Stress responses play a vital role in cellular survival against environmental challenges, often exploited by cancer cells to proliferate, counteract genomic instability, and resist therapeutic stress. Heat shock factor protein 1 (HSF1), a central transcription factor in stress response pathways, exhibits markedly elevated activity in cancer. Despite extensive research into the transcriptional role of HSF1, the mechanisms underlying its activation remain elusive. Upon exposure to conditions that induce protein damage, monomeric HSF1 undergoes rapid conformational changes and assembles into trimers, a key step for DNA binding and transactivation of target genes. This study investigates the role of HSF1 as a sensor of proteotoxic stress conditions. Our findings reveal that purified HSF1 maintains a stable monomeric conformation independent of molecular chaperones in vitro. Moreover, while it is known that heat stress triggers HSF1 trimerization, a notable increase in trimerization and DNA binding was observed in the presence of protein-based crowders. Conditions inducing protein misfolding and increased protein crowding in cells directly trigger HSF1 trimerization. In contrast, proteosynthesis inhibition, by reducing denatured proteins in the cell, prevents HSF1 activation. Surprisingly, HSF1 remains activated under proteotoxic stress conditions even when bound to Hsp70 and Hsp90. This finding suggests that the negative feedback regulation between HSF1 and chaperones is not directly driven by their interaction but is realized indirectly through chaperone-mediated restoration of cytoplasmic proteostasis. In summary, our study suggests that HSF1 serves as a molecular crowding sensor, trimerizing to initiate protective responses that enhance chaperone activities to restore homeostasis.

Department of Biochemistry Faculty of Science Masaryk University Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Research Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Brno Czech Republic

Zobrazit více v PubMed

Prince TL, Lang BJ, Guerrero-Gimenez ME, Fernandez-Munoz JM, Ackerman A, Calderwood SK. HSF1: Primary Factor in Molecular Chaperone Expression and a Major Contributor to Cancer Morbidity. Cells. 2020;9(4). doi: 10.3390/cells9041046 PubMed DOI PMC

Dai C, Whitesell L, Rogers AB, Lindquist S. Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis. Cell. 2007;130(6):1005–18. doi: 10.1016/j.cell.2007.07.020 PubMed DOI PMC

Cyran AM, Zhitkovich A. Heat Shock Proteins and HSF1 in Cancer. Front Oncol. 2022;12:860320. doi: 10.3389/fonc.2022.860320 PubMed DOI PMC

Hentze N, Le Breton L, Wiesner J, Kempf G, Mayer MP. Molecular mechanism of thermosensory function of human heat shock transcription factor Hsf1. eLife. 2016;5. Epub 2016/01/20. doi: 10.7554/eLife.11576 PubMed DOI PMC

Su KH, Cao J, Tang Z, Dai S, He Y, Sampson SB, et al.. HSF1 critically attunes proteotoxic stress sensing by mTORC1 to combat stress and promote growth. Nature cell biology. 2016;18(5):527–39. Epub 2016/04/05. doi: 10.1038/ncb3335 PubMed DOI PMC

Budzynski MA, Puustinen MC, Joutsen J, Sistonen L. Uncoupling Stress-Inducible Phosphorylation of Heat Shock Factor 1 from Its Activation. Molecular and cellular biology. 2015;35(14):2530–40. Epub 2015/05/13. doi: 10.1128/MCB.00816-14 PubMed DOI PMC

Raychaudhuri S, Loew C, Korner R, Pinkert S, Theis M, Hayer-Hartl M, et al.. Interplay of acetyltransferase EP300 and the proteasome system in regulating heat shock transcription factor 1. Cell. 2014;156(5):975–85. Epub 2014/03/04. doi: 10.1016/j.cell.2014.01.055 PubMed DOI

Zou J, Guo Y, Guettouche T, Smith DF, Voellmy R. Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell. 1998;94(4):471–80. Epub 1998/09/04. doi: 10.1016/S0092-8674(00)81588-3 PubMed DOI

Berggren WT, Lutz M, Modesto V. General Spinfection Protocol. StemBook. Cambridge (MA)2008. PubMed

Zhang R, Mayhood T, Lipari P, Wang Y, Durkin J, Syto R, et al.. Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction. Anal Biochem. 2004;331(1):138–46. doi: 10.1016/j.ab.2004.03.009 PubMed DOI

Kavan D, Man P. MSTools—Web based application for visualization and presentation of HXMS data. International Journal of Mass Spectrometry. 2011;302(1):53–8. doi: 10.1016/j.ijms.2010.07.030 DOI

