FUNGAL TRANSPORTOSOMICS

Andre GoffeauUniversite de Louvain

Bioinformatic and Comparative Genome Analysis Course

HKU-Pasteur Research Centre - Hong Kong, China

August 17 - August 29, 2009

Bioinformatic and Comparative Genome Analysis Course

HKU-Pasteur Research Centre - Hong Kong, China

August 17 - August 29, 2009

Genomesequencing

Transcriptomics Proteomics

Genomecomparison

Deletomics

Genomology

Databases

Interactomics

post-genomics, functional genomics, proteomics, transcriptomics,

pharmacogenomics, structural genomics, metagenomics,epigenomics,

patagenomics!!!

FUNGAL TRANSPORTOSOMICS

Nomenclature Committee of the International Union

of Biochemistry and Molecular Biology (NC-IUBMB)

In consultation with the IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (JCBN)

Enzyme Nomenclature

http://www.chem.qmul.ac.uk/iubmb/enzyme/

EC 1 Oxidoreductases

EC 1.1 Acting on the CH-OH group of donors

EC 1.1.1 With NAD or NADP as acceptor

EC 1.1.1.1 alcohol dehydrogenaseEC 1.1.1.2 alcohol dehydrogenase (NADP)EC 1.1.1.3 homoserine dehydrogenaseEC 1.1.1.4 (R,R)-butanediol dehydrogenase…EC 1.1.1.37 malate dehydrogenase

Enzyme Nomenclature

EC NOMENCLATURE

ONLY BASED ON CATALYTIC REACTIONS OF SOLUBLE ENZYMES WHICH ARE EXPERIMENTALY DEMONSTRATED

NO TRANSPORT REACTIONS

NO SEQUENCE /PHYLOGENY INFORMATION

A membrane protein

M.jannaschii

M.thermoautotrophicum

M.thermoautotrophicum

T. acidophilum

Proton-transporting P-type ATPases TMHMM

membrane span and topology prediction

Transporter Classification

TCDB Milton Saier

(validated by IUBMB)

1. A. 1. 1. 1Mechanism class

Mechanism subclass

Phylogenetic superfamily/family

Substrate(s)

Phylogenetic subfamily

First (number) :Second (letter) :Third

FourthFifth

1. Channels/Pores

2. Secondary Transporters

3. Primary Active Transporters

9. Incompletely Characterized Transport Systems

Mechanisms

2.A. uniporters, symporters, antiporters

2.A.1 The Major Facilitator Superfamily (MFS) 2.A.2 The Glycoside/Pentoside/Hexuronide (GPH): Cation Symporter

Family2.A.3 The Amino Acid-Polyamine-Organocation (APC) Superfamily2.A.4 The Cation Diffusion Facilitator (CDF) Family2.A.5 The Zinc (Zn2+)-Iron (Fe2+) Permease (ZIP) Family2.A.6 The Resistance-Nodulation-Cell Division (RND) Superfamily…2.A.73 The Inorganic Carbon (HCO3-) Transporter (ICT) Family 2.A.74 The 4 TMS Multidrug Endosomal Transporter (MET) Family 2.A.75 The L-Lysine Exporter (LysE) Family 2.A.76 The Resistance to Homoserine/Threonine (RhtB) Family

2.A.77 The Cadmium Resistance (CadD) Family

Superfamilies/Families

2.A. uniporters, symporters, antiporters

2.A.1 Major Facilitator Superfamily (MFS)

2.A.1.1 The Sugar Porter (SP) Family 2.A.1.2 The Drug:H+ Antiporter-1 (12 Spanner) (DHA1) Family 2.A.1.3 The Drug:H+ Antiporter-2 (14 Spanner) (DHA2) Family 2.A.1.4 The Organophosphate:Pi Antiporter (OPA) Family 2.A.1.5 The Oligosaccharide:H+ Symporter (OHS) Family 2.A.1.6 The Metabolite:H+ Symporter (MHS) Family 2.A.1.7 The Fucose:H+ Symporter (FHS) Family 2.A.1.8 The Nitrate/Nitrite Porter (NNP) Family 2.A.1.9 The Phosphate:H+ Symporter (PHS) Family 2.A.1.10 The Nucleoside:H+ Symporter (NHS) Family 2.A.1.11 The Oxalate:Formate Antiporter (OFA) Family 2.A.1.12 The Sialate:H+ Symporter (SHS) Family 2.A.1.13 The Monocarboxylate Porter (MCP) Family 2.A.1.14 The Anion:Cation Symporter (ACS) Family 2.A.1.15 The Aromatic Acid:H+ Symporter (AAHS) Family 2.A.1.16 The Siderophore-Iron Transporter (SIT) Family …2.A.1.30 The Putative Abietane Diterpenoid Transporter (ADT) Family

