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• W3015589319 abstract "Heat shock protein 70 (HSP70) chaperones play a central role in protein quality control and are crucial for many cellular processes, including protein folding, degradation, and disaggregation. Human HSP70s compose a family of 13 members that carry out their functions with the aid of even larger families of co-chaperones. A delicate interplay between HSP70s and co-chaperone recruitment is thought to determine substrate fate, yet it has been generally assumed that all Hsp70 paralogs have similar activities and are largely functionally redundant. However, here we found that when expressed in human cells, two highly homologous HSP70s, HSPA1A and HSPA1L, have opposing effects on cellular handling of various substrates. For example, HSPA1A reduced aggregation of the amyotrophic lateral sclerosis–associated protein variant superoxide dismutase 1 (SOD1)–A4V, whereas HSPA1L enhanced its aggregation. Intriguingly, variations in the substrate-binding domain of these HSP70s did not play a role in this difference. Instead, we observed that substrate fate is determined by differential interactions of the HSP70s with co-chaperones. Whereas most co-chaperones bound equally well to these two HSP70s, Hsp70/Hsp90-organizing protein (HOP) preferentially bound to HSPA1L, and the Hsp110 nucleotide-exchange factor HSPH2 preferred HSPA1A. The role of HSPH2 was especially crucial for the HSPA1A-mediated reduction in SOD1-A4V aggregation. These findings reveal a remarkable functional diversity at the level of the cellular HSP70s and indicate that this diversity is defined by their affinities for specific co-chaperones such as HSPH2. Heat shock protein 70 (HSP70) chaperones play a central role in protein quality control and are crucial for many cellular processes, including protein folding, degradation, and disaggregation. Human HSP70s compose a family of 13 members that carry out their functions with the aid of even larger families of co-chaperones. A delicate interplay between HSP70s and co-chaperone recruitment is thought to determine substrate fate, yet it has been generally assumed that all Hsp70 paralogs have similar activities and are largely functionally redundant. However, here we found that when expressed in human cells, two highly homologous HSP70s, HSPA1A and HSPA1L, have opposing effects on cellular handling of various substrates. For example, HSPA1A reduced aggregation of the amyotrophic lateral sclerosis–associated protein variant superoxide dismutase 1 (SOD1)–A4V, whereas HSPA1L enhanced its aggregation. Intriguingly, variations in the substrate-binding domain of these HSP70s did not play a role in this difference. Instead, we observed that substrate fate is determined by differential interactions of the HSP70s with co-chaperones. Whereas most co-chaperones bound equally well to these two HSP70s, Hsp70/Hsp90-organizing protein (HOP) preferentially bound to HSPA1L, and the Hsp110 nucleotide-exchange factor HSPH2 preferred HSPA1A. The role of HSPH2 was especially crucial for the HSPA1A-mediated reduction in SOD1-A4V aggregation. These findings reveal a remarkable functional diversity at the level of the cellular HSP70s and indicate that this diversity is defined by their affinities for specific co-chaperones such as HSPH2. The Hsp70 machinery is a central system of the protein quality control, and it is involved in many different processes including protein folding, degradation, aggregation prevention, and disaggregation (1Mayer M.P. Bukau B. Hsp70 chaperones: cellular functions and molecular mechanism.Cell Mol. Life Sci. 2005; 62 (15770419): 670-68410.1007/s00018-004-4464-6Crossref PubMed Scopus (1919) Google Scholar, 2Kim Y.E. Hipp M.S. Bracher A. Hayer-Hartl M. Hartl F.U. Molecular chaperone functions in protein folding and proteostasis.Annu. Rev. Biochem. 