Pierre Colas
Yeast Two-‐Hybrid Methods to Discover, Analyze and Modulate Protein Interac>ons Pierre Colas CR INSERM The Yeast Two-‐Hybrid (Y2H) System First sketch of the Y2H assay, submiCed in a grant applicaHon (1987) from Stanley Fields (2009) Proteomics • 13,000 Medline entries • Work horse to build (sub-‐)proteome scale interactomes: Human, animal models (Drosophila, C.elegans), Bacteria, Viruses, … The Y2H System “Prey”! A D Z Interaction X “Bait”! D B Blue! on X-Gal! lacZ / LEU2 Phenotype Growth without ! Leucine! A Number of Y2H Methods from Suter et al. (2008) Curr. Opin. Biotechnol. Cyclin-‐Dependent Kinases (CDKs) Cell cycle TranscripHon AlternaHve splicing Cycle + Apoptosis Cycle + Splicing from Malumbres & Barbacid (2009) Nature reviews Cancer & Satyanarayana et al. (2009) Oncogene Cyclin-‐Dependent Kinases (CDKs) Cyclins A1 A2 B1 B2 B3 E1 E2 D1 D2 D3 p25 C L H K T1 T2a from Malumbres & Barbacid (2009) Nature reviews Cancer & Satyanarayana et al. (2009) Oncogene T2b CDKs interact with mulHple protein partners AcHvaHng proteins Cyclins CKS TranscripHon factors CDKs CKIs (Ink4, Cip/Kip) PhosphorylaHon substrates Cyclin-‐Dependent Kinases (CDKs) Cyclins A1 A2 B1 B2 B3 E1 E2 D1 D2 D3 p25 C L H K T1 T2a from Malumbres & Barbacid (2009) Nature reviews Cancer & Satyanarayana et al. (2009) Oncogene T2b CDK10: a mysterious CDK… • Discovered as early as 1994 -‐ ≈20 Medline hits • Suspected role in the G2/M transiHon (anH-‐sense; dn mutant) • Interacts with ETS2 oncoprotein and inhibits its transcripHonal acHvity • RNAi-‐mediated knockdown of CDK10 -‐> Confers Tamoxifen resistance to breast tumor cells -‐> AcHvates MAPK pathway -‐> Increases c-‐RAF (MAPKKK) expression -‐> De-‐represses ETS2 (which controls c-‐RAF expression) • CDK10 expression level: biomarker to predict breast tumor response to endocrine therapy ? • Recent reports suggest that CDK10 would be a tumor suppressor in hepatocellular carcinomas & billiary tract cancers DeorphanizaHon of CDK10 • Y2H screens (interactome walking) by M. Flajolet (Rockefeller U., NY): -‐> interacHon Mm CDK10 – Mm « Cyckel » • No strict human ortholog of Cyckel, but very strong sequence homology between Cyckel C-‐ter region and FAM58A protein • Strong Y2H interacHon phenotype between Hs CDK10 et Hs FAM58A • Hs FAM58A sequence bears a cyclin signature -‐> Cyclin M (nomenclature proposed by A. Murray aoer human genome annotaHon) • Cyclin M, orphan protein, is even more mysterious than CDK10, but… FAM58A mutaHons cause STAR syndrome • STAR: syndactyly, télécanthus, ano-‐genital, renal, craniofacial anomalies, … • Heterozygous mutaHons (X chromosome): deleHons of first exons, mutaHons on splice sites or frameshio mutaHons -‐> N-‐ and C-‐ter truncaHons of Cyclin M Unger et al. 2008 Nature GeneKcs Can STAR-‐causing CycM mutant forms interact with CDK10 ? Y2H maHng assays to analyze protein interacHons a haploid α haploid Empty plasmid Bait 1 Bait 2 Bait 3 a/α diploid Y2H maHng assays between CDK10 and CycM isoforms The CDK10-‐CycM interacHon does not exist in STAR syndrome paHents from Guen et al. 2013 PNAS CDK10 is a Cyclin-‐Dependent Kinase from Guen et al. 2013 PNAS CDK10/CycM: major findings • CDK10/CycM phosphorylates ETS2 in vitro • CycM stabilizes CDK10 in cells -‐> is CDK10 monogamous ? • CycM silencing acHvates the MAPK pathway and causes tamoxifen resistance to breast tumor cells • CDK10/CycM posiHvely controls ETS2 degradaHon by the proteasome • STAR paHent-‐derived cells present higher ETS2 protein levels from Guen et al. 2013 PNAS CDK10/CycM: an interesHng case study to list truisms • Despite (sub-‐) proteome scale