PLEIOTROPIC DRUG TRANSPORTERS in Fungi Andr Goffeau, Bruxelles, - - PowerPoint PPT Presentation

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PLEIOTROPIC DRUG TRANSPORTERS in Fungi Andr Goffeau, Bruxelles, - - PowerPoint PPT Presentation

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PLEIOTROPIC DRUG TRANSPORTERS in Fungi Andr Goffeau, Bruxelles, 2012 PDR RANK & BECH-HANSEN MGG 1973 PDR1 BALZI, CHEN ULASZEWSKI, BOUTRY, DI PIETRO JBC 1987 PDR5 GOLIN 1992 PDR5 BALZI et al , JBC 1994 ASYMETRIC NBDs

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SLIDE 1

PLEIOTROPIC DRUG TRANSPORTERS in Fungi André Goffeau, Bruxelles, 2012

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SLIDE 2
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SLIDE 3

PDR RANK & BECH-HANSEN MGG 1973

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SLIDE 4

PDR1 BALZI, CHEN ULASZEWSKI, BOUTRY, DI PIETRO

JBC 1987

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SLIDE 5
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SLIDE 6
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SLIDE 7
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SLIDE 8

PDR5

GOLIN 1992

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SLIDE 9

PDR5

BALZI et al, JBC 1994

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SLIDE 10
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SLIDE 11

Walker A Linker region Walker B

SGGQKQRIAIA GNSGCGKST

ASYMETRIC NBD’s in Pdr5p

LLLDE

NBD2 NBD1

GRPGSGCTT * * * QCWDN * SGGERKRVSIA *** *** **

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SLIDE 12

Yor1 Yor1 Bpt1 Bat1 Yhl035 Yhl035 Ycf1 Ycf1 Ste6 Ynr070 Yor011 Pdr11 Pdr11 Ykr103/4 Yol075 Pdr12 Snq2 Snq2 Pdr10 Pdr10 Pdr15 Pdr15 Pdr5 Pdr5

MULTIPLE disruptions SINGLE disruptions

PDR5 PDR5 -

  • like

like NEW NEW MDR MDR

  • like

like MRP MRP -

  • like

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

The 16 "full - size" ABC transporters

Bat1 Ste6 Ynr070

MULTIPLE disruptions MULTIPLE disruptions SINGLE disruptions

PDR5 PDR5 -

  • like

like NEW NEW MDR MDR -

  • like

like MRP MRP -

  • like

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

NO disruptions yet

The 16 "full - size" ABC transporters

Yor011 Yhl035 Yhl035 Pdr12 Pdr10 Pdr10 Yol075 Pdr11 Pdr11 Snq2 Snq2 Pdr15 Pdr15 Pdr5 Pdr5 Yor1 Yor1 Ykr103/4 Bpt1 Ycf1 Ycf1 Ste6

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SLIDE 13

ATP

PDR-like: Pdr5p MRP/MDR-like: Yor1p

ATP ATP ATP

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SLIDE 14

pdr1-3

CARVAJAL et al MGG 1997

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SLIDE 15
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SLIDE 16
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SLIDE 17

The pdr1 The pdr1-

  • 3

3 mutant mutant overexpresses

  • verexpresses

a new protein band in the plasma membrane a new protein band in the plasma membrane

Decottignies et al , 1997

160kDa Pma

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SLIDE 18

GENETIC PURIFICATI GENETIC PURIFICATION ON

Snq2p Snq2p Pdr5p Pdr5p Yor1p Yor1p Δpdr5 Δ snq2 Δ pdr5,Δ snq2

160 kDa

Pma1p

Decottignies et al., 1997

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SLIDE 19

The mutant The mutant pdr1 pdr1-

  • 3

3 in the in the regulatory regulatory gene gene PDR PDR1 1 activates activates expression of expression of several several ABC ABC transporters transporters

PDR1 PDR5 SNQ2 YOR1 Others ?