2013; 82 (23746257): 323-35510.1146/annurev-biochem-060208-092442Crossref PubMed Scopus (864) Google Scholar, 3Mogk A. Bukau B. Kampinga H.H. Cellular handling of protein aggregates by disaggregation machines.Mol. Cell. 2018; 69 (29351843): 214-22610.1016/j.molcel.2018.01.004Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Hsp70 chaperones are among the most highly conserved proteins in evolution and in humans comprise a family of 13 members (4Radons J. The human HSP70 family of chaperones: where do we stand?.Cell Stress Chaperones. 2016; 21 (26865365): 379-40410.1007/s12192-016-0676-6Crossref PubMed Scopus (242) Google Scholar). They have been reported to interact with a wide range of substrates, both non-native and native, by recognizing exposed hydrophobic motifs found in most proteins (5Rüdiger S. Germeroth L. Schneider-Mergener J. Bukau B. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.EMBO J. 1997; 16 (9130695): 1501-150710.1093/emboj/16.7.1501Crossref PubMed Scopus (631) Google Scholar, 6Clerico E.M. Tilitsky J.M. Meng W. Gierasch L.M. How Hsp70 molecular machines interact with their substrates to mediate diverse physiological functions.J. Mol. Biol. 2015; 427 (25683596): 1575-158810.1016/j.jmb.2015.02.004Crossref PubMed Scopus (183) Google Scholar). Hsp70 proteins consist of an N-terminal nucleotide-binding domain (NBD or N), 3The abbreviations used are: NBDnucleotide-binding domainSBDsubstrate-binding domainALSamyotrophic lateral sclerosisCTDC-terminal domainJDPJ-domain proteinNEFnucleotide-exchange factorSODsuperoxide dismutaseaaamino acid(s)PTMpost-translational modificationT fractiontotal fractionS fractionsupernatant fractionP fractionpellet fractionHAhemagglutininMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumPDBProtein Data Bank. a substrate-binding domain (SBD or S), and a C-terminal domain (CTD or C) that forms a lid stabilizing bound substrates after ATP hydrolysis (1Mayer M.P. Bukau B. Hsp70 chaperones: cellular functions and molecular mechanism.Cell Mol. Life Sci. 2005; 62 (15770419): 670-68410.1007/s00018-004-4464-6Crossref PubMed Scopus (1919) Google Scholar). The Hsp70 activity is based on an ATP-dependent cycle, alternating between the low-substrate-affinity ATP-bound state and the high-substrate-affinity ADP-bound state. However, intrinsic ATPase activity of Hsp70 proteins is too low to function independently; that is why the cycle turnover is aided by the co-chaperones J-domain proteins (JDPs) and nucleotide-exchange factors (NEFs), which stimulate ATP hydrolysis and catalyze ADP/ATP exchange, respectively (7Mayer M.P. Hsp70 chaperone dynamics and molecular mechanism.Trends Biochem. Sci. 2013; 38 (24012426): 507-51410.1016/j.tibs.2013.08.001Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). In addition, the co-chaperones of the large JDP (also referred to as DNAJ) family (53 members in humans) act as recruiters of substrates via interaction with their versatile substrate-binding domains (8Kampinga H.H. Craig E.A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity.Nat. Rev. Mol. Cell Biol. 2010; 11 (20651708): 579-59210.1038/nrm2941Crossref PubMed Scopus (1015) Google Scholar). Upon interaction with the Hsp70s, via their conserved J-domain, JDPs together with the substrates stimulate Hsp70–ATPase activity, and substrates are transferred to Hsp70 (9Kityk R. Kopp J. Mayer M.P. Molecular mechanism of J-domain–triggered ATP hydrolysis by Hsp70 chaperones.Mol. Cell. 2018; 69 (29290615): 227-237.e410.1016/j.molcel.2017.12.003Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). To promote substrate release, four different types of co-chaperones can stimulate nucleotide exchange in human Hsp70s: BAG-domain proteins (6 members), Hsp110/Grp170 (HSPH, four members), HspBP1/Sil1 (two members), and GrpE (two members) (10Bracher A. Verghese J. The nucleotide exchange factors of Hsp70 molecular chaperones.Front. Mol. Biosci. 