Wittig I, Karas M, Schagger H. High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol Cell Proteomics. 2007;6(7):1215–25. doi: 10.1074/mcp.M700076-MCP200 PubMed DOI

Wittig I, Braun HP, Schagger H. Blue native PAGE. Nat Protoc. 2006;1(1):418–28. doi: 10.1038/nprot.2006.62 PubMed DOI

Li J, Labbadia J, Morimoto RI. Rethinking HSF1 in Stress, Development, and Organismal Health. Trends in cell biology. 2017;27(12):895–905. doi: 10.1016/j.tcb.2017.08.002 PubMed DOI PMC

Tokunaga Y, Otsuyama KI, Hayashida N. Cell Cycle Regulation by Heat Shock Transcription Factors. Cells. 2022;11(2). doi: 10.3390/cells11020203 PubMed DOI PMC

Dai S, Tang Z, Cao J, Zhou W, Li H, Sampson S, et al.. Suppression of the HSF1-mediated proteotoxic stress response by the metabolic stress sensor AMPK. The EMBO journal. 2015;34(3):275–93. doi: 10.15252/embj.201489062 PubMed DOI PMC

Santagata S, Mendillo ML, Tang YC, Subramanian A, Perley CC, Roche SP, et al.. Tight coordination of protein translation and HSF1 activation supports the anabolic malignant state. Science (New York, NY). 2013;341(6143):1238303. Epub 2013/07/23. doi: 10.1126/science.1238303 PubMed DOI PMC

Ota C, Takano K. Behavior of Bovine Serum Albumin Molecules in Molecular Crowding Environments Investigated by Raman Spectroscopy. Langmuir. 2016;32(29):7372–82. doi: 10.1021/acs.langmuir.6b01228 PubMed DOI

Alric B, Formosa-Dague C, Dague E, Holt LJ, Delarue M. Macromolecular crowding limits growth under pressure. Nature Physics. 2022;18(4):411–6. doi: 10.1038/s41567-022-01506-1 PubMed DOI PMC

Harada R, Tochio N, Kigawa T, Sugita Y, Feig M. Reduced native state stability in crowded cellular environment due to protein-protein interactions. J Am Chem Soc. 2013;135(9):3696–701. doi: 10.1021/ja3126992 PubMed DOI PMC

Delarue M, Brittingham GP, Pfeffer S, Surovtsev IV, Pinglay S, Kennedy KJ, et al.. mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding. Cell. 2018;174(2):338–49 e20. doi: 10.1016/j.cell.2018.05.042 PubMed DOI PMC

Iwasaki S, Iwasaki W, Takahashi M, Sakamoto A, Watanabe C, Shichino Y, et al.. The Translation Inhibitor Rocaglamide Targets a Bimolecular Cavity between eIF4A and Polypurine RNA. Mol Cell. 2019;73(4):738–48 e9. doi: 10.1016/j.molcel.2018.11.026 PubMed DOI PMC

Dmitriev SE, Vladimirov DO, Lashkevich KA. A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis. Biochemistry (Mosc). 2020;85(11):1389–421. doi: 10.1134/S0006297920110097 PubMed DOI PMC

Kmiecik SW, Le Breton L, Mayer MP. Feedback regulation of heat shock factor 1 (Hsf1) activity by Hsp70-mediated trimer unzipping and dissociation from DNA. The EMBO journal. 2020;39(14):e104096. doi: 10.15252/embj.2019104096 PubMed DOI PMC

Xu Y, Xie X, Duan Y, Wang L, Cheng Z, Cheng J. A review of impedance measurements of whole cells. Biosensors and Bioelectronics. Biosensors and Bioelectronics. 2016;77:824–36. doi: 10.1016/j.bios.2015.10.027 PubMed DOI

Lincon A, Das S, DasGupta S. Capturing protein denaturation using electrical impedance technique. Journal of Molecular Liquids. 2022;360:119301. doi: 10.1016/j.molliq.2022.119301 DOI

Gomez-Pastor R, Burchfiel ET, Thiele DJ. Regulation of heat shock transcription factors and their roles in physiology and disease. Nature reviews Molecular cell biology. 2018;19(1):4–19. Epub 2017/08/31. doi: 10.1038/nrm.2017.73 PubMed DOI PMC

Minton AP. The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media. J Biol Chem. 2001;276(14):10577–80. doi: 10.1074/jbc.R100005200 PubMed DOI

