scholarly journals Oligomeric forms of the 90-kDa heat shock protein

1998 ◽  
Vol 330 (2) ◽  
pp. 989-995 ◽  
Author(s):  
Takayuki NEMOTO ◽  
Nobuko SATO
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Mammalian Cells ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Native Page ◽  
Glycerol Gradient ◽  
Dimeric Form ◽  
Oligomeric Forms ◽  
Rat Liver Cytosol

Two isoforms of the 90-kDa heat shock protein, HSP90α and HSP90β, are present in the cytosol of mammalian cells. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions (native PAGE) revealed that HSP90α predominantly exists as a homodimer and that HSP90β 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 HSP90α sedimenting at 8-10S as predominant ones. On the other hand, the glycerol gradient centrifugation revealed multiple forms of HSP90β oligomers sedimenting at 6-12S. All of the HSP90β 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 HSP90β dimer to monomers during the electrophoresis. The same PAGE further revealed that the HSP90α oligomer also dissociated into dimers during the electrophoresis. Full-length form of bacterially-expressed human HSP90α 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.

scholarly journals RNA-binding proteins and heat-shock protein 90 are constituents of the cytoplasmic capping enzyme interactome

2018 ◽  
Vol 293 (43) ◽  
pp. 16596-16607 ◽  
Author(s):  
Jackson B. Trotman ◽  
Bernice A. Agana ◽  
Andrew J. Giltmier ◽  
Vicki H. Wysocki ◽  
Daniel R. Schoenberg
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Heat Shock Protein 90 ◽  
Hsp90 Inhibitor ◽  
Capping Enzyme ◽  
Eukaryotic Mrnas

The N7-methylguanosine cap is added in the nucleus early in gene transcription and is a defining feature of eukaryotic mRNAs. Mammalian cells also possess cytoplasmic machinery for restoring the cap at uncapped or partially degraded RNA 5′ ends. Central to both pathways is capping enzyme (CE) (RNA guanylyltransferase and 5′-phosphatase (RNGTT)), a bifunctional, nuclear and cytoplasmic enzyme. CE is recruited to the cytoplasmic capping complex by binding of a C-terminal proline-rich sequence to the third Src homology 3 (SH3) domain of NCK adapter protein 1 (NCK1). To gain broader insight into the cellular context of cytoplasmic recapping, here we identified the protein interactome of cytoplasmic CE in human U2OS cells through two complementary approaches: chemical cross-linking and recovery with cytoplasmic CE and protein screening with proximity-dependent biotin identification (BioID). This strategy unexpectedly identified 66 proteins, 52 of which are RNA-binding proteins. We found that CE interacts with several of these proteins independently of RNA, mediated by sequences within its N-terminal triphosphatase domain, and we present a model describing how CE-binding proteins may function in defining recapping targets. This analysis also revealed that CE is a client protein of heat shock protein 90 (HSP90). Nuclear and cytoplasmic CEs were exquisitely sensitive to inhibition of HSP90, with both forms declining significantly following treatment with each of several HSP90 inhibitors. Importantly, steady-state levels of capped mRNAs decreased in cells treated with the HSP90 inhibitor geldanamycin, raising the possibility that the cytotoxic effect of these drugs may partially be due to a general reduction in translatable mRNAs.

Adenovirus E1A requires synthesis of a cellular protein to establish a stable transcription complex in injected Xenopus laevis oocytes

1987 ◽  
Vol 7 (9) ◽  
pp. 3049-3056
Author(s):  
J D Richter ◽  
H C Hurst ◽  
N C Jones
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Mammalian Cells ◽  
Cellular Protein ◽  
Xenopus Laevis Oocytes ◽  
Protein Synthesis Inhibitors ◽  
Adenovirus E1a ◽  
High Level

The Escherichia coli-expressed adenovirus E1A 13S mRNA product injected into Xenopus oocytes was active, as assessed by its ability to stimulate the transcription of an injected gene which is normally responsive to E1A in mammalian cells. In the presence of the protein synthesis inhibitors pactamycin or cycloheximide, E1A was correctly posttranslationally modified (phosphorylated) and transported to the nucleus; but it failed to stimulate the transcription of an injected gene containing the human heat shock protein 70 promoter. The basal (unstimulated) level of transcription of the gene was unaffected by these inhibitors. If oocytes were cultured in the presence of cycloheximide after E1A stimulated transcription, however, the high level of transcription was maintained for several hours without new protein synthesis. Results of competition studies with the same promoter (the heat shock protein 70 promoter) linked to two marked genes demonstrated that once the induction of transcription by E1A took place, the stimulated levels of transcription were maintained, even when they were challenged with excess competitor DNA. Results of these studies suggest that E1A requires the synthesis of a cellular protein to form a stable transcription complex.

scholarly journals Adenovirus E1A requires synthesis of a cellular protein to establish a stable transcription complex in injected Xenopus laevis oocytes.

