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• W2039643915 abstract "By using a yeast functional complementation assay, we have identified AtTDX, a new Arabidopsis thaliana gene, encoding a two-domain 42-kDa protein. The amino-terminal domain of AtTDX is closely related to the co-chaperone Hsp70-interacting protein HIP, whereas its carboxyl-terminal part contains a thioredoxin domain. Both in vivo and in vitro assays showed that AtTDX is a protein-disulfide reductase. We next found that the HIP domain of AtTDX is capable of interacting with the ATPase domain of Ssb2, a yeast heat-shock protein 70 chaperone. Strikingly, the AtTDX-Ssb2 interaction can be released under oxidative stress, a redox-dependent regulation involving the thioredoxin activity of AtTDX. A mutation inactivating the cysteine 20 of the ATPase domain of Ssb2 was found to stabilize the AtTDX-Ssb2 interaction that becomes redox-insensitive. As cysteine 20 is conserved in virtually all the Hsp70 chaperones, our results suggest that this residue might be more generally the target of redox regulations of chaperone binding activity. By using a yeast functional complementation assay, we have identified AtTDX, a new Arabidopsis thaliana gene, encoding a two-domain 42-kDa protein. The amino-terminal domain of AtTDX is closely related to the co-chaperone Hsp70-interacting protein HIP, whereas its carboxyl-terminal part contains a thioredoxin domain. Both in vivo and in vitro assays showed that AtTDX is a protein-disulfide reductase. We next found that the HIP domain of AtTDX is capable of interacting with the ATPase domain of Ssb2, a yeast heat-shock protein 70 chaperone. Strikingly, the AtTDX-Ssb2 interaction can be released under oxidative stress, a redox-dependent regulation involving the thioredoxin activity of AtTDX. A mutation inactivating the cysteine 20 of the ATPase domain of Ssb2 was found to stabilize the AtTDX-Ssb2 interaction that becomes redox-insensitive. As cysteine 20 is conserved in virtually all the Hsp70 chaperones, our results suggest that this residue might be more generally the target of redox regulations of chaperone binding activity. Thioredoxin (TRX) 1The abbreviations used are: TRX, thioredoxin; ROS, reactive oxygen species; GST, glutathioneS-transferase; HA, hemagglutinin; TPR, tetratrico peptide repeat 1The abbreviations used are: TRX, thioredoxin; ROS, reactive oxygen species; GST, glutathioneS-transferase; HA, hemagglutinin; TPR, tetratrico peptide repeat is a small 12-kDa ubiquitous protein containing two redox-active half-cystine residues in an active center with conserved amino acid sequence Cys-X-X-Cys (where Xindicates various amino acids) that functions as a protein-disulfide reductase. The two cysteine residues in the active site provide the sulfhydryl groups involved in the thioredoxin-dependent reducing activity. Under an oxidized form, the TRX-S2protein contains a disulfide bridge within the active site that is reduced to a TRX-(SH2) dithiol by NADPH and the flavoprotein TRX reductase (for review, see Ref. 1Meyer Y. Miginiac-Maslow M. Schürmann P. Jacquot J.-P. McManus M.T. Laing W.A. Allan A.C. The Annual Plant Reviews. Sheffield Academic Press, Sheffield, UK2002: 1-23Google Scholar). Under this reduced form, thioredoxin becomes a very powerful reductant of disulfide bridges in target proteins.Thioredoxin was initially characterized from Escherichia coli extracts as a hydrogen donor for ribonucleotide reductase (2Holmgren A. Trends Biochem. Sci. 1981; 6: 26-29Abstract Full Text PDF Scopus (84) Google Scholar). Thioredoxins were later shown to act as hydrogen donors to peroxiredoxins that reduce hydrogen peroxide (3Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar) and to methionine-sulfoxide reductases that reactivate proteins damaged by stresses that generate reactive oxygen species (ROS) (4Fernando M.R. Nanri H. Yoshitake S. Nagato-Kuno K. Minakami S. Eur. J. Biochem. 1992; 209: 917-922Crossref PubMed Scopus (200) Google Scholar). They are necessary for a number of other metabolic enzymes that form a disulfide as part of their catalytic cycle (5Reitsch A. Beckwith J. Annu. Rev. Genet. 