efforts to build interactomes, “important” interacHons involving “important proteins” remain to be discovered • Interactome databases contain a wealth of precious informaHon and are under-‐exploited: the Cdc2rk/koko interacHon had been scored in Drosophila • The discovery of (some) protein interacHons is sHll probably the best way to understand protein funcHon • Y2H maHng assays offer an easy, straighuorward approach to link loss (or gain) of protein interacHon to diseases -‐> EdgeKcs EdgeHcs and Human Diseases ~ Half of the 50 000 mutaHons causing human diseases induce edgeHc perturbaHons (as opposed to nodal perturbaHons) from Zhong et al. 2009 Mol Syst Biol CDK2 WT L124D T182D 204-‐210 R50A I49A R157A R126A W167A K33A A31M E162G V156D V163S K88A A95D pEG202 Apt.C5 CKS1 Cyclin D Apt.8 Apt.10M p27 Apt.19 recognizes a surface on CDK2, in the vicinity of the patch delimited by K88, A95, W167 Apt.19 Apt.16 Apt.17 Y2H maHng assays to map protein binding sites • ATP cannot bind to this mutant • Apt.19 can bind to this mutant (not Apt.17) -‐> Apt. 19 binds close to the ATP site but does not « plunge » into it CDK2 WT L124D T182D 204-‐210 R50A I49A R157A R126A W167A K33A A31M E162G V156D V163S K88A A95D pEG202 Apt.C5 CKS1 Cyclin D Apt.8 Apt.10M p27 • M31 fills up the boCom of the ATP binding site Apt.19 Apt.16 Apt.17 Y2H maHng assays to map protein binding sites Screening of protein interacHon inhibitors by Y2H “Prey”! A D “Prey”! A D Y Y Interaction X “Bait”! X “Bait”! D B lacZ / LEU2 Interaction! inhibited D B lacZ / LEU2 Phenotype : blue on X-Gal /! Growth without Leucine! Phenotype : white on X-Gal /! No growth without Leucine! AptaScreen: a Y2H duplex screening assay ruc luc No Hit luc Toxic Hit Act Bait1 Prey 1 LexA Bait 2 Prey 2 CDKs interact with mulHple protein partners AcHvaHng proteins Cyclins CKS TranscripHon factors CDKs CKIs (Ink4, Cip/Kip) PhosphorylaHon substrates CKS Proteins: Important Regulators of CDK1/2 • Cell cycle regulaHon • Key role in Cyc A and p27Kip1 destrucHon • Overexpression-‐> suppression of S phase checkpoint • InteracHon with mtSSB, to control replicaHon of mitochondrial DNA Bourne et al. (1996) • Overexpression of CKS1 detected and associated to poor prognosis in mulHple cancers: breast, colon/rectum, prostate , kidney, non-‐small cell lung carcinomas • Knockout of CKS1 -‐> -‐ Decreased cell proliferaHon, migraHon and invasion -‐ Increased apoptosis Hit A selecHvely inhibits CDK/CKS interacHon phenotypes Hit B selecHvely inhibits CDK/CKS interacHon phenotypes Molecules A and B inhibit CDK2-‐CKS1 interacHon in vitro CKS1 CKS1 CDK2 DMSO CKS1 CDK2 Molecules pre-‐incubated CDK2 Preformed complex IdenHficaHon of acHve and inacHve close analogs Microscale Thermophoresis: QuanHficaHon of CDK2 binding from NanoTemper Technologies website A series Kd (µM) Molecule type B series Kd (µM) A 28.3 +/-‐ 3 Hits B 9.6 +/-‐ 0.2 A1 32.5 +/-‐ 6.7 AcHve analogs B1 17.8 +/-‐ 2.8 A2 287 +/-‐ 8.5 InacHve analogs B2 undetected Specificity of CDK/CKS interacHon inhibitors CKS1 GST GST CDK2 CDK2 CKS2 Specificity of CDK/CKS interacHon inhibitors CycA GST CDK2 Molecular Docking of Molecules A and B on CDK2 A! Cyclin! ATP-! binding site! Mol. A! Allosteric site A! CDK2! CKS1! Mol. B! Allosteric site B! B! C! Mol. A! CDK2! V164! W167! V163! L166! R200! CKS1! F203! CDK2! Q246! D206! Mol. B! AutoDock 4 CKS1! Inhibitors of CDK/CKS interacHon inhibit cell proliferaHon A-‐series MTS assay on MCF-‐7 breast tumor cell line B-‐series CDK/CKS interacHon inhibitors decrease CDK2 and CycA2 levels CDK/CKS interacHon inhibitors decrease p27KIP1 levels Skp1 Cul1 Roc1 Skp2 Skp1 Ub Cdc34 