1987 ◽  
Vol 7 (9) ◽  
pp. 3049-3056 ◽  
Author(s):  
J D Richter ◽  
H C Hurst ◽  
N C Jones
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Mammalian Cells ◽  
Cellular Protein ◽  
Xenopus Laevis Oocytes ◽  
Protein Synthesis Inhibitors ◽  
Adenovirus E1a ◽  
High Level

The Escherichia coli-expressed adenovirus E1A 13S mRNA product injected into Xenopus oocytes was active, as assessed by its ability to stimulate the transcription of an injected gene which is normally responsive to E1A in mammalian cells. In the presence of the protein synthesis inhibitors pactamycin or cycloheximide, E1A was correctly posttranslationally modified (phosphorylated) and transported to the nucleus; but it failed to stimulate the transcription of an injected gene containing the human heat shock protein 70 promoter. The basal (unstimulated) level of transcription of the gene was unaffected by these inhibitors. If oocytes were cultured in the presence of cycloheximide after E1A stimulated transcription, however, the high level of transcription was maintained for several hours without new protein synthesis. Results of competition studies with the same promoter (the heat shock protein 70 promoter) linked to two marked genes demonstrated that once the induction of transcription by E1A took place, the stimulated levels of transcription were maintained, even when they were challenged with excess competitor DNA. Results of these studies suggest that E1A requires the synthesis of a cellular protein to form a stable transcription complex.

scholarly journals Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity

2004 ◽  
Vol 381 (2) ◽  
pp. 379-387 ◽  
Author(s):  
Tirumala Kumar CHOWDARY ◽  
Bakthisaran RAMAN ◽  
Tangirala RAMAKRISHNA ◽  
Chintalagiri Mohan RAO
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Quaternary Structure ◽  
Citrate Synthase ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Nucleotide Sequence Analysis ◽  
Heat Shock Factor 1 ◽  
Induced Aggregation

A newly identified 22 kDa protein that interacts with Hsp27 (heat-shock protein 27) was shown to possess the characteristic α-crystallin domain, hence named Hsp22, and categorized as a member of the sHsp (small Hsp) family. Independent studies from different laboratories reported the protein with different names such as Hsp22, H11 kinase, E2IG1 and HspB8. We have identified, on the basis of the nucleotide sequence analysis, putative heat-shock factor 1 binding sites upstream of the Hsp22 translation start site. We demonstrate that indeed Hsp22 is heat-inducible. We show, in vitro, chaperone-like activity of Hsp22 in preventing dithiothreitol-induced aggregation of insulin and thermal aggregation of citrate synthase. We have cloned rat Hsp22, overexpressed and purified the protein to homogeneity and studied its structural and functional aspects. We find that Hsp22 fragments on storage. MS analysis of fragments suggests that the fragmentation might be due to the presence of labile peptide bonds. We have established conditions to improve its stability. Far-UV CD indicates a randomly coiled structure for Hsp22. Quaternary structure analyses by glycerol density-gradient centrifugation and gel filtration chromatography show that Hsp22 exists as a monomer in vitro, unlike other members of the sHsp family. Hsp22 exhibits significantly exposed hydrophobic surfaces as reported by bis-8-anilinonaphthalene-l-sulphonic acid fluorescence. We find that the chaperone-like activity is temperature dependent. Thus Hsp22 appears to be a true member of the sHsp family, which exists as a monomer in vitro and exhibits chaperone-like activity.

scholarly journals Simian virus 40 and polyoma virus induce synthesis of heat shock proteins in permissive cells.