Subfamilies

Phylogenetic criteria

-Identify S.cerevisiae ORFs of TMS > 2

-Identify TC # using BLAST

-Apply the following general criteria:

-for 3th digit (Super)Families :

10% identity (E < 10 –20)

-for 4th digit Subfamilies:

20% identity (E < 10 –35)

-for 5th digit Clusters:

35% identity (E < 10-65)

• Explore Transport Protein Diseases.doc

YETI

YEAST TRANSPORTOSOME INFORMATION

BLAST QUERY OF P39980 S.cerevisiae UMP

P39980 YEH5_YEAST (YEL065W)HYPOTHETICAL 70.6 KDA PROTEIN 1195 0.0

P38731 YHE0_YEAST (YHL040C)HYPOTHETICAL 70.9 KDA PROTEIN 530 e-149

P38724 YHE7_YEAST (YHL047C)HYPOTHETICAL 71.6 KDA PROTEIN 431 e-119

P36173 YK86_YEAST (YKR106W)HYPOTHETICAL 69.0 KDA PROTEIN 393 e-108

P25596 YCH0_YEAST (YCL073C..)HYPOTHETICAL 68.9 KDA PROTEIN 392 e-108

Q08299 ENB1_CHROMOSOME XV READING FRAME ORF YOL158C 280 4e-74

BLASTFASTA

The Siderophore-Iron Transporters (SIT)

Sit1/Arn3 (YEL065w) S.cerevisiae (spP39980)

Subfamily 2.A.1.16

Taf1 (YHL047c) S.cerevisiae (spP38724)

2.A.1.16.3 The ferric triacetylfusarinine:H+ symporter

Enb1 (YOL158c) S.cerevisiae (pirS66857)

2.A.1.16.2 The ferric enterobactin:H+ symporter

2.A.1.16.1 The ferrioxamine: H+ symporter

Clusters

ABC TRANSPORTERS in HUMAN DISEASESABC TRANSPORTERS in HUMAN DISEASES

CFTR human Cystic Fibrosis YCF1cMOAT " Dubin-Johnson Syndrome YCF1SUR " Hyperinsulinemic glycemia of infancy YCF1TAP1/2 " Multiple Sclerosis, Behcet's Syndrome MDL1/2ALD " Adrenoleukodistrophy PXA1PMP70,ALDR " Zellweger Syndrome PXA1

DEFECTIVE FUNCTIONDEFECTIVE FUNCTION

AMPLIFICATIONAMPLIFICATIONMDR1 human Drug resistant cancers STE6MRP " Drug resistant cancers YCF1, YOR1pfMDR2 Plasmodium f. Drug resistant malaria ATM1CaCDR1 Candida albicans Drug resistant candidosis PDR5, PDR10, PDR15EhPgp1 Entamoeba h. Drug resistant amoebiasis STE6ldMDR Leishmania d. Drug resistant Kala azar STE6SMDR2 Schistosoma m. Drug resistant schistosomiasis STE6

PLEIOTROPIC DRUG TRANSPORTERS (PDR)

in YEASTS

also called Multiple Drug Resistance (MDR)

