Stress response and protein folding Petr Müller Molecular and Cellular Pathophysiology 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… 1962 by Ferruccio Ritossa PUFFS Stress proteins Dinitrophenol-mitochondrialní uncoupler Proteins induced by increased temperature, mainly represented by chaperones Hsp90 Hsp70, DnaK Hsp60, GroEL Hsp40 DnaJ Hsp27, Crystalins, Hsp10,chaperonins, GroES 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, …. Sensors of proteotoxic stress HSF1 HSF2 Heat shock factors Stress UPR “Unfolded Protein Response” Increased temperature Oxidative stress Metabolic stress PERK kinase ATF6 ATF6 XBP1 NUCLEUS ER Endoplasmic Reticulum Mutations and genomic instability IRE1a Mitochondria KEAP1 NRF2 NRF2 NRF2 GolgiATF6 Oxidativestress 15-120 DNA binding domain HR-A HR-B HR-C TA domain regulatory domain 130 – 203 221 – 310 384 – 409 410 – 529 P S303 S307 S326 P K208 K224 K298 S S S S S S121 K126 K131 N C SUMO S307 P S326 P S303 P DBDHR-AHR-BHR-C TAD SUMO GAA n TTC n GAA P P A B Chaperones Heat shock proteins Ubiquitin Proteasome subunits HSF1 is the main regulator of gene expression responsible for maintaining protein homeostasis HSF1 HSF2 HSF4 HSF-X-linked HSF-Y-linked HSF-5 NRF2 NRF1 HIF1 ARNT DNA binding domain Hydrophobic repeats Leucine zippers Regulatory domain Transactivation domain C CC N C NNN Regulation of chaperone gene expression STRESS HSF1 Isoform X1 Isoform X3 Isoform X2 Isoform c Isoform a Isoform b Isoform a Isoform b HSF1 HSF2 HSF4 HSF5 130-203 Hydrophobic repeat HR-A/B 15-120 DNA binding 221-310 Regulatory domain HSF Y-linked HSF X-linked Lidské HSF Geny a isoformy Analysis of HSF1 activation HSF1-mCherry in A375 and H1299 Nuclear stress bodies Cell fractionation Native gel, detection of trimers Native gel - mCherry Monomer Trimer Typhoon FLA 9500 HSF1-mCherry + SBP-HSF1 Measurement of DNA binding capacity mCherryfluorescence Fluorescence polarization WB • Crosslinkink • Fractionation • phosphorylation Mechanisms of HSF1 activation HSF1 is essential for carcinogenesis and tumour progression A Homozygous Splice Mutation in the HSF4 Gene Is Associated with an Autosomal Recessive Congenital Cataract Congenital Cataract in Australian Shepard Mutation in HSF4 leads to decreased expression of crystalline genes in the lens, resulting in congenital cataracts Crystalline alpha/beta (CRYAB, CRYAA) HSF4 Unfolded protein response and autophagy • ATF6 is an endoplasmic reticulum (ER) stress-regulated transmembrane transcription factor that activates the transcription of ER molecules. • Accumulation of misfolded proteins in the Endoplasmic Reticulum results in the proteolytic cleavage of ATF6. • The cytosolic portion of ATF6 will move to the nucleus and act as a transcription factor to cause the transcription of ER chaperones. ATF6 SH KEAP1 -ON NRF2 -OFF SH KEAP1 -OFF NRF2 -ON S-S Oxidation NRF2 is a transcription factor that regulates the expression of antioxidant proteins that protect against oxidative damage triggered by injury and inflammation NRF2