1998; 32: 163-184Crossref PubMed Scopus (237) Google Scholar). In addition, thioredoxins induce conformational changes of the targeted proteins by disulfide bond reduction and assist in the protein folding pathway (6Pigiet V.P. Schuster B.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7643-7647Crossref PubMed Scopus (123) Google Scholar). Thioredoxins directly or indirectly interact with several nuclear factors, such as the mammalian transcription factor NFκB and Ref-1 (see Ref. 7Hirota K. Matsui M. Iwata S. Nishiyama A. Mori K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3633-3638Crossref PubMed Scopus (719) Google Scholar and references therein). Thioredoxins play important roles in cell cycle and division and embryogenesis (8Salz H.K. Flickinger T.W. Mittendorf E. Pellicena-Palle A. Petschek J.P. Albrecht E.B. Genetics. 1994; 136: 1075-1086Crossref PubMed Google Scholar) and inhibit spontaneous apoptosis in tumoral cells (9Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2056) Google Scholar). Mammalian thioredoxin has also been shown to associate directly with nuclear receptors such as the glucocorticoid receptor GR (10Makino Y. Yoshikawa N. Okamoto K. Hirota K. Yodoi J. Makino I. Tanaka H. J. Biol. Chem. 1999; 274: 3182-3188Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), whose regulation requires cooperation between heat-shock proteins such as Hsp70 and Hsp90 (11Buchner J. Trends Biochem. Sci. 1999; 24: 136-141Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar).Hsp70 and Hsp90 are molecular chaperones that are involved in many important biological processes by appropriately folding nascent polypeptides on ribosomes as well as by assembling multisubunit protein complexes (12Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3090) Google Scholar). Chaperones also play a pivotal role by renaturing proteins after exposure to various stresses, driving protein translocation across membranes, and disassembling protein complexes prior to protein degradation. Molecular chaperones often function together, and in this respect, members of the Hsp70 family are of prime importance. They are highly conserved from bacteria to human and exhibit a well defined structure as follows: the 44-kDa amino-terminal part of the protein binds nucleotides, whereas the 28-kDa carboxyl-terminal domain interacts with misfolded or partially unfolded polypeptides (13Boorstein W.R. Ziegelhoffer T. Craig E.A. J. Mol. Evol. 1994; 38: 1-17Crossref PubMed Scopus (420) Google Scholar).The chaperone activity of Hsp70 is regulated by cofactors that catalyze the interconversion between the ATP and ADP states (14Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2404) Google Scholar). In bacteria, cycling of the Hsp70 homologue DnaK between different nucleotide states is regulated by the chaperone cofactors DnaJ and GrpE (15McCarty J.S. Buchberger A. Reinstein J. Bukau B. J. Mol. Biol. 1995; 249: 126-137Crossref PubMed Scopus (349) Google Scholar). DnaJ stimulates the Hsp70 ATPase activity, and the conversion of Hsp70 into the ADP-bound state allows it to interact with polypeptide substrates (16Liberek K. Marszalek J. Ang D. Georgopoulos C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Crossref PubMed Scopus (684) Google Scholar). In contrast, GrpE binds to the ATPase domain of DnaK and triggers the release of ADP, hence accelerating substrate dissociation upon ATP re-binding (17Szabo A. Langer T. Schroder H. Flanagan J. Bukau B. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10345-10349Crossref PubMed Scopus (443) Google Scholar). In eukaryotes, the yeast DnaJ homologue Hsp40/Zuo1 (18Yan W. Schilke B. Pfund C. Walter W. Kim S. Craig E.A. EMBO J. 1998; 17: 4809-4817Crossref PubMed Scopus (131) Google Scholar) and the mammalian Hsp40/Hdj (19Michels A.A. Kanon B. Konings A.W.T. Ohtsuka K. Bensaude O. Kampinga H.H. J. Biol. Chem. 1997; 272: 33283-33289Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) have also been shown to associate with Hsp70. However, structural homologues of GrpE are limited to compartments of prokaryotic origin, i.e. mitochondria and chloroplasts (20Stuart R.A. Cyr D.M. Craig E.A. Neupert W. Trends Biochem. Sci. 1994; 19: 87-92Abstract Full Text PDF PubMed Scopus (157) Google Scholar). Mammals contain other classes of Hsp70 co-chaperones, such as the p48/Hip (Hsc70-interacting protein), BAG-1/Hap (Hsp70-activatingprotein), and CHIP (carboxyl terminus ofHsc70-interacting protein) (21Höhfeld J. Minami Y. Hartl F.U. Cell. 1995; 83: 589-598Abstract Full Text PDF PubMed Scopus (378) Google Scholar, 22Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (336) Google Scholar, 23Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.J. Yin L.Y. Patterson C. Mol. Cell. Biol. 1999; 19: 4535-4545Crossref PubMed Scopus (741) Google Scholar) cofactors. BAG-1 and Hop accelerate ADP release and stimulate the re-binding of ATP. CHIP decreases net ATPase activity and reduces chaperone efficiency, thus negatively regulating the Hsp-substrate binding cycle. In contrast, Hip stabilizes the ADP-bound form of Hsp70, extending the time window during which Hsc70 interacts stably with a polypeptide substrate. All these co-chaperones have a spatial distribution that is critical to the coordination of Hsp70 functions, because they ensure that substrates are both bound and released at an appropriate place and time by regulating the ATP/ADP cycle (24Pilon M. Schekman R. Cell. 1999; 97: 679-682Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).In this report, we describe the molecular cloning and characterization of a novel bipartite protein from Arabidopsis thaliana that may be a new component of the Hsp70 chaperone system. This protein exhibits a unique domain structure with a carboxyl-terminal thioredoxin domain, the amino-terminal domain containing three tetratricopeptide repeats similar to that of the rat and human Hip (21Höhfeld J. Minami Y. Hartl F.U. Cell. 1995; 83: 589-598Abstract Full Text PDF PubMed Scopus (378) Google Scholar). We termed this new protein AtTDX for Tetratricopeptidedomain-containing thioredoxin. We present evidence that AtTDX displays a disulfide reductase activity bothin vitro and in vivo due to its thioredoxin domain, whereas its amino terminus interacts specifically with the yeast Hsp70 Ssb2 protein. We show that the interaction between AtTDX and Ssb2 is sensitive to the redox status, and we demonstrate that the thioredoxin domain of AtTDX acts as a redox switch that turns the complex with Ssb2 on and off. We also show that a conserved cysteine in the ATPase domain of Ssb2 is required for disruption of the complex with AtTDX. The work we present here provides the first example of a redox-dependent interaction of a thioredoxin-like protein with a member of the Hsp70 family.DISCUSSIONDuring the last 10 years, an increasing number of genes encoding members of the thioredoxin family have been reported in all prokaryotic and eukaryotic organisms. In plants, the complexity of the thioredoxin reductase/thioredoxin systems was first shown by the sequencing ofA. thaliana expressed sequence tags that demonstrated the presence of at least five thioredoxin h-encoding genes in this organism (35Rivera-Madrid R. Mestres D. Marinho P. Jacquot J.-P. Decottignies P. Miginiac-Maslow M. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5620-5624Crossref PubMed Scopus (113) Google Scholar). Today, more than 18 thioredoxin and thioredoxin-like sequences have been found in A. thaliana (43Meyer Y. Vignols F. Reichheld J.-P. Methods Enzymol. 2001; 347: 394-402Crossref Scopus (118) Google Scholar). Here, we report the cloning and the characterization of AtTDX, a novel and striking member of the thioredoxin family from A. thaliana. AtTDX is the first member of the thioredoxin family described to date that possesses an extra domain with tetratricopeptide repeats. This domain interacts strongly with the ATPase domain of Ssb2, a member of the Hsp70 family. Moreover, the thioredoxin active site of AtTDX and a conserved cysteine residue within the ATPase domain of Ssb2 were shown to mediate the release of the AtTDX-Ssb2 interaction under oxidative stress conditions.AtTDX, a New Thioredoxin-like TPR-containing ProteinBy using yeast mutant cells that do not express endogenous thioredoxin, we have isolated by functional complementation AtTDX, a novel thioredoxin containing protein from A. thaliana. The AtTDX protein displays an interesting bipartite structure encompassing both a thioredoxin domain and a TPR repeat containing domain is highly similar to the co-chaperone Hip protein. Both in vitro and in vivo assays demonstrated that AtTDX has disulfide reductase activity and therefore that AtTDX could be assigned to the thioredoxin superfamily. In both prokaryotic and eukaryotic cells, several proteins containing a thioredoxin catalytic domain fused to an unrelated extra domain have been described (43Meyer Y. Vignols F. Reichheld J.