in MAMMALIAN CELLS

Yor1Yor1

Bpt1

Bat1

Yhl035Yhl035

Ycf1Ycf1

Ste6

Ynr070

Yor011

Pdr11Pdr11

Ykr103/4

Yol075

Pdr12Snq2Snq2 Pdr10Pdr10

Pdr15Pdr15

Pdr5Pdr5

PDR5 - likePDR5 - like

NEWNEW MDRMDR - like - like

MRP - likeMRP - like

(NBF-TM)2 (TM-NBF)2

The 16 "full - size" ABC transporters

Bat1

Ste6

Ynr070

NEWNEW MDR - likeMDR - like(NBF-TM)2 (TM-NBF)2

The 16 "full - size" ABC TRANSPORTERS

Yor011

Yhl035Yhl035

Pdr12 Pdr10Pdr10

Yol075

Pdr11Pdr11

Snq2Snq2Pdr15Pdr15

Pdr5Pdr5

Yor1Yor1 Ykr103/4

Bpt1

Ycf1Ycf1

Ste6

Yor1Yor1

Bpt1

Bat1

Yhl035Yhl035

Ycf1Ycf1

Ste6

Ynr070

Yor011

Pdr11Pdr11

Ykr103/4

Yol075

Pdr12Snq2Snq2 Pdr10Pdr10

Pdr15Pdr15

Pdr5Pdr5

MULTIPLE disruptions SINGLE disruptions

PDR5 - likePDR5 - like

NEWNEW MDRMDR - like - like

MRP - likeMRP - like

(NBF-TM)2 (TM-NBF)2

The 16 "full - size" ABC

Bat1

Ste6

Ynr070

MULTIPLE disruptionsMULTIPLE disruptions SINGLE disruptions

PDR5 - likePDR5 - like

NEWNEW MDR - likeMDR - like

MRP - likeMRP - like

(NBF-TM)2 (TM-NBF)2

NO disruptions yet

The 16 "full - size" ABC

Yor011

Yhl035Yhl035

Pdr12Pdr10Pdr10

Yol075

Pdr11Pdr11

Snq2Snq2Pdr15Pdr15

Pdr5Pdr5

Yor1Yor1 Ykr103/4

Bpt1

Ycf1Ycf1

Ste6

PDR5 PDR5

Antibiotics chloramphenicol tetracycline anisomycinantimycin A1 cycloheximidelincomycinoligomycin tunicamycinthiolutin

Steroids and their structural analogues5 androstan 3 ol 17 one5 androstan 3 ol 17 one5 androstan 17 ol 3 one5 androstane 3,17 dione5 androstane 3,17 dione1,4 androstadiene 3,17 dione5 pregnan 3 ol 20 one4 pregnen 20 ol 3 onetrans dehydroandrosterone4 androstene 3,17 dioneepiandrosterone acetateepiandrosteroneestriol 16, 17 diacetate 17 ethynyl estradioldeoxycorticosteronepregnenoloneprogesteronetestosteronediethylstilbestrolhexestrol4 hydroxytamoxifen

Ionophores valinomycin nigericinmonensinA23187

Others2,3,5 triphenyltetrazoliumchloride4 nitroquinoline n oxide8 hydroxyquinolinediphenylaminep aminodiphenylaminen decylamine4 nitrophenolanilineanthrontetranitrotetrazoliumchloride blueresazurinesporidesminstaurosporinefluphenazinecerulenincompactin

Anticancer drugs chemosensitizerstamoxifendaunorubicindoxorubicin trifluoperazine

Detergentstetradecyltrimethyl--ammonium bromidehexadecyltrimethyl--ammoniumbromidezwittergent 3-10zwittergent 3-12zwittergent 3-14brij 58brij 35n octylglucosiden dodecylglucosiden dodecylmaltoside

FungicidesitraconazoleketoconazoleeconazolemiconazoletriadimenolbitertanolnuarimolfenarimolcarbendazimdiclobutrazolphenapronilchlorothalonilcyprodinildodineimazalilprochlorazazoxystrobinflusilazoltebuconazoleCGA642512 mercapto--benzothiazolekrezoxim methylnystatinsoraphen Aepoxiconazole

Herbicidessulfomethuron methylfluometuronchloroxurondifenoxuronchlorbromurondiuron

Fluorescent dyesrhodamine 6Grhodamine 123

THE YEAST ABC TRANSPORTER PDR5 MEDIATES RESISTANCE TO MANY DIFFERENT COMPOUNDS

Kolaczkowski et al., 1997

Pdrp are activated in antifungal-resistant

human and plant pathogens such as Candida albicans ,Candida glabrata,

Candida krusei, Cryptococcus neoformans

and others ??