CKS1 Cyc E/A CDK2 P p27 Skp2 P Cul1 Roc1 CKS1 Cdc34 Ub Skp1 Cul1 Roc1 Skp2 Ub Cdc34 CKS1 Cyc E/A CDK2 Ub Ub Ub Ub AptaScreen: a proven Y2H duplex screening assay • Screening against CDK2-‐Apt.19 interacHon -‐> discovery of a potent CDK2 small-‐molecule inhibitor (Aptanomics SA, unpublished) • Screening against A20-‐D4 and A20-‐D5 protein interacHons, essenHal for vaccinia virus DNA replicaHon -‐> discovery of two molecules inhibiHng the replicaHon of orthopoxviruses (Flusin et al. 2012 AnKviral Research) • Screening against CDK2-‐CKS1 interacHon -‐> discovery of two series of small-‐ molecule inhibitors that inhibit cell proliferaHon (Hamdi et al. SubmiXed) B B A A DBD luc X ACT ACT Y Firefly luminescence ACT DBD ruc Renilla luminescence from Hamdi & Colas (2012) Trends Pharmacol Sci Forward duplex assay DBD luc X Y No Firefly luminescence ACT DBD ruc Renilla luminescence Y2H screening assays to discover inhibitors of protein interacHons B B ACT ACT A DBD reporter B Forward assays Growth Blue Luminescence DBD reporter B ACT Reverse assays A DBD reporter B A ACT DBD reporter DBD reporter Repressed transacHvator assay A ACT A No Growth REP No Growth from Hamdi & Colas (2012) Trends Pharmacol Sci No growth White No light B Growth REP A ACT DBD reporter Growth Y2H screening assays to discover inhibitors of protein interacHons from Hamdi & Colas (2012) Trends Pharmacol Sci ApplicaHons of Y2H methods Y2H methods Fundamental Biology Therapeu>c Research Systems Biology PPI discovery Y2H screening of cDNA libraries Understanding protein funcHon Target idenHficaHon InteracHon networks PPI analysis Y2H maHng assays IdenHficaHon of loss & gain of funcHon mutaHons Molecular eHology of diseases EdgeHcs PPI modulator discovery Y2H screening of Chemical Biology tools chemical libraries Target validaHon; EdgeHc TherapeuHc perturbaHons candidates on networks Combinatorial protein binders Y2H screening of CP libraries Understanding protein funcHon Target validaHon; Guides to small-‐ molecule drugs EdgeHc perturbaHons on networks Iden>fica>on of Y3H screening of small-‐molecule cDNA libraries targets Assessing specificity of chemical biology probes Assessing specificity of drug candidates -‐ ApplicaHons of Y2H methods Y2H methods Fundamental Biology Therapeu>c Research Systems Biology PPI discovery Y2H screening of cDNA libraries Understanding protein funcHon Target idenHficaHon InteracHon networks PPI analysis Y2H maHng assays IdenHficaHon of loss & gain of funcHon mutaHons Molecular eHology of diseases EdgeHcs PPI modulator discovery Y2H screening of Chemical Biology tools chemical libraries Target validaHon; EdgeHc TherapeuHc perturbaHons candidates on networks Combinatorial protein binders Y2H screening of CP libraries Understanding protein funcHon Target validaHon; Guides to small-‐ molecule drugs EdgeHc perturbaHons on networks Iden>fica>on of Y3H screening of small-‐molecule cDNA libraries targets Assessing specificity of chemical biology probes Assessing specificity of drug candidates -‐ 25 years of Y2H to explore protein interacHons Stanley Fields , 1987 CDK10/CycM Vincent Guen Carly Gamble Marc Flajolet Yoan Ferandin Aurélie Thollet Pascale Cohen Sheila Unger Andrea SuperH-‐Furga CDK/CKS Amel Hamdi Thomas Robert Aurélien Lesnard Peggy Suzanne Sylvain Rault Bruno Didier Annelise Lobstein Marcel Hibert Maria Miteva Morgan Pellerano May Morris AptaScreen AptaPrint Christophe Borie Carine Bardou Marc Bickle Mathieu Schapira Yeast Two-‐Hybrid Methods to Discover, Analyze and Modulate Protein Interac>ons Pierre Colas CR INSERM
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