-P. Methods Enzymol. 2001; 347: 394-402Crossref Scopus (118) Google Scholar). However, the function of the associated domain has been established in two instances only, the human cytosolic PICOT, a 37-kDa protein kinase C-interacting protein with amino-terminal thioredoxin domain (44Witte S. Villalba M. Bi K. Liu Y. Isakov N. Altman A. J. Biol. Chem. 2000; 275: 1902-1909Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), and the plant APS reductases, three 50-kDa exhibiting a carboxyl-terminal thioredoxin domain (45Gutierrez-Marcos J.F. Roberts M.A. Campbell E.I. Wray J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13377-13382Crossref PubMed Scopus (125) Google Scholar). The functional characterization of such proteins was of particular interest because it demonstrated coordinated functions between each domain.Among the members of the thioredoxin family, AtTDX is the first described as comprising TPR repeats associated to the thioredoxin domain. TPRs are 34-amino acid motifs that were originally identified in yeast (46Hirano T.N. Kinoshita N. Morikawa K. Yanagida M. Cell. 1990; 60: 319-328Abstract Full Text PDF PubMed Scopus (238) Google Scholar, 47Sikorski R.S. Boguski M.S. Goebl M. Hieter P. Cell. 1990; 60: 307-317Abstract Full Text PDF PubMed Scopus (391) Google Scholar) and then found in a large number of both prokaryotic and eukaryotic proteins (36Lamb J.R. Tugendreich S. Heiter P. Trends Biochem. Sci. 1995; 20: 257-259Abstract Full Text PDF PubMed Scopus (547) Google Scholar). TPR repeats mediate protein-protein interactions. TPR-containing proteins play diverse roles in many cellular processes like cell cycle regulation, transcriptional repression, heat shock response, protein kinase inhibition, and peroxisomal protein transport (36Lamb J.R. Tugendreich S. Heiter P. Trends Biochem. Sci. 1995; 20: 257-259Abstract Full Text PDF PubMed Scopus (547) Google Scholar). It is noteworthy that the TPR domain of AtTDX is more particularly related to TPR proteins known to interact with members of the heat-shock protein family. In particular, we found that the TPR repeats of AtTDX are highly related to those of Hip and Sti1p (the yeast Hop counterpart), with Hip being a co-chaperone that specifically binds to Hsp70 and Hop providing a physical link between Hsp70 and Hsp90 (48Johnson B.D. Schumacher R.J. Ross E.D. Toft O. J. Biol. Chem. 1998; 273: 3679-3686Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). In addition, AtTDX displays a significant degree of similarity with TPR domains of protein phosphatase 5, cyclophilin CyP40, and FKBP52, all factors known to interact with Hsp90 chaperones (11Buchner J. Trends Biochem. Sci. 1999; 24: 136-141Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar).AtTDX Is Capable of Interacting with a Yeast Hsp70 ChaperoneThe homology of AtTDX with HIP suggested that AtTDX could interact with heat-shock proteins through its TPR repeats. By using a heterologous two-hybrid strategy, we indeed isolated one yeast protein capable of interacting with AtTDX, the Ssb2 protein, a member of the yeast Hsp70 family (38Nelson R.J. Ziegelhoffer T. Nicolet C. Werner-Washburne M. Craig E.A. Cell. 1992; 71: 97-105Abstract Full Text PDF PubMed Scopus (426) Google Scholar).Interaction domain mapping experiments favor the hypothesis that the AtTDX-Ssb2 interaction is specific and does not result from the targeting of AtTDX by Ssb2 in response to overproduction and/or misfolding. Indeed, we show here that the ATPase domain of Ssb2 mediates its interaction with AtTDX, whereas it is well established that misfolded proteins are selectively recognized by the peptide-binding domain of Hsp70 chaperones (13Boorstein W.R. Ziegelhoffer T. Craig E.A. J. Mol. Evol. 1994; 38: 1-17Crossref PubMed Scopus (420) Google Scholar). Furthermore, we show that a single cysteine replacement within the thioredoxin active site of AtTDX strongly enhances the AtTDX-Ssb2 interaction, whereas it was shown that such a mutation has a very limited impact on thioredoxin structure (49Qin J. Clore G.M. Poindexter Kennedy W.M. Huth J.R. Gronenborn A.M. Structure. 