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. 1997 Jan 15;16(2):221–229. doi: 10.1093/emboj/16.2.221

Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation.

M Ehrnsperger 1, S Gräber 1, M Gaestel 1, J Buchner 1
PMCID: PMC1169629  PMID: 9029143

Abstract

Small heat shock proteins (sHsps) are a conserved and ubiquitous protein family. Their ability to convey thermoresistance suggests their participation in protecting the native conformation of proteins. However, the underlying functional principles of their protective properties and their role in concert with other chaperone families remain enigmatic. Here, we analysed the influence of Hsp25 on the inactivation and subsequent aggregation of a model protein, citrate synthase (CS), under heat shock conditions in vitro. We show that stable binding of several non-native CS molecules to one Hsp25 oligomer leads to an accumulation of CS unfolding intermediates, which are protected from irreversible aggregation. Furthermore, a number of different proteins which bind to Hsp25 can be isolated from heat-shocked extracts of cells. Under permissive folding conditions, CS can be released from Hsp25 and, in cooperation with Hsp70, an ATP-dependent chaperone, the native state can be restored. Taken together, our findings allow us to integrate sHsps functionally in the cellular chaperone system operating under heat shock conditions. The task of sHsps in this context is to efficiently trap a large number of unfolding proteins in a folding-competent state and thus create a reservoir of non-native proteins for an extended period of time, allowing refolding after restoration of physiological conditions in cooperation with other chaperones.

