Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.
Chaperone Fusion protein Small heat shock protein (sHSP) Protein aggregation Protein-protein interaction Light scattering assay Glutathione-S-transferase (GST)
This is a preview of subscription content, log in to check access.
We thank Caroline Weber Kinn for initial cloning work and Alina Smithe for assistance with the reading of the manuscript and running assays. We thank the Clare Boothe Luce Foundation for supporting this work and for providing summer support for H.E.A and C.S.B. The National Institutes of Health (NIH) R15 GM120654-01 provided funding for this investigation to K.A.M.
Basha E, O’Neill H, Vierling E (2011) Small heat shock proteins and α-crystallins: dynamic proteins with flexible functions. Trends Biochem Sci:1–12. doi:10.1016/j.tibs.2011.11.005
Bova MP, Yaron O, Huang Q et al (1999) Mutation R120G in alphaB-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function. Proc Natl Acad Sci U S A 96:6137–6142CrossRefPubMedPubMedCentralGoogle Scholar
Carra S, Rusmini P, Crippa V et al (2013) Different anti-aggregation and pro-degradative functions of the members of the mammalian sHSP family in neurological disorders. Philos Trans R Soc Lond Ser B Biol Sci 368:20110409–20110409. doi:10.1098/rstb.2011.0409CrossRefGoogle Scholar
Cha J-Y, Lee S-H, Seo KH et al (2016) N-terminal arm of orchardgrass Hsp17.2 (DgHsp17.2) is essential for both in vitro chaperone activity and in vivo thermotolerance in yeast. Arch Biochem Biophys 591:18–27. doi:10.1016/j.abb.2015.12.011CrossRefPubMedGoogle Scholar
Diaz-Latoud C, Buache E, Javouhey E, Arrigo A-P (2005) Substitution of the unique cysteine residue of murine Hsp25 interferes with the protective activity of this stress protein through inhibition of dimer formation. Antioxid Redox Signal 7(3–4):436–445. doi:10.1089/ars.2005.7.436
Evgrafov OV, Mersiyanova I, Irobi J, Van Den Bosch L, Dierick I, Leung CL et al (2004) Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy. Nat Genet 36(6):602–606. doi:10.1038/ng1354
Houlden H, Laura M, Wavrant-De Vrièze F, Blake J, Wood N, Reilly MM (2008) Mutations in the HSP27 (HSPB1) gene cause dominant, recessive, and sporadic distal HMN/CMT type 2. Neurology 71(21):1660–1668. doi:10.1212/01.wnl.0000319696.14225.67
Niedziela-Majka A, Rymarczyk G, Kochman M, Ożyhar A (1998) GST-Induced Dimerization of DNA-Binding Domains Alters Characteristics of Their Interaction with DNA. Protein Expr Purif 14(2):208–220. doi:10.1006/prep.1998.0932
Peschek J, Braun N, Rohrberg J, Back KC, Kriehuber T, Kastenmüller A et al (2013) Regulated structural transitions unleash the chaperone activity of αB-crystallin. Proceedings of the National Academy of Sciences of the United States of America 110(40):E3780–9. doi:10.1073/pnas.1308898110
Raju M, Santhoshkumar P, Sharma KK (2011) Cataract-causing αAG98R-crystallin mutant dissociates into monomers having chaperone activity. Mol Vis 17:7–15PubMedPubMedCentralGoogle Scholar
Regini JW, Ecroyd H, Meehan S et al (2010) The interaction of unfolding α-lactalbumin and malate dehydrogenase with the molecular chaperone αB-crystallin: a light and X-ray scattering investigation. Mol Vis 16:2446–2456PubMedPubMedCentralGoogle Scholar
Shashidharamurthy R, Koteiche HA, Dong J, Mchaourab HS (2005) Mechanism of chaperone function in small heat shock proteins: dissociation of the HSP27 oligomer is required for recognition and binding of destabilized T4 lysozyme. J Biol Chem 280:5281–5289. doi:10.1074/jbc.M407236200CrossRefPubMedGoogle Scholar
Sheluho D, Ackerman SH (2001) An accessible hydrophobic surface is a key element of the molecular chaperone action of Atp11p. J Biol Chem 276(43):39945–39949. doi:10.1074/jbc.M107252200
Skouri-Panet F, Quevillon-Cheruel S, Michiel M et al (2006) sHSPs under temperature and pressure: the opposite behaviour of lens alpha-crystallins and yeast HSP26. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1764:372–383. doi:10.1016/j.bbapap.2005.12.011CrossRefGoogle Scholar
Skouri-Panet F, Michiel M, Férard C et al (2012) Structural and functional specificity of small heat shock protein HspB1 and HspB4, two cellular partners of HspB5: role of the in vitro hetero-complex formation in chaperone activity. Biochimie 94:975–984. doi:10.1016/j.biochi.2011.12.018CrossRefPubMedGoogle Scholar
Sokołowska I, Piłka ES, Sandvig K, Węgrzyn G, Słomińska-Wojewódzka M (2015) Hydrophobicity of protein determinants influences the recognition of substrates by EDEM1 and EDEM2 in human cells. BMC Cell Biol 16(1):1. doi:10.1186/s12860-015-0047-7
Sudnitsyna MV, Mymrikov EV, Seit-Nebi AS, Gusev NB (2012) The Role of Intrinsically Disordered Regions in the Structure and Functioning of Small Heat Shock Proteins. Curr Protein Pept Sci 13(1):76–85. doi:10.2174/138920312799277875
Sudnitsyna MV, Mymrikov EV, Seit-Nebi AS, Gusev NB (2012) The role of intrinsically disordered regions in the structure and functioning of small heat shock proteins. Curr Protein Pept Sci 13:76–85CrossRefPubMedGoogle Scholar
Tudyka T, Skerra A (1997) Glutathione S-transferase can be used as a C-terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli. Protein Science: a Publication of the Protein Society 6(10):2180–2187. doi:10.1002/pro.5560061012