Miklos AC, Li C, Sharaf NG, Pielak GJ. Volume exclusion and soft interaction effects on protein stability under crowded conditions. Biochemistry. 2010;49(33):6984–91. doi: 10.1021/bi100727y PubMed DOI PMC

Gorensek-Benitez AH, Smith AE, Stadmiller SS, Perez Goncalves GM, Pielak GJ. Cosolutes, Crowding, and Protein Folding Kinetics. J Phys Chem B. 2017;121(27):6527–37. doi: 10.1021/acs.jpcb.7b03786 PubMed DOI PMC

Cohen RD, Pielak GJ. Quinary interactions with an unfolded state ensemble. Protein Sci. 2017;26(9):1698–703. doi: 10.1002/pro.3206 PubMed DOI PMC

Sarkar M, Li C, Pielak GJ. Soft interactions and crowding. Biophys Rev. 2013;5(2):187–94. doi: 10.1007/s12551-013-0104-4 PubMed DOI PMC

Miklos AC, Sarkar M, Wang Y, Pielak GJ. Protein crowding tunes protein stability. J Am Chem Soc. 2011;133(18):7116–20. doi: 10.1021/ja200067p PubMed DOI

Timr S, Sterpone F. Stabilizing or Destabilizing: Simulations of Chymotrypsin Inhibitor 2 under Crowding Reveal Existence of a Crossover Temperature. J Phys Chem Lett. 2021;12(6):1741–6. doi: 10.1021/acs.jpclett.0c03626 PubMed DOI

Al-Ayoubi SR, Schummel PH, Golub M, Peters J, Winter R. Influence of cosolvents, self-crowding, temperature and pressure on the sub-nanosecond dynamics and folding stability of lysozyme. Phys Chem Chem Phys. 2017;19(22):14230–7. doi: 10.1039/C7CP00705A PubMed DOI

Nayar D. Small crowder interactions can drive hydrophobic polymer collapse as well as unfolding. Phys Chem Chem Phys. 2020;22(32):18091–101. doi: 10.1039/D0CP02402C PubMed DOI

Harrison PM, Chan HS, Prusiner SB, Cohen FE. Conformational propagation with prion-like characteristics in a simple model of protein folding. Protein Sci. 2001;10(4):819–35. doi: 10.1110/ps.38701 PubMed DOI PMC

Ma Q, Hu JY, Chen J, Liang Y. The role of crowded physiological environments in prion and prion-like protein aggregation. International journal of molecular sciences. 2013;14(11):21339–52. doi: 10.3390/ijms141121339 PubMed DOI PMC

Siddiqui GA, Naeem A. Connecting the Dots: Macromolecular Crowding and Protein Aggregation. J Fluoresc. 2023;33(1):1–11. doi: 10.1007/s10895-022-03082-2 PubMed DOI

Hartl FU, Hayer-Hartl M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science (New York, NY). 2002;295(5561):1852–8. doi: 10.1126/science.1068408 PubMed DOI

Sherman MY, Qian SB. Less is more: improving proteostasis by translation slow down. Trends Biochem Sci. 2013;38(12):585–91. doi: 10.1016/j.tibs.2013.09.003 PubMed DOI

Rodriguez-Galan O, Garcia-Gomez JJ, Rosado IV, Wei W, Mendez-Godoy A, Pillet B, et al.. A functional connection between translation elongation and protein folding at the ribosome exit tunnel in Saccharomyces cerevisiae. Nucleic Acids Res. 2021;49(1):206–20. doi: 10.1093/nar/gkaa1200 PubMed DOI PMC

Stein KC, Frydman J. The stop-and-go traffic regulating protein biogenesis: How translation kinetics controls proteostasis. J Biol Chem. 2019;294(6):2076–84. doi: 10.1074/jbc.REV118.002814 PubMed DOI PMC

Kinjo AR, Takada S. Competition between protein folding and aggregation with molecular chaperones in crowded solutions: insight from mesoscopic simulations. Biophys J. 2003;85(6):3521–31. doi: 10.1016/S0006-3495(03)74772-9 PubMed DOI PMC

Farkas Z, Kalapis D, Bodi Z, Szamecz B, Daraba A, Almasi K, et al.. Hsp70-associated chaperones have a critical role in buffering protein production costs. eLife. 2018;7. doi: 10.7554/eLife.29845 PubMed DOI PMC

SUMOylation is not a prerequisite for HSF1's role in stress protection and transactivation

Role of HSF1 in cell division, tumorigenesis and therapy: a literature review