Molekulární chaperony a jejich úloha v patogenezi lidských chorob Petr Muller The native structure is determined only by the protein's amino acid sequence Christian Boehmer Anfinsen, Jr. (March 26, 1916 – May 14, 1995) Nobel Prize in Chemistry (1972) At the environmental conditions (temperature, solvent concentration and composition, etc.) at which folding occurs, the native structure is a unique, stable and kinetically accessible minimum of the free energy Entropy Hydratation Folding is entropy driven process Autophagy Ubiquitin proteasome system Chaperones Protein homeostasis / proteostasis Stress proteins / Chaperones Foldases are chaperones that accompany other proteins to help them to overcome the energy barriers during folding to native conformation (ATP dependent) Hsp70, Hsp90, GroEL… Holdases bind folding intermediates to prevent their aggregation Crystalins, p23, Hsp40… Approximate molecular weight(kDa) Prokaryotic proteins Eukaryotic proteins Function 10 kDa GroES Hsp10 20-30 kDa GrpE The HspB group of Hsp. Eleven members in mammals including Hsp27, HSPB6 or HspB1[28] 40 kDa DnaJ Hsp40 Co-factor of Hsp70 60 kDa GroEL, 60kDa antigen Hsp60 Involved in protein folding after its post-translational import to the mitochondrion/chloroplast 70 kDa DnaK The HspA group of Hsp including Hsp71,Hsp70, Hsp72, Grp78 (BiP), Hsx70 found only in primates Protein folding and unfolding, provides thermotolerance to cell on exposure to heat stress. Also prevents protein folding during post-translational import into the mitochondria/chloroplast. 90 kDa HtpG, C62.5 The HspC group of Hsp including Hsp90, Grp94 Maintenance of steroid receptors and transcription factors 100 kDa ClpB, ClpA, ClpX Hsp104, Hsp110 Tolerance of extreme temperature Ubiquitin-like Crystallins Small Hsps Prevent aggregation Thermotolerance Hsp27 HspB group/ small chaperones ATP ADP Hsp70 (DnaK, Grp78,..) chaperone machinery BAG NEF-Nucleotide exchange factor Hsp40 DnaJ J-proteins Chaperonins (GroEL-GroES, Hsp60, CCT-TRiC) Folding of cytoskeletal proteins (tubulin) Protein transport AAA+ proteases Proteasome Hsp104 Converts ATP to “mechanical” energy (molecular motors) Hsp104 (ClpB, ClpX,..) AAA+ ATPases Thermotolerance Aggregate refolding Prion folding (yeast Psi+/-) Hsp90 chaperone machinery • Conserved from procarytes to mammals • ATPase aktivity (like gyrase) • Mitochndrial, ER, cytoplasmic • Redundant isoformes Stress proteins/ Chaperones/Hsp90 Genetic instability Enhanced proteosynthesis Production of mutated, conformational instable protins The tumor cells demand high quality and amount of protein Hanahan D, Weinberg RA.: Cell. 2000 Jan 7;100(1):57-70. CDK4/CyclinD AKT Tyrosin kinase receptors VEGF, HIF hTRT BRAF MMP HSP90 client proteins Activity of Hsp90 is essential for expression of cancer phenotype Specific inhibitors Hsp90 inhibitor No of studies phase Company 1 tanespimycin (17AAAG) 36 III Bristol-Myers Squibb, Kosan 2 retaspimycin (IPI-504) 11 II/III* Infinity Pharmaceuticals 3 alvespimycin (17DMAG) 7 II Bristol-Myers Squibb, Kosan 4 STA-9090 14 II Synta Pharmaceuticals Corp. 5 AUY922 11 II Novartis Pharmaceuticals 6 CNF2024 (BIIB021) 7 II Biogen Idec 7 SNX-5422 4 I Pfizer, Serenex, Inc. 8 AT13387 3 I Astex Therapeutics 9 KW-2478 2 I/II Kyowa Hakko Kirin Pharma, Inc. 10 IPI-493 2 I Infinity Pharmaceuticals 11 HSP990 2 I Novartis Pharmaceuticals 12 MPC-3100 1 I Myrexis Inc. 13 Debio 0932 1 I Debiopharm S.A. 15 BIIB028 1 I Biogen Idec Isolation of Geldanamycin (1970) Geldanamycin binds ATP cavity of Hsp90 (1997) Clinical trials with Geldanamycin(2000) Hsp90 is unique therapeutic target for anti-cancer therapy more than 17 different molecules in clinical trials Variable response need for predictive markers Different assembly of Hsp90 machinery ? • posttranlational modifications • expression pattern of co-chaperones Client spectrum ? What does kill the cells: • apoptosis, aggregation, …. ATPATP N N C MD