2015; 2 (26913285): 10Crossref PubMed Scopus (112) Google Scholar). Despite differences in structure and working mechanism, all four types of NEFs interact with the Hsp70-NBD at different but partially overlapping sites. Other co-chaperones of Hsp70 facilitate the handover to other protein quality control systems. These include the Hsc70-interacting protein (HIP, also called ST13) and Hsp70/Hsp90-organizing protein (HOP, also called STIP1) that facilitate the cooperation of the Hsp70 and Hsp90 systems. Finally, co-factors such as the C terminus of Hsp70-interacting protein (CHIP, also called STUB1) are involved in regulating substrate degradation via the ubiquitin proteasome system (11Dekker S.L. Kampinga H.H. Bergink S. DNAJs: more than substrate delivery to HSPA.Front. Mol. Biosci. 2015; 2 (26176011): 35Crossref PubMed Scopus (30) Google Scholar). nucleotide-binding domain substrate-binding domain amyotrophic lateral sclerosis C-terminal domain J-domain protein nucleotide-exchange factor superoxide dismutase amino acid(s) post-translational modification total fraction supernatant fraction pellet fraction hemagglutinin 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium Protein Data Bank. Because of their high conservation both in evolution and within the Hsp70 family, (human) Hsp70 proteins are often regarded as largely interchangeable (12Warrick J.M. Chan H.Y. Gray-Board G.L. Chai Y. Paulson H.L. Bonini N.M. Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70.Nat. Genet. 1999; 23 (10581028): 425-42810.1038/70532Crossref PubMed Scopus (715) Google Scholar, 13Fernandez-Funez P. Sanchez-Garcia J. de Mena L. Zhang Y. Levites Y. Khare S. Golde T.E. Rincon-Limas D.E. Holdase activity of secreted Hsp70 masks amyloid-β42 neurotoxicity in Drosophila.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27531960): E5212-E522110.1073/pnas.1608045113Crossref PubMed Scopus (36) Google Scholar, 14Auluck P.K. Chan H.Y. Trojanowski J.Q. Lee V.M. Bonini N.M. Chaperone suppression of α-synuclein toxicity in a Drosophila model for Parkinson's disease.Science. 2002; 295 (11823645): 865-86810.1126/science.1067389Crossref PubMed Scopus (1010) Google Scholar, 15Wong S.L. Chan W.M. Chan H.Y. Sodium dodecyl sulfate-insoluble oligomers are involved in polyglutamine degeneration.FASEB J. 2008; 22 (18559990): 3348-335710.1096/fj.07-103887Crossref PubMed Scopus (26) Google Scholar, 16McLear J.A. Lebrecht D. Messer A. Wolfgang W.J. Combinational approach of intrabody with enhanced Hsp70 expression addresses multiple pathologies in a fly model of Huntington's disease.FASEB J. 2008; 22 (18199697): 2003-201110.1096/fj.07-099689Crossref PubMed Scopus (35) Google Scholar, 17Sojka D.R. Gogler-Pigłowska A. Vydra N. Cortez A.J. Filipczak P.T. Krawczyk Z. Scieglinska D. Functional redundancy of HSPA1, HSPA2 and other HSPA proteins in non–small cell lung carcinoma (NSCLC): an implication for NSCLC treatment.Sci. Rep. 2019; 10.1038/s41598-019-50840-7Crossref Scopus (10) Google Scholar). However, specificity between Hsp70 machines does exist, and different effects of the various Hsp70s have been reported (6Clerico E.M. Tilitsky J.M. Meng W. Gierasch L.M. How Hsp70 molecular machines interact with their substrates to mediate diverse physiological functions.J. Mol. Biol. 2015; 427 (25683596): 1575-158810.1016/j.jmb.2015.02.004Crossref PubMed Scopus (183) Google Scholar, 8Kampinga H.H. Craig