MIYAKAMA et al, 2005

Balzi et al., JBC (1994)

ECL3 ECL6

Walker A

Linker region

Walker B

GASGAGKTT* * * * *

Pdr5-NH2 GRPGSGCTT

Pdr5-COOH

The catalytic Lys from NH2 Walker A is substituted by Cys in Pdr sensu stricto

Fig. 1

N

C1 2 3 4 5 6 7 8 9 10 11 12

EL1 EL2

EL3EL4 EL5

EL6

IL1 IL2 IL3 IL4

NBD1 NBD2

TMD1 TMD2

Walker A2(GX2GXGK(S/T))

Walker B2(hhhhD)

ABC 2(LNVEQ)

Helical domain

Q-loop (Q)

H-loop(H)

D-loop(E)

P-loop C-loop pro-loop(P)

Walker A1(GX4GC(S/T))

Walker B1(X3WD)

ABC 1(VSGGE)

Helical domain

Q-loop(E)

H-loop(Y)

D-loop(D)

P-loop C-loop pro-loop(X)

Pdr Transportersonly in fungi, plants and protists

• Sensu stricto Pdr (Pdr5/10/12/15/18, Snq2) Reverse topology Cysteine in N-terminal Walker A Expanded ECL3 and ECL6 Efflux pump

Honorary Pdr (Aus1, Pdr11, YOL075) Reverse topology Influx pump?

Pdr10

Pdr15

Pdr5

Pdr12

Snq2

YNR070w (Pdr18)

Sensu stricto Pdrp in SACE

COMPARATOMICS

Hemiascomycetes

Saccharomyces cerevisiaeSaccharomyces cerevisiae

Dujon et al.

The Yeast ZIP Family (ZINC Iron Porters). TC # 2.A.5.

DEHA-0B16335g

DEHA-0E25388g

SACE-ZRT1

YALI-0F21659gDEHA-0B07337g

CAGL-0E01353gKLLA-0D16434g

CAGL-0M04301g

SACE-ZRT2

YALI-0D00759g

YALI-0E00748g

YALI-0F15411g

SACE-YKE4

KLLA-0F17886g

YALI-0D19008g

DEHA-0E06105g

KLLA-0A07601g

SACE-ATX2

CAGL-0K05577g

2.A.5.Y1GolgiMn

2.A.5.2ERZn

2.A.5.1plasma membraneZn

2.A.5.Y2no datano data

CONCLUSIONS

REDONDANT TRANSPORTERS ARE LOCATED IN DIFFERENT MEMBRANES

SOME TRANSPORTER SUBFAMILLIES HAVE NO SACE MEMBERS

In practice – the most variable families

13.26.2715711Yeast Metal Channel ( Cyt B-FRE )1.A.20.5

17.217.21814162414AminoAcid-Polyamine-Organocation Yeast Transporter( APC-YAT )

2.A.3.10

19.23.200088NADH Dehydrogenase I (NDH 1)3.D.1.2

21.06.0553314The Putative Fatty Acid Transporter (FAT-1) 9.B.17.1

21.35.6031285Fructose uniporter (FRU)2.A.1.13

23.56.0614514Ferrioxamine H+ symporter (SIT)2.A.1.16

45.75.2203417Oligopeptide Transporter (OPT)2.A.67.1

63.513.0101110727Peroxisomal Protein Importer (PPI) 9.A.5.1

114.817.4121082433Drug Proton Antiporter 1 (DHA-1)2.A.1.2

187.519.0106132739Anion Cation Symporter (ACS)2.A.1.14

153.729.23417204827Sugar Porter (SP)2.A.1.1

VarianceMeanSACECAGLKLLADEHAYALI Subfamily

BLAST of Snq2 in SACE and KLLA

BLAST E-value is not discriminating

BLAST score distinguishes four Snq2 homologs in KLLA

KlSnq2a clusters with ScSnq2

KlSnq2b clusters with ScPdr12

KlSnq2 c and d cluster with Sc Pdr5/10/15

ScSnq2

ScYNR070 KlSnq2a ScPdr12 KlSnq2b ScPdr5 KlSnq2c ScPdr15 ScPdr10 KlSnq2d

Neighbors analysisPdr5/15 filiation

ZYRO

CAGL

SACE

SACE

CAGL0F02585g CAGL0F02607g CAGL0F02651g CAGL0F02673g CAGL0F02695g CAGL0F02717g

YDR400 YDR402 YDR403 YDR404 YDR405 PDR15

PDR5

CAGL0F02739g

YDR407

CAGL0F2761g

YDR408

CAGL0F02783g

YDR409

YOR156

CAGL0F02805g

YDR410

CAGL0F02827g

YDR411

RO5530 RO5529 RO5523 RO5513 RO5509 RO5508

YOR150 YOR151

RO5532

YOR155

RO5533 RO5505

Ustilaginom.