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SLIDE 20

Elements of the Elements of the PDR Network PDR Network responsive responsive to the to the pdr1 pdr1-

  • 3

3 Transcription Transcription Regulator Regulator De RISI et al, De RISI et al, 2002 2002

> >

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SLIDE 21

GENE TARGETS OVEREXPRESSED GENE TARGETS OVEREXPRESSED BY THE pdr1 BY THE pdr1-

  • 3 DRUG

3 DRUG-

  • RESISTANCE REGULATORY MUTATION

RESISTANCE REGULATORY MUTATION

  • 1. ABC
  • 2. MFS
  • 3. LIPIDS
  • 4. WALLS
  • 5. STRESS
  • 6. UNKNOWN

PDR5 / SNQ2 / YOR1 / PDR10/ PDR15 TPO1 / RTA1 / YOR049c PDR16 / IPT1 / YLR099c YPL088w / YGL028c / YLR413w / YNR067c YLR346c / YGR035c / COS1

De Risi, van den Hazel et al., 1999

GRE2 / MET17 / FRE4 / REV1 / YGR243w

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SLIDE 22

PDR3/YAP1/YRR1

  • JULIUS SUBIC
  • CLAUDE JACQ
  • BERNARD TURCOTTE
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SLIDE 23

PDR Network

  • C. Jacq -
  • F. Devaux –

E.N.S

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SLIDE 24

TRANSPORT SPECIFICITY

  • f Pdr5p

Kolaszkowski et al JBC 1996

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SLIDE 25

PDR5 PDR5 Δ

Antibiotics

chloramphenicol tetracycline anisomycin antimycin A1 cycloheximide lincomycin

  • ligomycin

tunicamycin thiolutin

Steroids and their structural analogues

5 β androstan 3 α

  • l

17 one 5 β androstan 3 β

  • l

17 one 5 β androstan 17β

  • l

3 one 5 α androstane 3,17 dione 5 β androstane 3,17 dione 1,4 androstadiene 3,17 dione 5 β pregnan 3 β ol 20 one 4 pregnen 20 α ol 3 one trans dehydroandrosterone 4 androstene 3,17 dione epiandrosterone acetate epiandrosterone estriol 16, 17 diacetate 17 α ethynyl estradiol deoxycorticosterone pregnenolone progesterone testosterone diethylstilbestrol hexestrol 4 hydroxytamoxifen

Ionophores

valinomycin nigericin monensin A23187

Others

2,3,5 triphenyltetrazoliumchloride 4 nitroquinoline n oxide 8 hydroxyquinoline diphenylamine p aminodiphenylamine n decylamine 4 nitrophenol aniline anthron tetranitrotetrazoliumchloride blue resazurine sporidesmin staurosporine fluphenazine cerulenin compactin

Anticancer drugs chemosensitizers

tamoxifen daunorubicin doxorubicin trifluoperazine

Detergents

tetradecyltrimethyl-

  • ammonium bromide

hexadecyltrimethyl-

  • ammoniumbromide

zwittergent 3-10 zwittergent 3-12 zwittergent 3-14 brij 58 brij 35 n octylglucoside n dodecylglucoside n dodecylmaltoside

Fungicides

itraconazole ketoconazole econazole miconazole triadimenol bitertanol nuarimol fenarimol carbendazim diclobutrazol phenapronil chlorothalonil cyprodinil dodine imazalil prochloraz azoxystrobin flusilazol tebuconazole CGA64251 2 mercapto-

  • benzothiazole

krezoxim methyl nystatin soraphen A epoxiconazole

Herbicides

sulfomethuron methyl fluometuron chloroxuron difenoxuron chlorbromuron diuron

Fluorescent dyes

rhodamine 6G rhodamine 123

THE YEAST ABC TRANSPORTER PDR5 MEDIATES RESISTANCE TO MANY DIFFERENT COMPOUNDS

Kolaczkowski et al., 1997

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SLIDE 26

Membranes with Membranes with overexpressed

  • verexpressed

Pdr5p Pdr5p show ATP show ATP-

  • dependent

dependent fluorescence quenching of fluorescence quenching of Rhodamine Rhodamine 6G 6G