E.A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity.Nat. Rev. Mol. Cell Biol. 2010; 11 (20651708): 579-59210.1038/nrm2941Crossref PubMed Scopus (1015) Google Scholar, 18Kakkar V. Meister-Broekema M. Minoia M. Carra S. Kampinga H.H. Barcoding heat shock proteins to human diseases: looking beyond the heat shock response.Dis. Model. Mech. 2014; 7 (24719117): 421-43410.1242/dmm.014563Crossref PubMed Scopus (78) Google Scholar, 19Kampinga H.H. Bergink S. Heat shock proteins as potential targets for protective strategies in neurodegeneration.Lancet Neurol. 2016; 15 (27106072): 748-75910.1016/S1474-4422(16)00099-5Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 20Lotz S.K. Knighton L.E. Nitika Jones G.W. Truman A.W. Not quite the SSAme: unique roles for the yeast cytosolic Hsp70s.Curr. Genet. 2019; 65 (31020385): 1127-113410.1007/s00294-019-00978-8Crossref PubMed Scopus (13) Google Scholar). The recognition of substrates by Hsp70 is quite generic, and because there is a lot more variability in JDPs and NEFs, the last two families have been suggested as the ones that confer specificity to the Hsp70 system (8Kampinga H.H. Craig E.A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity.Nat. Rev. Mol. Cell Biol. 2010; 11 (20651708): 579-59210.1038/nrm2941Crossref PubMed Scopus (1015) Google Scholar). However, it has not been experimentally explored whether and to what extent (human) Hsp70 are indeed interchangeable. Also, whether different Hsp70s interact with specific co-chaperone partners and, if so, what determines the functional outcome of these different Hsp70 complexes have remained unclear. Various members of the Hsp70 machinery have been identified as suppressors of protein aggregation (18Kakkar V. Meister-Broekema M. Minoia M. Carra S. Kampinga H.H. Barcoding heat shock proteins to human diseases: looking beyond the heat shock response.Dis. Model. Mech. 2014; 7 (24719117): 421-43410.1242/dmm.014563Crossref PubMed Scopus (78) Google Scholar, 19Kampinga H.H. Bergink S. Heat shock proteins as potential targets for protective strategies in neurodegeneration.Lancet Neurol. 2016; 15 (27106072): 748-75910.1016/S1474-4422(16)00099-5Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). From the Hsp70 family, only few members have been tested, mainly HSPA1A (stress-inducible Hsp70) and HSPA8 (constitutive Hsp70, HSC70), but mostly not in a comparative way. In particular, HSPA1A up-regulation has been reported as highly effective in withstanding global protein aggregation induced by unfolding events such as heat shock (21Stege G.J. Li L. Kampinga H.H. Konings A.W. Li G.C. Importance of the ATP-binding domain and nucleolar localization domain of HSP72 in the protection of nuclear proteins against heat-induced aggregation.Exp. Cell Res. 1994; 214 (8082731): 279-28410.1006/excr.1994.1259Crossref PubMed Scopus (37) Google Scholar) or aggregation of specific thermosensitive proteins such as luciferase (22Nollen E.A. Brunsting J.F. Roelofsen H. Weber L.A. Kampinga H.H. In vivo chaperone activity of heat shock protein 70 and thermotolerance.Mol. Cell Biol. 1999; 19 (10022894): 2069-207910.1128/MCB.19.3.2069Crossref PubMed Scopus (186) Google Scholar). However, neither HSPA1A nor HSPA8 up-regulation is very effective in preventing aggregation of disease-associated amyloidogenic proteins in cells, although results may vary depending on the system or the type of the substrate (18Kakkar V. Meister-Broekema M. Minoia M. Carra S. Kampinga H.H. Barcoding heat shock proteins to human diseases: looking beyond the heat shock response.Dis. Model. Mech. 2014; 7 (24719117): 421-43410.1242/dmm.014563Crossref PubMed Scopus (78) Google Scholar, 19Kampinga H.H. Bergink S. Heat shock proteins as potential targets for protective strategies in neurodegeneration.Lancet Neurol. 