1995; 3: 289-297Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 50Wynn R. Cocco M.J. Richards F.M. Biochemistry. 1995; 34: 11807-11813Crossref PubMed Scopus (35) Google Scholar). Finally, we searched for Hsp70 genes inA. thaliana data bases and found that theAtHsc70-1 gene (51Wu S.H. Wang C. Chen J. Lin B.L. Plant Mol. Biol. 1994; 25: 577-583Crossref PubMed Scopus (26) Google Scholar) encodes the closest A. thaliana homologue of Ssb2. The two proteins Ssb2 and AtHsc70-1 share 53% identical residues (68% similarity). We cloned theAtHsc70-1 gene into the pADGal4 vector and succeeded in obtaining a two-hybrid interaction with AtTDX, even weaker than that involving Ssb2 (not shown). These results, considered as a whole, are strong evidence that AtTDX is a new Hsp70 interactant.Surprisingly, we did not isolate any clone encoding Hsp70 proteins from the Ssa family in our two-hybrid screens. Ssa and Ssb share 65% identity (more than 72% of similarity) in their ATPase domain, which is precisely the region of Ssb2 that interacts with AtTDX. In our screens, only positive clones strongly interacting with AtTDX were selected, and one can imagine that proteins interacting with AtTDX with a low specificity were not retained. We also did not isolate clones encoding members of the Hsp90 proteins, because of the high degree of similarity between the TPR repeats of AtTDX and the Hop/Sti1 proteins. The absence of such proteins among the positives clones suggests that AtTDX does not act as a physical link between Hsp70 and Hsp90 proteins, as Hop does (48Johnson B.D. Schumacher R.J. Ross E.D. Toft O. J. Biol. Chem. 1998; 273: 3679-3686Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar).The fact that AtTDX interacts with Ssb2 and with an A. thaliana Ssb homologue raises the question as to how the AtTDX function correlates with Hsp70 chaperone activity. Ssb proteins are ribosome-associated chaperones that interact with nascent chain and likely play an important role in early protein folding events (38Nelson R.J. Ziegelhoffer T. Nicolet C. Werner-Washburne M. Craig E.A. Cell. 1992; 71: 97-105Abstract Full Text PDF PubMed Scopus (426) Google Scholar, 52Pfund C. Huang P. Lopez-Hoyo N. Craig E.A. Mol. Biol. Cell. 2001; 12: 3773-3782Crossref PubMed Scopus (58) Google Scholar). Several partner proteins that regulate Hsp70 function as a molecular chaperone have already been identified, including positive (Hsp40 and HIP) and negative (CHIP and Bag-1) regulators (18Yan W. Schilke B. Pfund C. Walter W. Kim S. Craig E.A. EMBO J. 1998; 17: 4809-4817Crossref PubMed Scopus (131) Google Scholar, 21Höhfeld J. Minami Y. Hartl F.U. Cell. 1995; 83: 589-598Abstract Full Text PDF PubMed Scopus (378) Google Scholar, 22Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (336) Google Scholar, 23Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.J. Yin L.Y. Patterson C. Mol. Cell. Biol. 1999; 19: 4535-4545Crossref PubMed Scopus (741) Google Scholar). HIP and Bag-1, like AtTDX, interact with the Hsp70 ATPase domain (21Höhfeld J. Minami Y. Hartl F.U. Cell. 1995; 83: 589-598Abstract Full Text PDF PubMed Scopus (378) Google Scholar, 54Takayama S. Bimston D.N. Matsuzawa S. Freeman B.C. Aime-Sempe C. Xie Z. Morimoto R.I. Reed J.C. EMBO J. 1997; 16: 4887-4896Crossref PubMed Scopus (435) Google Scholar) and functionally compete in regulating the in vivo chaperone activity of Hsp70 (55Nollen E.A. Kabakov A.E. Brunsting J.F. Kanon B. Hohfeld J. Kampinga H.H. J. Biol. Chem. 2001; 276: 4677-4682Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), within the cycle between the ADP- and ATP-bound states. Interestingly, AtTDX and AtHIP are highly homologous in their amino-terminal interacting TPR region. Because both are expressed inA. thaliana, one could imagine that they sequentially interact with some common Hsp70 at different times and/or in different circumstances during the plant life. The next step in understanding AtTDX function will be to investigate in which of the ATP/ADP states Ssb2 interacts with AtTDX and whether AtTDX binds to