GWENAELLE CONSEIL,2003 GWENAELLE CONSEIL,2003

fluorescence intensity

time [min]

8 6 4 2

PDR5 deleted

ATP

PDR5 overexpressed

  • ligomycin

10 20 30

Kolaczkowski et al., 1997

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SLIDE 27

Competitive Competitive inhibition of inhibition of prenyl prenyl-

  • flavonoids

flavonoids

  • n rhodamine 6G
  • n rhodamine 6G

interaction interaction

Ki (chrysin) = 2.7 µM Ki (galangin) = 1.1 µM Ki (6,6-DMA-galangin) = 0.18 µM

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SLIDE 28

Partial and non Partial and non competitive competitive inhibition of the inhibition of the nucleotide nucleotide hydrolytic hydrolytic activity activity

  • f
  • f

Pdr5p Pdr5p-

  • enriched

enriched yeast yeast plasma membranes plasma membranes

Chrysin derivatives

Ki (6-DMA-galangin = 5.4 µM Ki (6-DMA-galangin = 62 µM

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SLIDE 29
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SLIDE 30

HETEROLOGOUS EXPRESSION OF MEMBRANE PROTEINS A.DECOTTIGNIES/B.MONK

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SLIDE 31

MULTIPLE DELETIONS OF YEAST MULTIPLE DELETIONS OF YEAST ABC TRANSPORTERS AND REGULATORS ABC TRANSPORTERS AND REGULATORS

AD123456789 AD123456789

Δ YOR1 Δ PDR15 Δ PDR1 Δ PDR3 Δ PDR11 Δ YCF1 Δ PDR10 Δ PDR5 Δ SNQ2

AD12345678 AD12345678 AD1234567 AD1234567 AD123456 AD123456 AD12346 AD12346 AD1234 AD1234 AD123 AD123 AD12 AD12 AD1 AD1

pdr1-3

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SLIDE 32

HYPERSENSITIVITY TO DRUGS OF THE YEAST STRAIN AD1 HYPERSENSITIVITY TO DRUGS OF THE YEAST STRAIN AD1-

  • 9,

9, DELETED OF MULTIPLE ABC TRANSPORTERS DELETED OF MULTIPLE ABC TRANSPORTERS

INHIBITOR INHIBITOR WILD WILD-

  • TYPE

TYPE AD1 AD1-

  • 9

9 Fold-

MIC ( MIC (µ µg/ml) MIC ( g/ml) MIC (µ µg/ml) g/ml) Difference

Brefeldin A 100 6.25 16 Clotrimazole 1.56 0.0012 128 128 Crystal Violet 0.073 0.049 1.5 Cycloheximide 0.049 0.012 4.1 Fluconazole 12.5 0.391 32 Fluphenazine 12.5 6.25 2 8-Hydroxyquinoline 25 6.25 4 Imazalil 0.78 0.025 31 Ketokonazole 0.781 0.006 130 130 Miconazole 0.391 0.0031 126 126 Nocodazole 6.25 0.781 8 Propiconazole 1.56 0.003 5.2 Rhodamine 6G 25 0.098 255 255 Sanguinarine 3.13 0.781 4 Staurosporine 0.781 0.024 33 Sulfometuron methyl 6.25 1.56 4 Thiabendazole >100 14 7.2

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SLIDE 33
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SLIDE 34

STRAINS made by Anabelle Decottignies and Elvira Carvajal STRAINS curated by Stan Ulaszewski and Michel Ghislain michel.ghislain@uclouvain.bee

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SLIDE 35

Yor1p

Pma1

PDR5p

pdr1-3

YEAST PDR AS A SYSTEM FOR YEAST PDR AS A SYSTEM FOR OVEREXPRESSION OF MEMBRANE PROTEINS OVEREXPRESSION OF MEMBRANE PROTEINS

  • Regulatory

pdr1 mutant

  • Membrane protein

gene under PDR5 promoter

  • Host deleted of major contaminant

ABC transporter genes (PDR5, SNQ2, YOR1, PDR11) YOR1

Decottignies et al., 1997

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SLIDE 36
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SLIDE 37