2016; 15 (27106072): 748-75910.1016/S1474-4422(16)00099-5Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). At least one exception to this is the aggregation of superoxide dismutase 1 (SOD1) mutants that cause amyotrophic lateral sclerosis (ALS) (23Prudencio M. Hart P.J. Borchelt D.R. Andersen P.M. Variation in aggregation propensities among ALS-associated variants of SOD1: correlation to human disease.Hum. Mol. Genet. 2009; 18 (19483195): 3217-322610.1093/hmg/ddp260Crossref PubMed Scopus (166) Google Scholar). Elevated expression of one Hsp70 (HSPA1A) has been reported to suppress mutant SOD1 aggregation (24Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. Up-regulation of protein chaperones preserves viability of cells expressing toxic Cu/Zn-superoxide dismutase mutants associated with amyotrophic lateral sclerosis.J. Neurochem. 1999; 72 (9930742): 693-69910.1046/j.1471-4159.1999.0720693.xCrossref PubMed Scopus (219) Google Scholar). Here, using two different Hsp70 clients, mutant SOD1 and a folding-impaired, mutant luciferase (25Gupta R. Kasturi P. Bracher A. Loew C. Zheng M. Villella A. Garza D. Hartl F.U. Raychaudhuri S. Firefly luciferase mutants as sensors of proteome stress.Nat. Methods. 2011; 8 (21892152): 879-88410.1038/nmeth.1697Crossref PubMed Scopus (108) Google Scholar) (R188Q,R261Q referred to as LucDM), we dissect the effects of all cytosolic and nuclear Hsp70 orthologs on protein aggregation in cells. In particular, we found that two highly homologous Hsp70s, HSPA1A and HSPA1L, have opposing effects on the fate of these two clients. Strikingly, this differential activity is explicitly attributed to differences in the NBDs of the two Hsp70s, which subsequently affect their ability to functionally interact with specific co-chaperones. These data suggest another layer of functional diversification within the Hsp70 machines in human cells, which is directed by differential Hsp70–co-chaperone binding. The different cytosolic/nuclear Hsp70 members HSPA1A (stress-inducible HSP70, HSP70–1, HSP72), HSPA1L (HSP70-like), HSPA2 (HSP70–2), HSPA6 (HSP70B), and HSPA8 (constitutive HSP70, HSC70) show high sequence conservation (Fig. S1 and Table S1) and bind similar peptide motifs (5Rüdiger S. Germeroth L. Schneider-Mergener J. Bukau B. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.EMBO J. 1997; 16 (9130695): 1501-150710.1093/emboj/16.7.1501Crossref PubMed Scopus (631) Google Scholar, 6Clerico E.M. Tilitsky J.M. Meng W. Gierasch L.M. How Hsp70 molecular machines interact with their substrates to mediate diverse physiological functions.J. Mol. Biol. 2015; 427 (25683596): 1575-158810.1016/j.jmb.2015.02.004Crossref PubMed Scopus (183) Google Scholar, 26Fourie A.M. Sambrook J.F. Gething M.J. Common and divergent peptide binding specificities of hsp70 molecular chaperones.J. Biol. Chem. 1994; 269 (7982963): 30470-30478Abstract Full Text PDF PubMed Google Scholar) and hence have often been considered as functionally interchangeable. However, we noticed that the outcome in terms of client handling could differ significantly (27Hageman J. van Waarde M.A. Zylicz A. Walerych D. Kampinga H.H. The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities.Biochem. J. 2011; 435 (21231916): 127-14210.1042/BJ20101247Crossref PubMed Scopus (113) Google Scholar, 28Kakkar V. Kuiper E.F. Pandey A. Braakman I. Kampinga H.H. Versatile members of the DNAJ family show Hsp70 dependent anti-aggregation activity on RING1 mutant parkin C289G.Sci. Rep. 2016; 6 (27713507): 3483010.1038/srep34830Crossref PubMed Scopus (17) Google Scholar, 29Hageman J. Rujano M.A. van Waarde M.A. Kakkar V. Dirks R.P. Govorukhina