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Selected References

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  1. Allen S. P., Polazzi J. O., Gierse J. K., Easton A. M. Two novel heat shock genes encoding proteins produced in response to heterologous protein expression in Escherichia coli. J Bacteriol. 1992 Nov;174(21):6938–6947. doi: 10.1128/jb.174.21.6938-6947.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arrigo A. P., Suhan J. P., Welch W. J. Dynamic changes in the structure and intracellular locale of the mammalian low-molecular-weight heat shock protein. Mol Cell Biol. 1988 Dec;8(12):5059–5071. doi: 10.1128/mcb.8.12.5059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Becker J., Craig E. A. Heat-shock proteins as molecular chaperones. Eur J Biochem. 1994 Jan 15;219(1-2):11–23. doi: 10.1007/978-3-642-79502-2_2. [DOI] [PubMed] [Google Scholar]
  4. Behlke J., Lutsch G., Gaestel M., Bielka H. Supramolecular structure of the recombinant murine small heat shock protein hsp25. FEBS Lett. 1991 Aug 19;288(1-2):119–122. doi: 10.1016/0014-5793(91)81016-2. [DOI] [PubMed] [Google Scholar]
  5. Benndorf R., Hayess K., Ryazantsev S., Wieske M., Behlke J., Lutsch G. Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerization-inhibiting activity. J Biol Chem. 1994 Aug 12;269(32):20780–20784. [PubMed] [Google Scholar]
  6. Bhat S. P., Nagineni C. N. alpha B subunit of lens-specific protein alpha-crystallin is present in other ocular and non-ocular tissues. Biochem Biophys Res Commun. 1989 Jan 16;158(1):319–325. doi: 10.1016/s0006-291x(89)80215-3. [DOI] [PubMed] [Google Scholar]
  7. Bond U., Schlesinger M. J. Heat-shock proteins and development. Adv Genet. 1987;24:1–29. doi: 10.1016/s0065-2660(08)60005-x. [DOI] [PubMed] [Google Scholar]
  8. Buchner J. Supervising the fold: functional principles of molecular chaperones. FASEB J. 1996 Jan;10(1):10–19. [PubMed] [Google Scholar]
  9. Chiesa R., McDermott M. J., Mann E., Spector A. The apparent molecular size of native alpha-crystallin B in non-lenticular tissues. FEBS Lett. 1990 Jul 30;268(1):222–226. doi: 10.1016/0014-5793(90)81013-e. [DOI] [PubMed] [Google Scholar]
  10. Ciocca D. R., Oesterreich S., Chamness G. C., McGuire W. L., Fuqua S. A. Biological and clinical implications of heat shock protein 27,000 (Hsp27): a review. J Natl Cancer Inst. 1993 Oct 6;85(19):1558–1570. doi: 10.1093/jnci/85.19.1558. [DOI] [PubMed] [Google Scholar]
  11. Collier N. C., Heuser J., Levy M. A., Schlesinger M. J. Ultrastructural and biochemical analysis of the stress granule in chicken embryo fibroblasts. J Cell Biol. 1988 Apr;106(4):1131–1139. doi: 10.1083/jcb.106.4.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Crête P., Landry J. Induction of HSP27 phosphorylation and thermoresistance in Chinese hamster cells by arsenite, cycloheximide, A23187, and EGTA. Radiat Res. 1990 Mar;121(3):320–327. [PubMed] [Google Scholar]
  13. Freeman B. C., Morimoto R. I. The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding. EMBO J. 1996 Jun 17;15(12):2969–2979. [PMC free article] [PubMed] [Google Scholar]
  14. Gaestel M., Gross B., Benndorf R., Strauss M., Schunk W. H., Kraft R., Otto A., Böhm H., Stahl J., Drabsch H. Molecular cloning, sequencing and expression in Escherichia coli of the 25-kDa growth-related protein of Ehrlich ascites tumor and its homology to mammalian stress proteins. Eur J Biochem. 1989 Jan 15;179(1):209–213. doi: 10.1111/j.1432-1033.1989.tb14542.x. [DOI] [PubMed] [Google Scholar]
  15. Gernold M., Knauf U., Gaestel M., Stahl J., Kloetzel P. M. Development and tissue-specific distribution of mouse small heat shock protein hsp25. Dev Genet. 1993;14(2):103–111. doi: 10.1002/dvg.1020140204. [DOI] [PubMed] [Google Scholar]
  16. Gething M. J. Molecular chaperones: individualists or groupies? Curr Opin Cell Biol. 1991 Aug;3(4):610–614. doi: 10.1016/0955-0674(91)90030-3. [DOI] [PubMed] [Google Scholar]
  17. Horwitz J. Alpha-crystallin can function as a molecular chaperone. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10449–10453. doi: 10.1073/pnas.89.21.10449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Höhfeld J., Minami Y., Hartl F. U. Hip, a novel cochaperone involved in the eukaryotic Hsc70/Hsp40 reaction cycle. Cell. 1995 Nov 17;83(4):589–598. doi: 10.1016/0092-8674(95)90099-3. [DOI] [PubMed] [Google Scholar]
  19. Inaguma Y., Shinohara H., Goto S., Kato K. Translocation and induction of alpha B crystallin by heat shock in rat glioma (GA-1) cells. Biochem Biophys Res Commun. 1992 Jan 31;182(2):844–850. doi: 10.1016/0006-291x(92)91809-5. [DOI] [PubMed] [Google Scholar]
  20. Ingolia T. D., Craig E. A. Four small Drosophila heat shock proteins are related to each other and to mammalian alpha-crystallin. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2360–2364. doi: 10.1073/pnas.79.7.2360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jaenicke R., Creighton T. E. Protein folding: junior chaperones. Curr Biol. 1993 Apr 1;3(4):234–235. doi: 10.1016/0960-9822(93)90342-l. [DOI] [PubMed] [Google Scholar]
  22. Jakob U., Buchner J. Assisting spontaneity: the role of Hsp90 and small Hsps as molecular chaperones. Trends Biochem Sci. 1994 May;19(5):205–211. doi: 10.1016/0968-0004(94)90023-x. [DOI] [PubMed] [Google Scholar]
  23. Jakob U., Gaestel M., Engel K., Buchner J. Small heat shock proteins are molecular chaperones. J Biol Chem. 1993 Jan 25;268(3):1517–1520. [PubMed] [Google Scholar]
  24. Jakob U., Lilie H., Meyer I., Buchner J. Transient interaction of Hsp90 with early unfolding intermediates of citrate synthase. Implications for heat shock in vivo. J Biol Chem. 1995 Mar 31;270(13):7288–7294. doi: 10.1074/jbc.270.13.7288. [DOI] [PubMed] [Google Scholar]