Decottignies et al., 1997

Pma1p

Yor1p

Δ ABC

pdr1-3 pdr1-3 YOR1 PDR5 pr::YOR1

OVERPRODUCTION OF YOR1p OVERPRODUCTION OF YOR1p

OLIGOMYCIN RESISTANCE

0.1

4 30 µg/ml

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SLIDE 38

MEMBRANE TRANSPORTERS OVEREXPRESSED IN S.CEREVISIAE

  • CaCDR1p
  • CaCDR2p
  • CgCDR1p
  • CgCDR2p
  • ABCA1
  • BenRp

ABC MFS

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SLIDE 39

SUPER CDR1 and Super PDR5 SUPER CDR1 and Super PDR5

in in S.cerevisiae S.cerevisiae plasma membranes plasma membranes

MULTIDELETIONS MULTIDELETIONS SUPER CDR1 SUPER CDR1 SUPER PDR5 SUPER PDR5

B.MONK 2001

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SLIDE 40

EM structural analysis

  • f the S.cerevisiae

Pdr5p

MARCO/RIGAUD

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SLIDE 41

PURIFIED Pdr5p PARTICLES

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Pdr5p Particles Projection

50 Å

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SLIDE 43

A B A B A B A B

Dimeric Pdr5p

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ATP binding requires two « head to tail » subunits

A B B A C C A B B A C C

subunit 1 subunit 2

Walker motifs

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Pgp structure, Chang et al ,2009

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SLIDE 46

PHYLOGENY

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SLIDE 47

0.1

YALI005PDR YALI019PDR YALI007PDR YALI009PDR NCU05591.2 KLLA011PDR ASGO002PDR ASGO012PDR DEHA007PDR DEHA017PDR DEHA018PDR DEHA019PDR DEHA013PDR DEHA024PDR DEHA025PDR KLLA017PDR PDR10 CAGL017PDR PDR5 CAGL009PDR PDR15 SCPO012YNR SCPO006YNR NCU09830.2 NCU08056.2 NCU10009.2 YALI024PDR KLLA004PDR CAGL019PDR PDR12 DEHA001PDR ASGO001PDR KLLA010PDR YNR070W CAGL011PDR SNQ2

Neurospora crassa Schizo pombe Yarrowia lipolytica Debaryomyces hansenii Ashbya gossypii Kluyveromyces lactis Candida glabrata Saccharomyces cerevisiae

Neurospora S Pombe SNQ2 PDR5

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SLIDE 48

Conclusion We can now envisage

  • crystallization
  • f each

catalytic step

  • f Pdr5p
  • verexpression, purification, crystallization

and phenotypic caracterization

  • f each

subfamily

  • f

fungal and plant’s Pdr

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SLIDE 49
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SLIDE 50

LYON

  • DI PIETRO attilio
  • JAULT jean-michel

OTAGO

  • MONK brian
  • CANNON richard
  • NIMMI kyoko

WROCLAW

  • ULASZEWSKI stan

LLN/GENA/GENE

  • BARET philippe
  • DE HERTOG benoit
  • DIFFELS julie
  • HANCY frederic

PARIS ENS

  • JACQ

claude

  • MARC philippe
  • DEVAUX frédéric

IOWA

  • MOYE-ROWLEY

scott

ODENSE

  • FEY stephen
  • MOSE LARSEN peter
  • NAWROCKI arkadiusz

PARIS Curie

  • RIGAUD jean-louis
  • MARCO sergio

COLLABORATING LABORATORIES

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SLIDE 51

STILL GOING ON

Degradation

  • f Pdr5p by GHISLAIN michel

Control by kinases and phosphatase by MONTERO monica Crystallisation

  • f Pdr5p by XIA di

Plant Inhibitors

  • f Pdr5p by PEIRERA antonio

Crystallisation

  • f Yor1p by Falson

pierre Vmr1p a novel vacuolar yeast MRP1 by WAWRZYCKA donata Plant PDRs by BOUTRY marc et al