Abstract
The heat shock protein 70 (Hsp70, human HSPA1A) plays indispensable roles in cellular stress responses and protein quality control (PQC). In the framework of PQC, it cooperates with the ubiquitin-proteasome system (UPS) to clear damaged and dysfunctional proteins in the cell. Moreover, Hsp70 itself is rapidly degraded following the recovery from stress. It was demonstrated that its fast turnover is mediated via ubiquitination and subsequent degradation by the 26S proteasome. At the same time, the effect of Hsp70 on the functional state of proteasomes has been insufficiently investigated. Here, we characterized the direct effect of recombinant Hsp70 on the activity of 20S and 26S proteasomes and studied Hsp70 degradation by the 20S proteasome in vitro. We have shown that the activity of purified 20S proteasomes is decreased following incubation with recombinant human Hsp70. On the other hand, high concentrations of Hsp70 activated 26S proteasomes. Finally, we obtained evidence that in addition to previously reported ubiquitin-dependent degradation, Hsp70 could be cleaved independent of ubiquitination by the 20S proteasome. The results obtained reveal novel aspects of the interplay between Hsp70 and proteasomes.
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Abramova EB, Astakhova TM, Erokhov PA, Sharova NP (2004) Multiple forms of the proteasomes and some approaches to their separation Izvestiia Akademii nauk Seriia biologicheskaia / Rossiiskaia akademiia nauk:150–156
Abramova EB, Sharova NP, Karpov VL (2002) The proteasome: destroy to live. Mol Biol 36:761–776
Alberti S, Esser C, Hohfeld J (2003) BAG-1—a nucleotide exchange factor of Hsc70 with multiple cellular functions. Cell Stress Chaperones 8:225–231
Alexandrova A, Petrov L, Georgieva A, Kirkova M, Kukan M (2008) Effects of proteasome inhibitor, MG132, on proteasome activity and oxidative status of rat liver. Cell Biochem Funct 26:392–398. doi:10.1002/cbf.1459
Arlt A et al (2009) Increased proteasome subunit protein expression and proteasome activity in colon cancer relate to an enhanced activation of nuclear factor E2-related factor 2 (Nrf2). Oncogene 28:3983–3996. doi:10.1038/onc.2009.264
Asher G, Tsvetkov P, Kahana C, Shaul Y (2005) A mechanism of ubiquitin-independent proteasomal degradation of the tumor suppressors p53 and p73. Genes Dev 19:316–321. doi:10.1101/gad.319905
Ballinger CA, Connell P, Wu Y, Hu Z, Thompson LJ, Yin LY, Patterson C (1999) Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions. Mol Cell Biol 19:4535–4545
Bardwell JC, Jakob U (2012) Conditional disorder in chaperone action. Trends Biochem Sci 37:517–525. doi:10.1016/j.tibs.2012.08.006
Baugh JM, Viktorova EG, Pilipenko EV (2009) Proteasomes can degrade a significant proportion of cellular proteins independent of ubiquitination. J Mol Biol 386:814–827. doi:10.1016/j.jmb.2008.12.081
Ben-Nissan G, Sharon M (2014) Regulating the 20S proteasome ubiquitin-independent degradation pathway. Biomol Ther 4:862–884. doi:10.3390/biom4030862
Bendotti C, Marino M, Cheroni C, Fontana E, Crippa V, Poletti A, De Biasi S (2012) Dysfunction of constitutive and inducible ubiquitin-proteasome system in amyotrophic lateral sclerosis: implication for protein aggregation and immune response. Prog Neurobiol 97:101–126. doi:10.1016/j.pneurobio.2011.10.001
Bentea E, Verbruggen L, Massie A (2016) The proteasome inhibition model of Parkinson’s disease. J Park Dis. doi:10.3233/jpd-160921
Bobkova NV et al (2015) Exogenous Hsp70 delays senescence and improves cognitive function in aging mice. Proc Natl Acad Sci U S A 112:16006–16011. doi:10.1073/pnas.1516131112