Tremellom.

Homob.Agaricom.

Basidiomycetae

Ascomycetae.

Taphrinomycotina

Saccharomycotina

Pezizomycotina

Onygenales

Dothideomycetes

Eurotiomycetes

Leotiomycetes

Sordariamyces.

P

P

P

P

P

(P)

PP

P

PP

PP

PP

P P

P

PP

P

P

P

PP

P

PP

PP

P

P

PHYLOGENETIC ANALYSIS OF 78 PDR

(SENSU STRICTO AND HONORAY)FROM 11SPECIES

SACHARO/PEZIZO/BASIDIO

PHYLOGENETIC CLUSTERS OF FUNGAL PDRs

CONCLUSIONS

A total of 8 clusters (A to H)

Three clusters contain only SACE members (A,D,E)

Two honorary clusters are conserved (E,F)

Four new clusters not present in SACE(B,C,G,H)

The four new clusters are sensu stricto Pdrp (see Cys in Walker A1)

Are the new clusters drug efflux pumps? Which specificity?

•

PHYLOGENETIC ANALYSIS of 349 Pdr from 56 species distributed in 5 subphyla

H1

DSNQ2

FYOL075

B

APDR5

G-----

EAUS1

C

H2

Fig. 3

ConclusionOnly one more cluster H1/H2 that

belongs to sensu stricto PDR

STRUCTURE PREDICTOMICS

CONCLUSION

1.Combined to analysis of mutants,the elucidated sequence motifs and the prediced topology will become important for

interpretation of crystallographic data and for elucidation of the catalytic mechanism of the efflux pumps including the amazing pleiotropic specificity for substrates and the asymetry of the

two NBD.

2.This infornation must be taken into account to understand the molecular evolutionmolecular evolution of this important protein family (>500

millions years?)

3.This molecular information should facilitate the design of more sensitive antifungals.

COLLABORATORS

OTAGO

• MONK brian• CANNON richard• NIMMI kyoko• LAMPING erwin

LLN/GENA

• BARET philippe• DE HERTOG benoit • DIFFELS julie• SERET marie line

LLN/FYSA

• BALZI elizabetta• KOLASCZKOWSKI marcin

SAN DIEGO• SAIER milton

ACKNOWLEDGEMENTS

BIBLIOGRAPHY REVUES PDR

Monk B.and Goffeau A. 2008, SCIENCE

321:367-369

Richard Cannon et al. 2009,CLINICAL MICROBIOLOGY REVIEWS

22:291-321

Homo sapiens

ZYGOMYCETESRhizopus oryzae

CHYTRIDS

Cryptococcus neoformans

Pneumocystis carenii

Saccharomyces cerevisiaeCandida glabrata

Candida parapsilosisCandida tropicalisCandida dubliniensisCandida albicans

Candida lusitaniaeCandida guillermondii

Aspergillus nidulansAspergillus fumigatus

Aspergillus oryzae

Aspergillus terreus

Coccidioides immitis

Histoplasma capsulatum

Chytridium comfervae

Candida Cluster

Saccharomyces Cluster

Pneumocystis Cluster

Coccidoides-Histoplasma Cluster

Cryptococcus Cluster

1000 800 600 400 200 0

Aspergillus Cluster

Phylogeny of human fungal pathogens

WGD

GTG

Millions of years

Snq2p in Hemiascomycetes

WA12924WA12921

TH11581TH11579TH11568TH11564

RO1395RO1393RO1392RO1387

WA12920

TH11563

RO1386RO1385

WA12917

TH11559

WA12916

TH11558

RO1382

WA12069WA12062

K. thermotolerans

Z. rouxii

K. waltii

KLLA0D03432gKLLA0D03454gKLLA0D03476gKLLA0D03498gKLLA0D03520gKLLA0D03542gK. lactis