N. Oosterveld-Hut H.M. Lubsen N.H. Kampinga H.H. A DNAJB chaperone subfamily with HDAC-dependent activities suppresses toxic protein aggregation.Mol. Cell. 2010; 37 (20159555): 355-36910.1016/j.molcel.2010.01.001Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 30Rujano M.A. Kampinga H.H. Salomons F.A. Modulation of polyglutamine inclusion formation by the Hsp70 chaperone machine.Exp. Cell Res. 2007; 313 (17822698): 3568-357810.1016/j.yexcr.2007.07.034Crossref PubMed Scopus (43) Google Scholar). To study this in more detail, we used the well-known aggregating ALS disease–associated SOD1 mutant A4V (SOD1A4V) (23Prudencio M. Hart P.J. Borchelt D.R. Andersen P.M. Variation in aggregation propensities among ALS-associated variants of SOD1: correlation to human disease.Hum. Mol. Genet. 2009; 18 (19483195): 3217-322610.1093/hmg/ddp260Crossref PubMed Scopus (166) Google Scholar), a reported Hsp70 client (24Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. Up-regulation of protein chaperones preserves viability of cells expressing toxic Cu/Zn-superoxide dismutase mutants associated with amyotrophic lateral sclerosis.J. Neurochem. 1999; 72 (9930742): 693-69910.1046/j.1471-4159.1999.0720693.xCrossref PubMed Scopus (219) Google Scholar) as a model substrate. First, we developed a quantifiable fractionation method (Fig. 1A) to monitor aggregation of mCherry-tagged SOD1A4V. mCherry-SOD1A4V formed visible inclusions in cells and was partially detergent-insoluble after fractionation in contrast to mCherry-SOD1WT that showed diffuse expression and remained in the soluble fraction (Fig. 1, B and C, and Fig. S2A). Interestingly, expression of most Hsp70s in HEK293 cells enhanced rather than reduced SOD1A4V aggregation, and only HSPA1A showed a significant aggregation suppressing effect (Fig. 1D and Fig. S2B). Largely similar results were obtained in U2OS cells, with most Hsp70 members having either no effect or enhancing SOD1A4V aggregation and only HSPA1A leading to a significant reduction in SOD1A4V aggregation (Fig. 1E and Fig. S2C). The most striking observation was the consistent opposing effects of two of the closest paralogs HSPA1A and HSPA1L, with the former significantly reducing and the latter greatly enhancing SOD1A4V aggregation in both HEK293 and U2OS cells (Fig. 1, D and E). This opposing effect is not due to a difference in the increased expression because both Hsp70s are similarly (over)expressed (Fig. 1, D–F). SOD1A4V expression did not lead to any growth disadvantages, and neither HSPA1A nor HSPA1L influenced this (Fig. S2D). To explore whether the differential behavior of HSPA1A and HSPA1L is not limited to SOD1A4V aggregation, we investigated the impact of these two Hsp70s on the folding of GFP-tagged double mutant luciferase (GFP-LucDM) in cells (Fig. 1F). This model substrate is partly insoluble (Fig. 1F) and a Hsp70 client (Fig. S2E). When expressed in cells the GFP-LucDM fusion only displays minimal luminescence activity as compared with WT luciferase (Fig. S2F). Co-expression with HSPA1A but not HSPA1L leads to a reduction in the fraction of insoluble luciferase and in parallel to a drop in total protein levels of cellular GFP-LucDM (Fig. 1F) pointing toward a HSPA1A-driven degradation process. Because the total luminescence activity is nearly unaffected by co-expression of either HSPA1A or HSPA1L (Fig. S2F), this implies that the activity per amount of luciferase present in the cell (the “specific activity”) is elevated by the co-expression of HSPA1A (Fig. S2, G and H). Therefore, similar to mutant SOD1, in cells only HSPA1A can assist in the cellular handling of misfolded luciferase. HSPA1L and HSPA1A are 89% identical in their amino acid sequence, and most differences lie in the substrate-locking C-terminal lid domain (Fig. S1 and Table S1). HSPA1A is one of the most studied human Hsp70s, whereas not much is known about HSPA1L and its cellular functions. In contrast to HSPA1A, HSPA1L lacks a HSF-binding element in its promoter and is indeed less heat stress–inducible (31Milner C.M. Campbell R.D. Structure and expression of the three MHC-linked HSP70 genes.Immunogenetics. 