Bobkova NV et al (2014) Therapeutic effect of exogenous hsp70 in mouse models of Alzheimer’s disease. J Alzheimer’s Dis: JAD 38:425–435. doi:10.3233/jad-130779
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Chen B, Retzlaff M, Roos T, Frydman J (2011) Cellular strategies of protein quality control. Cold Spring Harb Perspect Biol 3:a004374. doi:10.1101/cshperspect.a004374
Ciechanover A, Kwon YT (2015) Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies. Exp Mol Med 47:e147. doi:10.1038/emm.2014.117
Evans CG, Wisen S, Gestwicki JE (2006) Heat shock proteins 70 and 90 inhibit early stages of amyloid beta-(1-42) aggregation in vitro. J Biol Chem 281:33182–33191. doi:10.1074/jbc.M606192200
Evgen’ev MB, Garbuz DG, Zatsepina OG (2014) Heat shock proteins and whole body adaptation to extreme environments. Springer,
Fabre B et al (2014) Label-free quantitative proteomics reveals the dynamics of proteasome complexes composition and stoichiometry in a wide range of human cell lines. J Proteome Res 13:3027–3037. doi:10.1021/pr500193k
Ferrington DA, Gregerson DS (2012) Immunoproteasomes: structure, function, and antigen presentation. Prog Mol Biol Transl Sci 109:75–112. doi:10.1016/b978-0-12-397863-9.00003-1
Gestwicki JE, Garza D (2012) Protein quality control in neurodegenerative disease. Prog Mol Biol Transl Sci 107:327–353. doi:10.1016/b978-0-12-385883-2.00003-5
Gifondorwa DJ et al (2007) Exogenous delivery of heat shock protein 70 increases lifespan in a mouse model of amyotrophic lateral sclerosis. J Neurosci Off J Soc Neurosci 27:13173–13180. doi:10.1523/jneurosci.4057-07.2007
Grune T, Catalgol B, Licht A, Ermak G, Pickering AM, Ngo JK, Davies KJ (2011) HSP70 mediates dissociation and reassociation of the 26S proteasome during adaptation to oxidative stress. Free Radic Biol Med 51:1355–1364. doi:10.1016/j.freeradbiomed.2011.06.015
Gurskiy YG et al (2016) The development of modified human Hsp70 (HSPA1A) and its production in the milk of transgenic mice. Cell Stress Chaperones 21:1055–1064. doi:10.1007/s12192-016-0729-x
Hartl FU, Bracher A, Hayer-Hartl M (2011) Molecular chaperones in protein folding and proteostasis. Nature 475:324–332. doi:10.1038/nature10317
Jung T, Grune T (2013) The proteasome and the degradation of oxidized proteins: part I-structure of proteasomes. Redox Biol 1:178–182. doi:10.1016/j.redox.2013.01.004
Kalmar B, Edet-Amana E, Greensmith L (2012) Treatment with a coinducer of the heat shock response delays muscle denervation in the SOD1-G93A mouse model of amyotrophic lateral sclerosis. Amyotroph Lateral Scler : Off Publ World Fed Neurol Res Group on Motor Neuron Dis 13:378–392. doi:10.3109/17482968.2012.660953
Kisselev AF, Akopian TN, Woo KM, Goldberg AL (1999) The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes. Implications for understanding the degradative mechanism and antigen presentation. J Biol Chem 274:3363–3371
Kisselev AF, Kaganovich D, Goldberg AL (2002) Binding of hydrophobic peptides to several non-catalytic sites promotes peptide hydrolysis by all active sites of 20 S proteasomes. Evidence for peptide-induced channel opening in the alpha-rings. J Biol Chem 277:22260–22270. doi:10.1074/jbc.M112360200
Li CY, Lee JS, Ko YG, Kim JI, Seo JS (2000) Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation. J Biol Chem 275:25665–25671. doi:10.1074/jbc.M906383199
Li Z, Srivastava P (2004) Heat-shock proteins. Current protocols in immunology / edited by John E Coligan [et al] Appendix 1:Appendix 1T doi:10.1002/0471142735.ima01ts58