1983 ◽  
Vol 3 (1) ◽  
pp. 1-8 ◽  
Author(s):  
E W Khandjian ◽  
H Türler
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Gel Electrophoresis ◽  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyoma Virus ◽  
Two Dimensional Gel Electrophoresis ◽  
Mouse Cells ◽  
Stimulation Of

During the lytic infection of monkey and mouse cells with simian virus 40 and polyoma virus, respectively, the preferentially increased synthesis of two host proteins of 92,000 and 72,000 Mr was observed by 15 to 20 h after infection besides the general stimulation of most cellular proteins. The incubation of uninfected monkey and mouse cell cultures for 30 to 60 min at 43.5 degrees C induced the enhanced synthesis of at least three proteins of 92,000, 72,000 and 70,000 Mr, the last one being the major heat shock protein of mammalian cells. Two-dimensional gel electrophoresis and partial proteolytic digestion confirmed that the same 92,000- and 72,000-Mr proteins are stimulated by virus infection and thermal treatment. In simian virus 40-infected CV-1 cells, we also observed the weak stimulation of a 70,000-Mr protein comigrating in gel electrophoresis with the major heat shock protein. The 92,000-, 72,000- and 70,000-Mr proteins of monkey cells are structurally very similar to the corresponding proteins of mouse cells. In immunoprecipitations, no specific association of these proteins to simian virus 40 T antigens was noticed.

Glucocorticoids modulate the synthesis and expression of a 72 kDa heat shock protein in cultured Graves' retroocular fibroblasts

1993 ◽  
Vol 128 (1) ◽  
pp. 41-50 ◽  
Author(s):  
Armin E Heufelder ◽  
Bjoern E Wenzel ◽  
Rebecca S Bahn
Keyword(s):  
Heat Stress ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Thyroid Autoimmunity ◽  
Human Fibroblasts ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Simultaneous Exposure

Glucocorticoids modulate numerous proliferative, metabolic and immunological functions in human fibroblasts, some of which appear to be mediated via glucocorticoid receptors. We studied the influence of glucocorticosteroids on the synthesis and expression of a 72 kDa heat shock protein that is thought to play a role in thyroid autoimmunity. Experiments were performed using orbital fibroblasts derived from patients with Graves' ophthalmopathy and normal individuals. Cell monolayers were exposed to various concentrations of dexamethasone, the specific glucocorticoid agonist RU 28362, the glucocorticoid antagonist RU 38486, or combinations thereof, prior to heat stress or exposure to hydrogen peroxide. Heat shock protein 72 expression was assessed using sodium dodecylsulfate polyacrylamide-gel electrophoresis of cellular extracts, followed by autoradiography or immunoblotting with a mouse monoclonal antibody against the 72 kDa heat shock protein and quantitative scanning densitometry. In addition, cellular distribution of the immunoreactivity for the 72 kDa heat shock protein was studied using indirect immunofluorescence on parallel cultures. In other experiments, aimed at studying heat shock protein synthesis, cell cultures were pulse-labeled with [35S]-methionine prior to harvesting. Treatment with dexamethasone or RU 28362 markedly attenuated the heat stress-enhanced synthesis and expression of the 72 kDa heat shock protein and several other heat shock proteins both in normal and in Graves' retroocular fibroblasts (p<0.001). In addition, either treatment reduced baseline expression of the 72 kDa heat shock protein in Graves' retroocular fibroblasts (p<0.01). These effects were dose-dependent and appeared to be mediated via the glucocorticoid receptor, because combined exposure to dexamethasone or RU 28362 plus RU 38486 completely restored synthesis and expression of the 72 kDa heat shock protein. Baseline or stress-enhanced expression of the 72 kDa heat shock protein was not altered by treatment of monolayers with RU 38486 alone. As demonstrated by immunofluorescence, the characteristic intracellular shifting of the 72 kDa heat shock protein in response to cellular stress was partially inhibited by glucocorticoid agonists and restored by simultaneous exposure to glucocorticoid agonists and RU 38486. These results demonstrate that dexamethasone and the specific glucocorticoid agonist RU 28362 can modulate baseline- and stress-induced synthesis and expression of the 72 kDa heat shock protein, as well as its subcellular distribution. in cultured retroocular fibroblasts. Our studies suggest that these compounds exert these effects via the glucocorticoid receptor.

Specific proteins synthesized in human lymphocytes during hypoxia

1991 ◽  
Vol 261 (4) ◽  
pp. 92-96
Author(s):  
A. A. Aldashev ◽  
K. A. Agibetov ◽  
A. A. Yugai ◽  
A. T. Shamshiev
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Calcium Concentration ◽  
Culture Media ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Major Protein ◽  
Blood Lymphocytes

