![Page 4: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/4.jpg)
Japanese Encephalitis Virus(JEV)
Source: medwork84.com
Source: cdc.gov
30% fatality rate50,000 Cases10,000 Cases
![Page 5: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/5.jpg)
Prevention and treatment of JEV disease
Drug No drug exist
Vaccine development
Mosquitoes control Elimination of mosquitoes breeding places
Available vaccine
![Page 6: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/6.jpg)
Molecular biology of Japanese Encephalitis Virus
Source : molecular-virology.uni-hd.de
![Page 7: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/7.jpg)
The NS2BHypothetical model NS2B-NS3 complex
hydrophobicity plot
51 DMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 95
Brinkworth et al, 1999
• 130 aa• activating domain central hydrophilic region (Falgout et al, 1993)• 3 membrane spanning parts
![Page 8: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/8.jpg)
The NS3
•Chymotrypsin-like fold2-β barrel domains •Inactive alone•Enzyme’s pocket is small
NTPase
Protease
RNA Helicase
Theoretical model from PDB 2I84
![Page 9: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/9.jpg)
The NS3 protease
conformational change alteration of the enzyme pocket additional substrate binding site
•NS3 serine protease domain 20 kDa•catalytic residues His51, Asp75, Ser135
Complexation with NS2B cofactor
![Page 10: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/10.jpg)
![Page 11: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/11.jpg)
• The result shown the slightly differences in biochemical properties.
• Some physico-chemical properties shared by all the proteases.
![Page 12: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/12.jpg)
Homology Modelling of NS3 protease
![Page 13: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/13.jpg)
Homology Modelling of NS3 protease
JEVWNV DEN
![Page 14: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/14.jpg)
![Page 15: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/15.jpg)
![Page 16: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/16.jpg)
• NS2B-NS3 WNV can adopt two distinct conformation.• The NS2B-NS3 shown the induced fit mechanism.
![Page 17: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/17.jpg)
• Two component of protease has a substrate specificity.
• Two component of protease has a unique activation mechanism.
• The NS3 protease share the similar structure in the Flavivirus group but are different in cofactor binding.
![Page 18: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/18.jpg)
• To investigate the function determination in the JEV NS2B for the activation of NS3 protease.
![Page 19: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/19.jpg)
Method 1.Expression and purification of deleted and mutation forms
of NS2B-NS3 protease
Full length NS2B and NS3 protease
PCR to amply deleted and mutation forms of NS2B
Clone into vector
Expression and Purification
SDS-PAGE and Western Blot
![Page 20: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/20.jpg)
Method 1.
![Page 21: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/21.jpg)
Result
Lane 1- 5 = Washing Fraction with 100 mM ImidazoleLane 6 -10 = Eluted Fraction with 400 mM Imidazole
![Page 22: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/22.jpg)
Method II
NS2B-NS3 protease
Incubate with GKR-AMC
Measured the florescence Change
Trans-cleavage of fluorogenie peptide substrate
![Page 23: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/23.jpg)
Result
![Page 24: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/24.jpg)
Method III
JEV NS2B and NS3 protease protein
Based on WNV crystallographic structure
Perform the structural modelling by using Swiss modeling workstation & MEDock program
Molecular Modelling of a JEV NS2B-NS3 complex
![Page 25: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/25.jpg)
Result
![Page 26: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/26.jpg)
Result
Lane 1- 5 = Washing Fraction with 100 mM ImidazoleLane 6 -10 = Eluted Fraction with 400 mM Imidazole
![Page 27: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/27.jpg)
Discussion• Ser46 and Ile60 are essential region of JEV
NS2B for activation of NS3 protease.
• Alanine substitution demonstrate the functional conformation of Trp53, Glu55 and Arg56 in JEV NS2B for the cleavage ability of NS2B-NS3 protease.
• Residues Ala67 to Asp76 of JEV NS2B suggested to provide the additional β-strands to stabilize the fold of NS3 protease.
![Page 28: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/28.jpg)
Discussion• Residues Lys78 to Leu87 of JEV NS2B may
involved in the formation of the active site in the NS2B-NS3 protease.
![Page 29: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/29.jpg)
• Due to the substrate specificity of the NS2B-NS3 protease.
• Can we make a very specific anti-viral drug to Flavivirus ?
The pan-Flavivirus NS3 protease drugs may be developed for Flaviviral diseases.
![Page 30: Seminar Final.Key](https://reader035.vdocument.in/reader035/viewer/2022081403/5550212cb4c905af648b534d/html5/thumbnails/30.jpg)
Thank you for your attention.