Developmental regulation of murine mammary-gland 90 kDa heat-shock proteins.
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ABSTRACT:
SUBMITTER: Catelli MG
PROVIDER: S-EPMC1138448 | biostudies-other | 1989 Mar
REPOSITORIES: biostudies-other
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Project description:In the present study we investigated the substrate-binding characteristics of three members of the 90 kDa heat shock protein (HSP90) family, namely the alpha isoform of human HSP90 (HSP90alpha), human GRP94 (94 kDa glucose-regulated protein, a form of HSP90 from endoplasmic reticulum), and HtpG (the Escherichia coli homologue of HSP90) and the domain responsible for these characteristics. The recombinant forms of HSP90alpha, GRP94 and HtpG existed as dimers and became oligomerized at higher temperatures. Among the three family members, HtpG required the highest temperature (65 degrees C) for its transition to oligomeric forms. The precipitation of the substrate protein, glutathione S-transferase, which occurred at 55 degrees C, was efficiently prevented by the simultaneous presence of a sufficient amount of HSP90alpha or GRP94, but not by HtpG, which was still present as a dimer at that temperature. However, precipitation was stopped completely at 65-70 degrees C, at which temperature HtpG was oligomerized. Thus the transition of HSP90-family proteins to a state with self-oligomerization ability is essential for preventing the precipitation of substrate proteins. We then investigated the domain responsible for the substrate binding of HtpG on the basis of the three domain structures. The self-oligomerizing and substrate-binding activities towards glutathione S-transferase and citrate synthase were both located in a single domain, the N-terminal domain (residues 1-336) of HtpG. We therefore propose that the primary peptide-binding site is located in the N-terminal domain of HSP90-family proteins.
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Project description:Hsp90 and tubulin are among the most abundant proteins in the cytosol of eukaryotic cells. Although Hsp90 plays key roles in maintaining its client proteins in their active state, tubulin is essential for fundamental processes such as cell morphogenesis and division. Several studies have suggested a possible connection between Hsp90 and the microtubule cytoskeleton. Because tubulin is a labile protein in its soluble form, we investigated whether Hsp90 protects it against thermal denaturation. Both proteins were purified from porcine brain, and their interaction was characterized in vitro by using spectrophotometry, sedimentation assays, video-enhanced differential interference contrast light microscopy, and native polyacrylamide gel electrophoresis. Our results show that Hsp90 protects tubulin against thermal denaturation and keeps it in a state compatible with microtubule polymerization. We demonstrate that Hsp90 cannot resolve tubulin aggregates but that it likely binds early unfolding intermediates, preventing their aggregation. Protection was maximal at a stoichiometry of two molecules of Hsp90 for one of tubulin. This protection does not require ATP binding and hydrolysis by Hsp90, but it is counteracted by geldanamycin, a specific inhibitor of Hsp90.
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Project description:Two isoforms of the 90-kDa heat shock protein, HSP90alpha and HSP90beta, are present in the cytosol of mammalian cells. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions (native PAGE) revealed that HSP90alpha predominantly exists as a homodimer and that HSP90beta is present mainly as a monomer [Minami, Kawasaki, Miyata, Suzuki and Yahara (1991) J. Biol. Chem. 266, 10099-10103]. However, only the dimeric form has been observed under other analytical conditions such as gradient centrifugation. In this study, therefore, we investigated native forms of HSP90 by use of immunochemical techniques with isoform-specific monoclonal antibodies recently developed in our laboratory. Glycerol gradient centrifugation at the physiological salt concentration as well as native PAGE analysis of rat liver cytosol revealed oligomeric forms of HSP90alpha sedimenting at 8-10S as predominant ones. On the other hand, the glycerol gradient centrifugation revealed multiple forms of HSP90beta oligomers sedimenting at 6-12S. All of the HSP90beta oligomers, however, migrated at 100-kDa monomer and 190-kDa dimer positions on native PAGE. A novel two-dimensional double native PAGE revealed that the entity was converted from the HSP90beta dimer to monomers during the electrophoresis. The same PAGE further revealed that the HSP90alpha oligomer also dissociated into dimers during the electrophoresis. Full-length form of bacterially-expressed human HSP90alpha migrated as dimers, but a considerable amount did not penetrate into the gel under native PAGE conditions, indicating the existence of oligomeric forms. Electrophoretic studies of deletion mutants of HSP90 demonstrated that the C-terminal 200 amino acids were capable of forming oligomers. Taken together, we conclude that both of the HSP90 isoforms predominantly exist as oligomeric forms in the cytosol even under unstressed conditions but that they artificially dissociate into smaller forms when subjected to native PAGE.