Hypoxia is a severe stress factor to which man and most other mammalian species are capable of adapting. However, the cellular mechanisms that enable cells to tolerate decreases in ambient oxygen tension are still unknown. We have previously shown that hypoxia induces the synthesis of unique proteins (molecular mass 38, 52, 74, 76 kDa) in human aortic endothelial cells and lymphocytes. In this study we investigated the specificity of hypoxia on the upregulation of these hypoxic stress proteins (HYP) in human peripheral blood lymphocytes and the role of calcium in this response. 35S-methionine pulse-labeling studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis and autoradiography demonstrated that normobaric hypoxia (4% O2–5% CO2–91% N2) enhanced synthesis of HYP, whereas heat-shock protein synthesis was not affected. Heat shock (42°C) and cold stress (4°C) did, however, induce synthesis of heat-shock protein but not HYP. The 38-kDa HYP is the major protein for which synthesis is upregulated by hypoxia. Its isoelectric point (pI) is 3.5–4.0, and it is localized in the cytosol. The 52-kDa HYP has a pI of >6.5, and it is also localized in the cytosol. The 74- and 76-kDa HYPs appear to be membrane bound. In addition to hypoxia, an increase in calcium concentration in the culture media (25–50 mM) enhanced synthesis of HYP. An ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)/Ca2+ binding complex, when added to blood lymphocytes during exposure to hypoxia, significantly inhibited HYP synthesis. On the basis of the similarity of molecular masses, pI, localization in the cytosol, and Ca2+ dependence of HYP expression, we suggest that HYP 38 and 52 may be isoenzymes of inositol 1,4,5-triphosphate 3-kinase, an enzyme that regulates phosphoinositol turnover and intracellular calcium concentration in cells. calcium; internal localization; stress

scholarly journals Tissue-dependent expression of heat shock factor 2 isoforms with distinct transcriptional activities.

1995 ◽  
Vol 15 (10) ◽  
pp. 5288-5293 ◽  
Author(s):  
M L Goodson ◽  
O K Park-Sarge ◽  
K D Sarge
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Mammalian Cells ◽  
Protein Gene ◽  
Heat Shock Factor ◽  
Dependent Manner ◽  
Heat Shock Protein Gene ◽  
Mouse Tissues

Heat shock factor 2 (HSF2) functions as a transcriptional regulator of heat shock protein gene expression in mammalian cells undergoing processes of differentiation and development. Our previous studies demonstrated high regulated expression and unusual constitutive DNA-binding activity of the HSF2 protein in mouse testes, suggesting that HSF2 functions to regulate heat shock protein gene expression in spermatogenic cells. The purpose of this study was to test whether HSF2 regulation in testes is associated with alterations in the HSF2 polypeptide expressed in testes relative to other mouse tissues. Our results show that mouse cells express not one but two distinct HSF2 proteins and that the levels of these HSF2 isoforms are regulated in a tissue-dependent manner. The testes express predominantly the 71-kDa HSF2-alpha isoform, while the heart and brain express primarily the 69-kDa HSF2-beta isoform. These isoforms are generated by alternative splicing of HSF2 pre-mRNA, which results in the inclusion of an 18-amino-acid coding sequence in the HSF2-alpha mRNA that is skipped in the HSF2-beta mRNA. HSF2 alternative splicing is also developmentally regulated, as our results reveal a switch in expression from the HSF2-beta mRNA isoform to the HSF2-alpha isoform during testis postnatal developmental. Transfection analysis shows that the HSF2-alpha protein, the predominant isoform expressed in testis cells, is a more potent transcriptional activator than the HSF2-beta isoform. These results reveal a new mechanism for the control of HSF2 function in mammalian cells, in which regulated alternative splicing is used to modulate HSF2 transcriptional activity in a tissue-dependent manner.

Analysis of oligomeric transition of silkworm small heat shock protein sHSP20.8 using high hydrostatic pressure native PAGE

2013 ◽  
Vol 33 (2) ◽  
pp. 258-264 ◽  
Author(s):  
Tetsuro Fujisawa ◽  
Toshifumi Ueda ◽  
Keiichi Kameyama ◽  
Yoichi Aso ◽  
Ryo Ishiguro
Keyword(s):  
Hydrostatic Pressure ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
High Hydrostatic Pressure ◽  
Small Heat Shock Protein ◽  
Native Page

scholarly journals Heat Shock Protein (Hsp) 40 Mutants Inhibit Hsp70 in Mammalian Cells

1999 ◽  
Vol 274 (51) ◽  
pp. 36757-36763 ◽  
Author(s):  
Annemieke A. Michels ◽  
Bart Kanon ◽  
Olivier Bensaude ◽  
Harm H. Kampinga
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Mammalian Cells ◽  
Hsp 40
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