  25. Kato K., Hasegawa K., Goto S., Inaguma Y. Dissociation as a result of phosphorylation of an aggregated form of the small stress protein, hsp27. J Biol Chem. 1994 Apr 15;269(15):11274–11278. [PubMed] [Google Scholar]
  26. Kato K., Shinohara H., Kurobe N., Goto S., Inaguma Y., Ohshima K. Immunoreactive alpha A crystallin in rat non-lenticular tissues detected with a sensitive immunoassay method. Biochim Biophys Acta. 1991 Oct 25;1080(2):173–180. doi: 10.1016/0167-4838(91)90146-q. [DOI] [PubMed] [Google Scholar]
  27. Knauf U., Bielka H., Gaestel M. Over-expression of the small heat-shock protein, hsp25, inhibits growth of Ehrlich ascites tumor cells. FEBS Lett. 1992 Sep 14;309(3):297–302. doi: 10.1016/0014-5793(92)80793-g. [DOI] [PubMed] [Google Scholar]
  28. Knauf U., Jakob U., Engel K., Buchner J., Gaestel M. Stress- and mitogen-induced phosphorylation of the small heat shock protein Hsp25 by MAPKAP kinase 2 is not essential for chaperone properties and cellular thermoresistance. EMBO J. 1994 Jan 1;13(1):54–60. doi: 10.1002/j.1460-2075.1994.tb06234.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Landry J., Chrétien P., Lambert H., Hickey E., Weber L. A. Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells. J Cell Biol. 1989 Jul;109(1):7–15. doi: 10.1083/jcb.109.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lavoie J. N., Hickey E., Weber L. A., Landry J. Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of heat shock protein 27. J Biol Chem. 1993 Nov 15;268(32):24210–24214. [PubMed] [Google Scholar]
  31. Lee G. J., Pokala N., Vierling E. Structure and in vitro molecular chaperone activity of cytosolic small heat shock proteins from pea. J Biol Chem. 1995 May 5;270(18):10432–10438. doi: 10.1074/jbc.270.18.10432. [DOI] [PubMed] [Google Scholar]
  32. Mehlen P., Mehlen A., Guillet D., Preville X., Arrigo A. P. Tumor necrosis factor-alpha induces changes in the phosphorylation, cellular localization, and oligomerization of human hsp27, a stress protein that confers cellular resistance to this cytokine. J Cell Biochem. 1995 Jun;58(2):248–259. doi: 10.1002/jcb.240580213. [DOI] [PubMed] [Google Scholar]
  33. Merck K. B., Groenen P. J., Voorter C. E., de Haard-Hoekman W. A., Horwitz J., Bloemendal H., de Jong W. W. Structural and functional similarities of bovine alpha-crystallin and mouse small heat-shock protein. A family of chaperones. J Biol Chem. 1993 Jan 15;268(2):1046–1052. [PubMed] [Google Scholar]
  34. Miron T., Vancompernolle K., Vandekerckhove J., Wilchek M., Geiger B. A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein. J Cell Biol. 1991 Jul;114(2):255–261. doi: 10.1083/jcb.114.2.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nicholl I. D., Quinlan R. A. Chaperone activity of alpha-crystallins modulates intermediate filament assembly. EMBO J. 1994 Feb 15;13(4):945–953. doi: 10.1002/j.1460-2075.1994.tb06339.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nover L., Scharf K. D., Neumann D. Cytoplasmic heat shock granules are formed from precursor particles and are associated with a specific set of mRNAs. Mol Cell Biol. 1989 Mar;9(3):1298–1308. doi: 10.1128/mcb.9.3.1298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Patriarca E. J., Maresca B. Acquired thermotolerance following heat shock protein synthesis prevents impairment of mitochondrial ATPase activity at elevated temperatures in Saccharomyces cerevisiae. Exp Cell Res. 1990 Sep;190(1):57–64. doi: 10.1016/0014-4827(90)90143-x. [DOI] [PubMed] [Google Scholar]
  39. Pauli D., Tonka C. H., Tissieres A., Arrigo A. P. Tissue-specific expression of the heat shock protein HSP27 during Drosophila melanogaster development. J Cell Biol. 1990 Sep;111(3):817–828. doi: 10.1083/jcb.111.3.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Plesofsky-Vig N., Vig J., Brambl R. Phylogeny of the alpha-crystallin-related heat-shock proteins. J Mol Evol. 1992 Dec;35(6):537–545. doi: 10.1007/BF00160214. [DOI] [PubMed] [Google Scholar]
  41. Schirmer E. C., Lindquist S., Vierling E. An Arabidopsis heat shock protein complements a thermotolerance defect in yeast. Plant Cell. 1994 Dec;6(12):1899–1909. doi: 10.1105/tpc.6.12.1899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stege G. J., Li G. C., Li L., Kampinga H. H., Konings A. W. On the role of hsp72 in heat-induced intranuclear protein aggregation. Int J Hyperthermia. 1994 Sep-Oct;10(5):659–674. doi: 10.3109/02656739409022446. [DOI] [PubMed] [Google Scholar]
  43. Welch W. J. Phorbol ester, calcium ionophore, or serum added to quiescent rat embryo fibroblast cells all result in the elevated phosphorylation of two 28,000-dalton mammalian stress proteins. J Biol Chem. 1985 Mar 10;260(5):3058–3062. [PubMed] [Google Scholar]
  44. Wiech H., Buchner J., Zimmermann M., Zimmermann R., Jakob U. Hsc70, immunoglobulin heavy chain binding protein, and Hsp90 differ in their ability to stimulate transport of precursor proteins into mammalian microsomes. J Biol Chem. 1993 Apr 5;268(10):7414–7421. [PubMed] [Google Scholar]
  45. de Jong W. W., Leunissen J. A., Voorter C. E. Evolution of the alpha-crystallin/small heat-shock protein family. Mol Biol Evol. 1993 Jan;10(1):103–126. doi: 10.1093/oxfordjournals.molbev.a039992. [DOI] [PubMed] [Google Scholar]
  46. van den IJssel P. R., Overkamp P., Knauf U., Gaestel M., de Jong W. W. Alpha A-crystallin confers cellular thermoresistance. FEBS Lett. 1994 Nov 21;355(1):54–56. doi: 10.1016/0014-5793(94)01175-3. [DOI] [PubMed] [Google Scholar]

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