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Project description:The 90-kDa heat shock protein (Hsp90) is involved in the regulation and activation of numerous client proteins essential for diverse functions such as cell growth and differentiation. Although the function of cytosolic Hsp90 is dependent on a battery of cochaperone proteins regulating both its ATPase activity and its interaction with client proteins, little is known about the real Hsp90 molecular mechanism. Besides its highly flexible dimeric state, Hsp90 is able to self-oligomerize in the presence of divalent cations or under heat shock. In addition to dimers, oligomers exhibit a chaperone activity. In this work, we focused on Mg(2+)-induced oligomers that we named Type I, Type II, and Type III in increasing molecular mass order. After stabilization of these quaternary structures, we optimized a purification protocol. Combining analytical ultracentrifugation, size exclusion chromatography coupled to multiangle laser light scattering, and high mass matrix-assisted laser desorption/ionization time of flight mass spectrometry, we determined biochemical and biophysical characteristics of each Hsp90 oligomer. We demonstrate that Type I oligomer is a tetramer, and Type II is an hexamer, whereas Type III is a dodecamer. These even-numbered structures demonstrate that the building brick for oligomerization is the dimer up to the Type II, whereas Type III probably results from the association of two Type II. Moreover, the Type II oligomer structure, studied by negative stain transmission electron microscopy tomography, exhibits a "nest-like" shape that forms a "cozy chaperoning chamber" where the client protein folding/protection could occur.
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Project description:BackgroundSystemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of autoantibodies to multiple self-antigens, including heat shock proteins (HSP). Because of the increased expression of HSP90 and abnormal immune responses to it in SLE, we investigated whether an HSP90 DNA vaccine could modulate the development and clinical manifestations of SLE in lupus-prone mice.Methods(NZB x NZW)F1 (NZB/W) mice were vaccinated with DNA constructs encoding HSP90 or control plasmids or vehicle. The mice were then monitored for survival, circulating anti-dsDNA autoantibodies, and immune phenotypes. Renal disease was evaluated by immunohistochemistry and by the measurement of proteinuria.ResultsVaccination with HSP90 DNA reduced lupus disease manifestations and prolonged the survival of NZB/W mice. The protective effects of the HSP90 DNA vaccine associated with the induction of tolerogenic dendritic cells (DCs) and an expansion of T regulatory cells (Tregs).ConclusionsThe beneficial effects of DNA vaccination with HSP90 in murine SLE support the possibility of HSP90-based therapeutic modalities of intervention in SLE.
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Project description:A fundamental question in molecular evolution is how protein functional differentiation alters the ability of cells and organisms to cope with stress and survive. To answer this question we used two paralogous Hsp70s from mouse and explored whether these highly similar cytosolic molecular chaperones, which apart their temporal expression have been considered functionally interchangeable, are differentiated with respect to their lipid-binding function. We demonstrate that the two proteins bind to diverse lipids with different affinities and therefore are functionally specialized. The observed lipid-binding patterns may be related with the ability of both Hsp70s to induce cell death by binding to a particular plasma-membrane lipid, and the potential of only one of them to promote cell survival by binding to a specific lysosomal-membrane lipid. These observations reveal that two seemingly identical proteins differentially modulate cellular adaptation and survival by having acquired specialized functions via sequence divergence. Therefore, this study provides an evolutionary paradigm, where promiscuity, specificity, sub- and neo-functionalization orchestrate one of the most conserved systems in nature, the cellular stress-response.
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Project description:Deciphering the molecular basis of stem cell pluripotency is fundamental to the understanding of stem cell biology, early embryonic development, and to the clinical application of regenerative medicine. We report here that the molecular chaperone heat shock protein 90 (Hsp90) is essential for mouse embryonic stem cell (ESC) pluripotency through regulating multiple pluripotency factors, including Oct4, Nanog, and signal transducer and activator of transcription 3. Inhibition of Hsp90 by either 17-N-Allylamino-17-demethoxygeldanamycin or miRNA led to ESC differentiation. Overexpression of Hsp90? partially rescued the phenotype; in particular, the levels of Oct4 and Nanog were restored. Notably, Hsp90 associated with Oct4 and Nanog in the same cellular complex and protected them from degradation by the ubiquitin proteasome pathway, suggesting that Oct4 and Nanog are potential novel Hsp90 client proteins. In addition, Hsp90 inhibition reduced the mRNA level of Oct4, but not that of Nanog, indicating that Hsp90 participates in Oct4 mRNA processing or maturation. Hsp90 inhibition also increased expression of some protein markers for mesodermal lineages, implying that Hsp90 suppresses mesodermal differentiation from ESCs. These findings support a new role for Hsp90 in maintaining ESC pluripotency by sustaining the level of multiple pluripotency factors, particularly Oct4 and Nanog.
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Project description:We report the nucleotide sequence of a 2652 bp derived from a chicken 90-kDa heat shock protein (hsp 90) genomic clone. This fragment contains 890 bp of the 5' flanking region and 1762 bp of structural gene sequence encoding the first 85 amino acids of the protein. The start site of transcription was determined by primer extension and RNase mapping. Two introns have been identified. The first intron presents two features in common with the unique intron of the hsp 83 of drosophila: its location just before the ATG initiation codon and its length of approximately 1.3 Kb. The 5' flanking region contains a TATAA element, a CCAAT box and several putative cis-regulatory elements that might account for the basal level of expression and developmental regulation of the gene. Functional analyses show that hsp 90 gene expression is constitutive and heat inducible and that a full heat shock response requires the cooperativity of two distinct blocks of overlapping heat shock response elements.
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