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Lyupina YV, Bogatyrev ME, Orlova A, Marjukhnich EV, Kazansky DB, Sharova NP (2013) Proteasomes in the brain of beta2-microglobulin knockout mice. Biochemistry Biokhimiia 78:1124–1133. doi:10.1134/s0006297913100064
Mitchell HK, Petersen NS, Buzin CH (1985) Self-degradation of heat shock proteins. Proc Natl Acad Sci U S A 82:4969–4973
Moorthy AK, Savinova OV, Ho JQ, Wang VY, Vu D, Ghosh G (2006) The 20S proteasome processes NF-kappaB1 p105 into p50 in a translation-independent manner. EMBO J 25:1945–1956. doi:10.1038/sj.emboj.7601081
Morozov A, Kulikova AA, Astakhova TM, Mitkevich VA, Burnysheva KM, Adzhubei AA, Erokhov PA, Evgen’ev MB, Sharova NP, Karpov VL, Makarov AA (2016) Amyloid-β increases activity of proteasomes capped with 19S and 11S regulators. J Alzheimers Dis 54 doi:10.3233/JAD-160491
Morozov AV, Yurinskaya MM, Mitkevich VA, Garbuz DG, Preobrazhenskaia OV, Vinokurov MG, Evgen’ev MB, Karpov VL, Makarov AA (2017) Heat-shock protein HSP70 decreases activity of proteasomes in human neuroblastoma cells, treated by amyloid-beta 1-42 with isomerized Asp7. Mol Biol 51:143–147
Multhoff G, Hightower LE (2011) Distinguishing integral and receptor-bound heat shock protein 70 (Hsp70) on the cell surface by Hsp70-specific antibodies. Cell Stress Chaperones 16:251–255. doi:10.1007/s12192-010-0247-1
Murata S, Minami Y, Minami M, Chiba T, Tanaka K (2001) CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein. EMBO Rep 2:1133–1138. doi:10.1093/embo-reports/kve246
Nijhuis EH, Poot AA, Feijen J, Vermes I (2008) Hsp70- and p53-reponses after heat treatment and/or X-irradiation mediate the susceptibility of hematopoietic cells to undergo apoptosis. Int J Radiat Biol 84:99–105. doi:10.1080/09553000701817084
Pickering AM, Davies KJ (2012) Degradation of damaged proteins: the main function of the 20S proteasome. Prog Mol Biol Transl Sci 109:227–248. doi:10.1016/b978-0-12-397863-9.00006-7
Pickering AM, Koop AL, Teoh CY, Ermak G, Grune T, Davies KJ (2010) The immunoproteasome, the 20S proteasome and the PA28alphabeta proteasome regulator are oxidative-stress-adaptive proteolytic complexes. Biochem J 432:585–594. doi:10.1042/bj20100878
Qian SB, McDonough H, Boellmann F, Cyr DM, Patterson C (2006) CHIP-mediated stress recovery by sequential ubiquitination of substrates and Hsp70. Nature 440:551–555. doi:10.1038/nature04600
Radons J (2016) The human HSP70 family of chaperones: where do we stand? Cell Stress Chaperones 21:379–404. doi:10.1007/s12192-016-0676-6
Richter K, Haslbeck M, Buchner J (2010) The heat shock response: life on the verge of death. Mol Cell 40:253–266. doi:10.1016/j.molcel.2010.10.006
Ross CA, Poirier MA (2004) Protein aggregation and neurodegenerative disease. Nat Med 10(Suppl):S10–S17. doi:10.1038/nm1066
Rozhkova E et al (2010) Exogenous mammalian extracellular HSP70 reduces endotoxin manifestations at the cellular and organism levels. Ann N Y Acad Sci 1197:94–107. doi:10.1111/j.1749-6632.2009.05375.x
Samali A, Cotter TG (1996) Heat shock proteins increase resistance to apoptosis. Exp Cell Res 223:163–170. doi:10.1006/excr.1996.0070
Schmidt M, Finley D (2014) Regulation of proteasome activity in health and disease. Biochim Biophys Acta 1843:13–25. doi:10.1016/j.bbamcr.2013.08.012
Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860. doi:10.1038/nprot.2006.468
Shiber A, Ravid T (2014) Chaperoning proteins for destruction: diverse roles of Hsp70 chaperones and their co-chaperones in targeting misfolded proteins to the proteasome. Biomol Ther 4:704–724. doi:10.3390/biom4030704
Smock RG, Blackburn ME, Gierasch LM (2011) Conserved, disordered C terminus of DnaK enhances cellular survival upon stress and DnaK in vitro chaperone activity. J Biol Chem 286:31821–31829. doi:10.1074/jbc.M111.265835