Ssb2 by displacing Hsp70 cofactors or by synergy with other co-chaperones. This could bring new elements to determine whether AtTDX as a new Hsp70 co-chaperone.A Redox Switch Dissociates the AtTDX-Hsp70 ComplexOne major result from our two-hybrid experiments indicated that the AtTDX-Ssb2 interaction is specifically released upon oxidative stress. Both the removal of the thioredoxin domain from AtTDX and the replacement of one cysteine from the thioredoxin active site by a serine residue renders the AtTDX-Ssb2 interaction insensitive to oxidative stress. This is the demonstration that the thioredoxin domain of AtTDX is directly involved in the oxidative process that dissociates AtTDX from Ssb2. Furthermore, we showed that Cys-20 located in the ATPase domain of Ssb2 is not necessary for AtTDX-Ssb2 complex formation but is mandatory for its dissociation. Taken altogether, our results demonstrate that a redox switch governs the association/dissociation of AtTDX-Ssb2 complex.Oxidizing conditions like exposure to H2O2 are known to cause disulfide bonds to form and chaperone function to be turned on. Proteins from distinct Hsp families were also shown to be affected in their activity and/or conformation by the redox status. The murine small Hsp25 that carries a single Cys residue equilibrates between reduced protein and protein dimer, depending on the oxidoreduction conditions (56Zavialov A.V. Gaestel M. Korpela T. Zavialov V.P. Biochim. Biophys. Acta. 1998; 1388: 123-132Crossref PubMed Scopus (29) Google Scholar). The E. coli Hsp33 has been described recently (57Jakob U. Muse W. Eser M. Bardwell J.C.A. Cell. 1999; 96: 341-352Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar) as a chaperone with an on-off mode of activity that uses reactive disulfide bonds as molecular switches. Exposure to hydrogen peroxide causes zinc to be released from Hsp33 conserved cysteines and disulfide bond formation and the chaperone function to be turned on. Another example is given by Tsai et al. (58Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar) that showed that the human protein-disulfide isomerase is capable of redox-driven chaperone activity. All these examples are consistent with our results that show an in vivo redox regulation of a member of the Hsp70 family by a thioredoxin.Recently, Hsp70 chaperoning activity was shown to be increased by environments that mimics oxidative stress (59Callahan M.K. Chaillot D. Jacquin C. Clark P.R. Menoret A. J. Biol. Chem. 2002; 277: 33604-33609Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Because association with peptides under oxidative conditions is not reversible by reducing agents, it has been proposed that other chaperone-associated factors are required for substrate release. AtTDX may be one of these chaperone-associated factors, acting as an oxidative stress detector through its disulfide reductase activity. In the case of AtTDX/Ssb2 dissociation, involvement of AtTDX disulfide reductase activity and oxidation of Ssb2 cysteine 20 are two mandatory events demonstrated by the present work. At present, the exact function of Ssb2 Cys-20 under normal conditions and oxidative stress remains to be demonstrated. A survey of all Hsp70 of bacteria, fungi, animals, and plants shows the presence of a conserved cysteine in the position homologue to that of Ssb2 Cys-20. This suggests that the mechanism of a redox-dependent release is conserved. Moreover, analysis of the Ssb2 sequence with Swiss-PDB-Viewer and Rasmol programs proposed that Cys-20 is located in a small convex cavity near the protein surface and is thus easily accessible for disulfide bond formation/oxidation. These findings suggest that Ssb2 Cys-20 may be engaged in a disulfide bond with a third partner, which could be another Hsp70 molecule in a self-association (53Benaroudj N. Triniolles F. Ladjimi M.M. J. Biol. Chem. 1996; 271: 18471-18476Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) or another protein with conserved cysteine. How thioredoxin activity can affect, directly or indirectly, the redox state of Ssb2 Cys-20 under oxidative stress remains unsolved. Several hypotheses exist that require further experiments, including fine analysis of determinants and other partners involved in the