MD Client cdc37 p23 SGT1 C HOP Hsp70 Hsp90 Hsp90 C Multichaperone complex • Hsp90+Hsp70 • cochaperones TPR TPR C HSP70 HSP90 C p53 p53 HSP70 p53 U-Box U CHIP U U U Folding Stabilisation Degradation G1/G0 Nascent p53 proliferation TPR Hsp90 inhib. HOP What is the mechanism regulating folding degradation balance ? C C Strep B Hsp70/90 P Strep B Hsp70/90 CHIP HOP Cell lysate pulldown of HOP and CHIP • Biotinylated phospho/non phospho peptides of Hsp70/Hsp90 competition Lysate Biotin C-Hsp70 C-Hsp70phospho C-Hsp90a C-Hsp90aphospho HOP CHIP Actin HOP Hsp70 phospho GGS2.1 Hsp70 Hsp90 phospho GDD8.2 Hsp90 Control CIP Control CIP Serum starvation T N T N T N T N T N T N T N T N T N T N T N Hsp70 Hsp70 phos Hsp90a Hsp90a phos HOP CHIP Actin PCNA Detection of phosphorylated Hsp70 and Hsp90 phospho-specific monoclonal antibodies antibodies HSP70 HSP90CC PP GGS2.1 GGS8.2 HSP90 C U-Box U CHIP U U U Degradation of unstable proteins HSP70 C HSP70 HSP90C C Stabilization of mutated p53 P P HOP Oncogenic signalization Fosforylace Hsp70 / Hsp90 Normal differentiated cell Cancer cell Normal differentiated cell Cancer cell C-terminus Hsp70/90 non phosphorylated Phosphorylated Hsp90 Hsp70 Hsp bind preferentially CHIP Hsps bind preferentially HOP Designed to degrade unfolded protein High folding capacity of Hsp90 Higher expression of CHIP Increased level of HOP Lower sensitivity ti Hsp90 inhibitors High sensitivity to Hsp90 inhibitors TPR1 TPR2 Positively charged clamp TOMM34 protein – co-chaperone Tetratricopeptide repeat (TPR) domain TPR1 TPR2 De novo modeled structure of TOMM34 domains C-terminus Hsp70/Hsp90 EEVD HSP70 HSP90 TPR1 TPR2 N C C N TPR1 TRR2 Tomm34 - + - + - + Hsp70 Hsp90 TPR1 TPR2 FL Tomm34 HDX – basic basics Exchangeable hydrogens: 1) side chains containing –OH, – SH, –NH2, –COOH and –CONH2 groups and hydrogens from the amino and carboxy termini 2) carbon-bound aliphatic and aromatic hydrogens 3) hydrogens arising from the amide linkages between amino acids of the protein polypeptide chain HDX – HSP70/90-TOMM34 interaction without ATP TOMM34 HSP70 HSP90 ATP ADP ATP ATP EEVD ADP ADP Strep. Dynabeads SBP ATP HSP70 EEVD TPR2 The effect of ATP on HSP70 – Tomm34 interaction ADP ATP HDX – HSP70-TOMM34 interaction with ATP VEDE HSP70-TOMM34 interaction with ATP, the role of HSP40 TPR1 TPR2 C N ATP HSP70 TPR1 TPR2 C C N ADP HSP40 Cooperation of Hsp70/Hsp90 folding by Tomm34 Tomm34 Tomm34 Tomm34 Hsp70 binds TPR1 strongly at ATP bound state, client is released ATP hydrolysis releases Hsp70 from Tomm34 Hsp90 binds TPR2 at client free state Client binding to Hsp90 disrupt its interaction with Tomm34 Tomm34 nucleotide free Hsp70 binds TPR1 weakly through EEVD domain HSP40 Hsp90 client proteins Is there any structural/motif motif recognized by Hsp90 ? N N NATP ADP 17AAG 17AAG inhibition Degradation No consensus sequence of Hsp90 clients Ligand promotes conformational stabilization of steroid receptors Lysate •Proteins •DNA, RNA •ATP •Detergents •Lipids •LMW compounds Benzonase Gel filtration 0.5% CHAPS SBP-HSP90 SBP-HSP70 HSP90 Control4°C H1299 Lenti SBP-Hsp70 H1299 Lenti SBP-Hsp90 37°C +ATP +ATP +ATP HSP90 N N N N NATP ADP HSP90 MoO4 - 17AAG Functional proteomic to study Hsp90 complexes 0 1 2 3 4 5 1 2 3 4 5 6 HOP 0 0,5 1 1,5 1 2 3 4 5 6 HSP90b 0 0,5 1 1,5 1 2 3 4 5 6 HSP90a 0 1 2 3 4 1 2 3 4 5 6 RAS 0 1 2 3 4 5 6 1 2 3 4 5 6 Actin 0 1 2 3 4 1 2 3 4 5 6 CHIP Molybdate Hsp90 inh. Control Hsp90SBP N ATP ADP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP Weak Links The Universal Key to the Stability of Networks and Complex Systems Both water and chaperones provide a diffuse set of rapidly fluctuating weak links (low affinity and low probability interactions), which allow the generalization of all these statements to a multitude of networks. Any protein can be client of Hsp90 Děkuji za pozornost • Bořivoj Vojtěšek • Filip Trčka • Eva Růčková • Michal Ďurech • Kateřina Křivánková Ted R. Hupp