Soss SE, Rose KL, Hill S, Jouan S, Chazin WJ (2015) Biochemical and proteomic analysis of ubiquitination of Hsc70 and Hsp70 by the E3 ligase CHIP. PLoS One 10:e0128240. doi:10.1371/journal.pone.0128240
Tanahashi N, Murakami Y, Minami Y, Shimbara N, Hendil KB, Tanaka K (2000) Hybrid proteasomes. Induction by interferon-gamma and contribution to ATP-dependent proteolysis. J Biol Chem 275:14336–14345
Tseng BP, Green KN, Chan JL, Blurton-Jones M, LaFerla FM (2008) Abeta inhibits the proteasome and enhances amyloid and tau accumulation. Neurobiol Aging 29:1607–1618. doi:10.1016/j.neurobiolaging.2007.04.014
Turturici G, Sconzo G, Geraci F (2011) Hsp70 and its molecular role in nervous system diseases. Biochem Res Int 2011:618127. doi:10.1155/2011/618127
Uversky VN (2011) Flexible nets of malleable guardians: intrinsically disordered chaperones in neurodegenerative diseases. Chem Rev 111:1134–1166. doi:10.1021/cr100186d
Wacker JL, Zareie MH, Fong H, Sarikaya M, Muchowski PJ (2004) Hsp70 and Hsp40 attenuate formation of spherical and annular polyglutamine oligomers by partitioning monomer. Nat Struct Mol Biol 11:1215–1222. doi:10.1038/nsmb860
Wang AM et al (2013) Activation of Hsp70 reduces neurotoxicity by promoting polyglutamine protein degradation. Nat Chem Biol 9:112–118. doi:10.1038/nchembio.1140
Westhoff B, Chapple JP, van der Spuy J, Hohfeld J, Cheetham ME (2005) HSJ1 is a neuronal shuttling factor for the sorting of chaperone clients to the proteasome. Curr Biol 15:1058–1064. doi:10.1016/j.cub.2005.04.058
Yurinskaya M et al (2015a) The fate of exogenous human HSP70 introduced into animal cells by different means. Curr Drug Deliv 12:524–532
Yurinskaya MM, Mit’kevich VA, Barykin EP, Garbuz DG, Evgen’ev MB, Makarov AA, Vinokurov MG (2015b) Heat-shock protein HSP70 protects neuroblastoma cells SK-N-SH from the neurotoxic effects hydrogen peroxide and the beta-amyloid peptide. Mol Biol 49:1030–1034. doi:10.7868/s0026898415060233
Zhang P, Leu JI, Murphy ME, George DL, Marmorstein R (2014) Crystal structure of the stress-inducible human heat shock protein 70 substrate-binding domain in complex with peptide substrate. PLoS One 9:e103518. doi:10.1371/journal.pone.0103518
Zhao X, Yang J (2010) Amyloid-beta peptide is a substrate of the human 20S proteasome. ACS Chem Neurosci 1:655–660. doi:10.1021/cn100067e
Zierhut B et al (2004) Heat shock protein 70 (Hsp70) subtype expression in neuroendocrine tissue and identification of a neuroendocrine tumour-specific Hsp70 truncation. Endocr Relat Cancer 11:377–389
Acknowledgements
Authors would like to thank Dr. Vladimir Morozov for important suggestions and fruitful discussions. We are grateful to Dr. Marina Serebryakova for the assistance with MALDI MS. MALDI MS facility became available to us in the framework of the Moscow State University Development Program PNG 5.13.
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The investigation of proteasome activity in the culture cells and extracts was supported by Ministry of Education and Science of Russian Federation (agreement no. 14.Z50.31.0014) Russian Science Foundation grant (14-50-00060) and Russian President Foundation grant (МК-3613.2017.4) to A.M.
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Morozov, A.V., Astakhova, T.M., Garbuz, D.G. et al. Interplay between recombinant Hsp70 and proteasomes: proteasome activity modulation and ubiquitin-independent cleavage of Hsp70. Cell Stress and Chaperones 22, 687–697 (2017). https://doi.org/10.1007/s12192-017-0792-y
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DOI: https://doi.org/10.1007/s12192-017-0792-y