interaction. Thioredoxin (TRX) 1The abbreviations used are: TRX, thioredoxin; ROS, reactive oxygen species; GST, glutathioneS-transferase; HA, hemagglutinin; TPR, tetratrico peptide repeat 1The abbreviations used are: TRX, thioredoxin; ROS, reactive oxygen species; GST, glutathioneS-transferase; HA, hemagglutinin; TPR, tetratrico peptide repeat is a small 12-kDa ubiquitous protein containing two redox-active half-cystine residues in an active center with conserved amino acid sequence Cys-X-X-Cys (where Xindicates various amino acids) that functions as a protein-disulfide reductase. The two cysteine residues in the active site provide the sulfhydryl groups involved in the thioredoxin-dependent reducing activity. Under an oxidized form, the TRX-S2protein contains a disulfide bridge within the active site that is reduced to a TRX-(SH2) dithiol by NADPH and the flavoprotein TRX reductase (for review, see Ref. 1Meyer Y. Miginiac-Maslow M. Schürmann P. Jacquot J.-P. McManus M.T. Laing W.A. Allan A.C. The Annual Plant Reviews. Sheffield Academic Press, Sheffield, UK2002: 1-23Google Scholar). Under this reduced form, thioredoxin becomes a very powerful reductant of disulfide bridges in target proteins. Thioredoxin was initially characterized from Escherichia coli extracts as a hydrogen donor for ribonucleotide reductase (2Holmgren A. Trends Biochem. Sci. 1981; 6: 26-29Abstract Full Text PDF Scopus (84) Google Scholar). Thioredoxins were later shown to act as hydrogen donors to peroxiredoxins that reduce hydrogen peroxide (3Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar) and to methionine-sulfoxide reductases that reactivate proteins damaged by stresses that generate reactive oxygen species (ROS) (4Fernando M.R. Nanri H. Yoshitake S. Nagato-Kuno K. Minakami S. Eur. J. Biochem. 1992; 209: 917-922Crossref PubMed Scopus (200) Google Scholar). They are necessary for a number of other metabolic enzymes that form a disulfide as part of their catalytic cycle (5Reitsch A. Beckwith J. Annu. Rev. Genet. 1998; 32: 163-184Crossref PubMed Scopus (237) Google Scholar). In addition, thioredoxins induce conformational changes of the targeted proteins by disulfide bond reduction and assist in the protein folding pathway (6Pigiet V.P. Schuster B.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7643-7647Crossref PubMed Scopus (123) Google Scholar). Thioredoxins directly or indirectly interact with several nuclear factors, such as the mammalian transcription factor NFκB and Ref-1 (see Ref. 7Hirota K. Matsui M. Iwata S. Nishiyama A. Mori K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3633-3638Crossref PubMed Scopus (719) Google Scholar and references therein). Thioredoxins play important roles in cell cycle and division and embryogenesis (8Salz H.K. Flickinger T.W. Mittendorf E. Pellicena-Palle A. Petschek J.P. Albrecht E.B. Genetics. 1994; 136: 1075-1086Crossref PubMed Google Scholar) and inhibit spontaneous apoptosis in tumoral cells (9Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2056) Google Scholar). Mammalian thioredoxin has also been shown to associate directly with nuclear receptors such as the glucocorticoid receptor GR (10Makino Y. Yoshikawa N. Okamoto K. Hirota K. Yodoi J. Makino I. Tanaka H. J. Biol. Chem. 1999; 274: 3182-3188Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), whose regulation requires cooperation between heat-shock proteins such as Hsp70 and Hsp90 (11Buchner J. Trends Biochem. Sci. 1999; 24: 136-141Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). Hsp70 and Hsp90 are molecular chaperones that are involved in many important biological processes by appropriately folding nascent polypeptides on ribosomes as well as by assembling multisubunit protein complex" @default.
• W2039643915 created "2016-06-24" @default.
• W2039643915 creator A5040470049 @default.
• W2039643915 creator A5040573508 @default.
• W2039643915 creator A5073341292 @default.
• W2039643915 creator A5088292184 @default.
• W2039643915 date "2003-02-01" @default.
• W2039643915 modified "2023-09-27" @default.
• W2039643915 title "Redox Control of Hsp70-Co-chaperone Interaction Revealed by Expression of a Thioredoxin-like Arabidopsis Protein" @default.
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