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In higher eucaryotes, heat shock factor (HSF) exists in a cryptic form in unstressed cells. We investigated molecular forms of human HSF before and after activation by sucrose density gradient centrifugation and by gel mobility shift assay using a 32P-labeled heat shock element (HSE). We found that the in vivo or in vitro activated HSF, which is capable of binding to HSE, and its inactive form present in unstressed cells have different sedimentation coefficient; the former is 8 S whereas the latter is 4-5 S. Both the 8 S and 4-5 S forms contain the HSF polypeptide which has the ability to bind to HSE upon activation. The inactive 4-5 S form acquires HSE-binding ability when activated by heat shock or other stimuli. This HSF activity was greatly reduced, however, during recentrifugation in sucrose density gradient and, in addition, the residual activity was not recovered in 8 S fractions. Transformation of the inactive 4-5 S form of HSF to the stable, active 8 S form was achieved when the inactive form was activated and mixed with cytosols of unstressed cells.  相似文献   

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Complex modes of heat shock factor activation.   总被引:29,自引:15,他引:14       下载免费PDF全文
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This study represents the initial characterization of the heat shock factor (HSF) in filamentous fungi. We demonstrate that HSFs from Beauveria bassiana, Metarhizium anisopliae, Tolypocladium nivea, Paecilomyces farinosus, and Verticillium lecanii bind to the heat shock element (HSE) constitutively (non-shocked), and that heat shock resulted in increased quantities and decreased mobility of HSF-HSE complexes. The monomeric molecular mass of both heat-induced and constitutive HSFs was determined to be 85.8 kDa by UV-crosslinking and the apparent molecular masses of the native HSF-HSE complexes as determined by pore exclusion gradient gel electrophoresis was 260 and 300 kDa, respectively. Proteolytic band clipping assays using trypsin and chymotrypsin revealed an identical partial cleavage profile for constitutive and heat-induced HSF-HSE complexes. Thus, it appears that both constitutive and heat-inducible complexes are formed by trimers composed of the same HSF molecule which undergoes conformational changes during heat shock. The mobility difference between the complexes was not abolished by enzymatic dephosphorylation and deglycosylation, indicating that the reduced mobility of the heat-induced HSF is probably due to a post-translational modification other than phosphorylation or glycosylation.  相似文献   

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The mechanisms of sensing and signalling of heat and oxidative stresses are not well understood. The central question of this paper is whether in plant cells oxidative stress, in particular H2O2, is required for heat stress- and heat shock factor (HSF)-dependent expression of genes. Heat stress increases intracellular accumulation of H2O2 in Arabidopsis cell culture. The accumulation was greatly diminished using ascorbate as a scavenger or respectively diphenyleneiodonium chloride (DPI) as an inhibitor of reactive oxygen species production. The mRNA of heat shock protein (HSP) genes, exemplified by Hsp17.6, Hsp18.2, and the two cytosolic ascorbate peroxidase genes Apx1, Apx2, reached similar levels by moderate heat stress (37°C) or by treatment with H2O2, butylperoxide and diamide at room temperature. The heat-induced expression levels were significantly reduced in the presence of ascorbate or DPI indicating that H2O2 is an essential component in the heat stress signalling pathway. Rapid (15 min) formation of heat shock promoter element (HSE) protein-binding complex of high molecular weight in extracts of heat-stressed or H2O2-treated cells and the inability to form this complex after ascorbate treatment suggests that oxidative stress affects gene expression via HSF activation and conversely, that H2O2 is involved in HSF activation during the early phase of heat stress. The heat stress induction of a high mobility HSE-binding complex, characteristic for later phase of heat shock response, was blocked by ascorbate and DPI. H2O2 was unable to induce this complex suggesting that H2O2 is involved only in the early stages of HSF activation. Significant induction of the genes tested after diamid treatment and moderate expression of the sHSP genes in the presence of 50 mM ascorbate at 37°C occurred without activation of HSF, indicating that other mechanisms may be involved in stress signalling. Electronic Supplementary Material Supplementary material is available for this article at http//dx.doi.org/10.1007/s11103-006-0045-4 Roman A. Volkov and Irina I. Panchuk contributed equally  相似文献   

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Kim SA  Yoon JH  Kim DK  Kim SG  Ahn SG 《FEBS letters》2005,579(29):6559-6563
Heat shock factor 1 (HSF1) is a major transactivator of heat shock genes in response to stress and mediates cell protection against various harmful conditions. In this study, we identified the interaction of CHIP (carboxyl terminus of the heat shock cognate protein 70-interacting protein) with the N-terminus of HSF1. Using GST full-down assay, we found that CHIP directly interacts with C-terminal deleted HSF1 (a.a. 1-290) but not with full-length HSF1 under non-stressed conditions. Interestingly, interaction of CHIP with full-length HSF1 was induced by heat shock treatment. The structural change of HSF1 was observed under heat stressed conditions by CD spectra. These observations demonstrate the direct interaction between HSF1 and CHIP and this interaction requires conformational change of HSF1 by heat stress.  相似文献   

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The DNA-binding activity of a tobacco heat shock factor (HSF) was induced by heat treatment (37–40 °C) of a cell-free extract that contained extra-nuclear fraction, but not in an extract of isolated nuclei. These observations suggest that an inactive form of HSF can directly recognize and transduce the heat shock signal and that such transduction requires components of the extranuclear fraction. Addition of ATP or of most other nucleoside triphosphates reduced the binding of the HSF to the heat shock element (HSE) in the same extract, and removal of ATP by dialysis from the extract restored the ability of the HSF to bind to DNA. The restored activity of the HSF could be eliminated again by a second addition of ATP. Our observations provide the first example of the involvement of ATP in the regulation of the reversible changes in HSF that control its ability to bind to HSEs in a cell-free extract.Abbreviations AMP-PNP adenylyl imidodiphosphate - GUS -glucuronidase - HSE heat shock element - HSF heat shock factor  相似文献   

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