1990; 32 (1700760): 242-251Crossref PubMed Scopus (380) Google Scholar). HSPA1L is expressed at low levels in most tissues (32Daugaard M. Rohde M. Jäättelä M. The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions.FEBS Lett. 2007; 581 (17544402): 3702-371010.1016/j.febslet.2007.05.039Crossref PubMed Scopus (819) Google Scholar). To further investigate why two very similar Hsp70s show such opposing effects on substrate handling, we generated chimeras to identify which part of the protein is responsible for this difference. Exchanging the NBD of HSPA1A with that of HSPA1L (NLSACA) generated a protein with HSPA1L-like activity that enhanced SOD1A4V aggregation (Fig. 2, A and B). Inversely, the chimera with the NBD of HSPA1A and the SBD and CTD of HSPA1L (NASLCL) gained an HSPA1A-like activity in suppressing SOD1A4V aggregation (Fig. 2B). This pointed toward the NBD as being responsible for the opposing effect of HSPA1A and HSPA1L on SOD1A4V aggregation. This was further confirmed because exchanging the individual SBDs or CTDs generated chimeric proteins whose activity fully depended on their NBDs (Fig. S3A). These results indicate that neither the SBD nor the CTD play a role in the differential effect of these two Hsp70s on protein aggregation. Interestingly, the SBD and especially the CTD are the most disparate domains based on the amino acid sequence (Fig. S1). Because the SBD confers substrate binding, this suggests that the difference in substrate fate cannot be attributed to differential SOD1A4V binding. Consistently, mCherry-SOD1A4V co-immunoprecipitated efficiently with both GFP-HSPA1A and GFP-HSPA1L (Fig. 2C). Of note, the GFP-tagged Hsp70s behave similarly to their V5-tagged versions (Fig. S3B). The endogenous WT SOD1 did not co-immunoprecipitate with either Hsp70, despite the similar expression level as mCherry-SOD1A4V (Fig. 2C). This underscores the specificity of both HSPA1A and HSPA1L for the mutant SOD1 protein. In agreement with our findings, the importance of the NBD as a driver for functional specificity between Hsp70s has been previously noted for yeast (33James P. Pfund C. Craig E.A. Functional specificity among Hsp 70 molecular chaperones.Science. 1997; 275 (8994035): 387-38910.1126/science.275.5298.387Crossref PubMed Scopus (182) Google Scholar) and human Hsp70s (27Hageman J. van Waarde M.A. Zylicz A. Walerych D. Kampinga H.H. The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities.Biochem. J. 2011; 435 (21231916): 127-14210.1042/BJ20101247Crossref PubMed Scopus (113) Google Scholar). The reason for this importance of the NBD is unclear. The NBDs of HSPA1A and HSPA1L share 91% sequence identity (Fig. S1). Structural alignment utilizing previously published data (34Wisniewska M. Karlberg T. Lehtiö L. Johansson I. Kotenyova T. Moche M. Schüler H. Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70–2, HSPA6/Hsp70B′, and HSPA5/BiP/GRP78.PLoS One. 2010; 5 (20072699): e862510.1371/journal.pone.0008625Crossref PubMed Scopus (105) Google Scholar) revealed that the NBDs of HSPA1A and HSPA1L are almost identical (Fig. 2D), making such a different impact on a chaperone function really remarkable. Mapping the nonconserved residues between HSPA1A and HSPA1L on HSPA1A-NBD shows that they are spread over the entire NBD structure (Fig. S3C). The ATP/ADP-binding pocket, which resides in the middle of the NBD cleft, is fully conserved between the two Hsp70s (Fig. S3C). Highlighting these nonconserved amino acids pointed out that the accessible surface between the HSPA1A-NBD and HSPA1L-NBD was only slightly different (Fig. S3D). However, there were some subtle differences that could possibly affect the interaction interface with co-chaperones without significantly altering the core structure. Exchanging two subregions, aa 1–111 (N1) and aa 112–389 (N2), of HSPA1A with the homologous regions of HSPA1L (aa 1–113 and aa 114–391 for N1 and N2, respectively) and vice versa revealed that the effects on SOD1A4V aggregation were mainly coupled to the N2 region of the NBD (Fig. 2E). To identify potential intrinsic functional differences in the ATPase cycle and biochemical activity of HSPA1A and HSPA1L, we purified both Hsp70s and NBD swaps. The intrinsic ATPase activity of each Hsp70 alone was low and equal for all four Hsp70 variants tested (Fig. 3A). The addition of increasing amounts of HSPH2 (a canonical NEF) at concentrations used in protein refolding assays accelerated the ATP cycle in a concentration-dependent manner that was similar for all variants as well (Fig. 3A). Consistent with this, the binding affinities of HSPH2 to both HSPA1A and HSPA1L were identical within error (Fig. S4A; HSPA1A, 107 ± 31 nm, and HSPA1L, 85 ± 31 nm). To corroborate this in living cells, we generated glutamate-to-glutamine mutations in the conserved ATP interaction sites Glu175 and Glu177 of HSPA1A and HSPA1L, respectively, that abrogate their nucleotide cycle (35Fontaine S.N. Rauch J.N. Nordhues B.A. Assimon V.A. Stothert A.R. Jinwal U.K. Sabbagh J.J. Chang L. Stevens Jr., S.M. Zuiderweg E.R. Gestwicki J.E. Dickey C.A. Isoform-selective genetic inhibition of constitutive cytosolic Hsp70 activity promotes client Tau degradation using an altered co-chaperone complement.J. Biol. Chem. 2015; 290 (25864199): 13115-1312710.1074/jbc.M115.637595Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Expression of either the HSPA1AE175Q or the HSPA1LE177Q mutant in cells dramatically increased SOD1A4V aggregation (Fig. S4B), confirming that a functional Hsp70 ATP hydrolysis step is crucial for SOD1A4V processing by either HSPA1A or HSPA1L. Moreover, the biochemical activities of HSPA1A and HSPA1L and their NBD swaps were indistinguishable using an in vitro protein refolding assay as readout: both variants lead to similar rates of refolding of heat-denatured luciferase (Fig. S4C). These data confirm that both HSPA1A and HSPA1L are bona fide Hsp70s with similar ATPase and biochemical activities. This implies that the opposing effects on substrate handling observed in cells lies in the cellular context in which these Hsp70s operate. Handover of certain substrates from the Hsp70 cycle to the Hsp90 system can have dramatic consequences on the fate of substrates (36Karras G.I. Yi S. Sahni N. Fischer M. Xie J. Vidal M. D'Andrea A.D. Whitesell L. Lindquist S. HSP90 shapes the consequences of human genetic variation.Cell. 2017; 168 (28215707): 856-866.e1210.1016/j.cell.2017.01.023Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 37Morán Luengo T. Kityk R. Mayer M.P. Rüdiger" @default.
• W3015589319 created "2020-04-17" @default.
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• W3015589319 date "2020-05-01" @default.
• W3015589319 modified "2023-10-18" @default.
• W3015589319 title "Functional diversity between HSP70 paralogs caused by variable interactions with specific co-chaperones" @default.
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• W3015589319 doi "https://doi.org/10.1074/jbc.ra119.012449" @default.

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