title: The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities.
abstract: Humans contain many HSP (heat-shock protein) 70/HSPA- and HSP40/DNAJ-encoding genes and most of the corresponding proteins are localized in the cytosol. To test for possible functional differences and/or substrate specificity, we assessed the effect of overexpression of each of these HSPs on refolding of heat-denatured luciferase and on the suppression of aggregation of a non-foldable polyQ (polyglutamine)-expanded Huntingtin fragment. Overexpressed chaperones that suppressed polyQ aggregation were found not to be able to stimulate luciferase refolding. Inversely, chaperones that supported luciferase refolding were poor suppressors of polyQ aggregation. This was not related to client specificity itself, as the polyQ aggregation inhibitors often also suppressed heat-induced aggregation of luciferase. Surprisingly, the exclusively heat-inducible HSPA6 lacks both luciferase refolding and polyQ aggregation-suppressing activities. Furthermore, whereas overexpression of HSPA1A protected cells from heat-induced cell death, overexpression of HSPA6 did not. Inversely, siRNA (small interfering RNA)-mediated blocking of HSPA6 did not impair the development of heat-induced thermotolerance. Yet, HSPA6 has a functional substrate-binding domain and possesses intrinsic ATPase activity that is as high as that of the canonical HSPA1A when stimulated by J-proteins. In vitro data suggest that this may be relevant to substrate specificity, as purified HSPA6 could not chaperone heat-unfolded luciferase but was able to assist in reactivation of heat-unfolded p53. So, even within the highly sequence-conserved HSPA family, functional differentiation is larger than expected, with HSPA6 being an extreme example that may have evolved to maintain specific critical functions under conditions of severe stress.
authors.affiliation: Department of Cell Biology, Section of Radiation and Stress Cell Biology, University Medical Center Groningen, University of Groningen, Groningen 9700 AD, The Netherlands.
authors.forename: Jurre
authors.initials: J
authors.lastname: Hageman
authors.forename: Maria A W H
authors.initials: MA
authors.lastname: van Waarde
authors.forename: Alicja
authors.initials: A
authors.lastname: Zylicz
authors.forename: Dawid
authors.initials: D
authors.lastname: Walerych
authors.forename: Harm H
authors.initials: HH
authors.lastname: Kampinga
mesh_terms: D000818:Animals; D002460:Cell Line; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D020869:Gene Expression Profiling; D020868:Gene Silencing; D050956:HSP40 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature / Q000009:adverse effects; D006801:Humans; D049409:Luciferases, Firefly / Q000737:chemistry / Q000378:metabolism; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D058849:Protein Refolding; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C490786:HSPA1A protein, human; C544598:HSPA6 protein, human; D018832:Molecular Chaperones; D010455:Peptides; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; C097188:polyglutamine; D049409:Luciferases, Firefly; D002950:Citrate (si)-Synthase