共查询到20条相似文献,搜索用时 15 毫秒
1.
Wei Qi Matthew C. White Woonyoung Choi Charles Guo Colin Dinney David J. McConkey Arlene Siefker-Radtke 《PloS one》2013,8(7)
The proteasome inhibitor bortezomib (Velcade) is a promising new agent for bladder cancer therapy, but inducible cytoprotective mechanisms may limit its potential efficacy. We used whole genome mRNA expression profiling to study the effects of bortezomib on stress-induced gene expression in a panel of human bladder cancer cell lines. Bortezomib induced strong upregulation of the inducible HSP70 isoforms HSPA1A and HSPA1B isoforms of Hsp72 in 253J B-V and SW780 (HSPA1Ahigh) cells, but only induced the HSPA1B isoform in UM-UC10 and UM-UC13 (HSPA1Alow) cells. Bortezomib stimulated the binding of heat shock factor-1 (HSF1) to the HSPA1A promoter in 253JB-V but not in UM-UC13 cells. Methylation-specific PCR revealed that the HSPA1A promoter was methylated in the HSPA1Alow cell lines (UM-UC10 and UM-UC13), and exposure to the chromatin demethylating agent 5-aza-2′-deoxycytidine restored HSPA1A expression. Overexpression of Hsp72 promoted bortezomib resistance in the UM-UC10 and UM-UC13 cells, whereas transient knockdown of HSPA1B further sensitized these cells to bortezomib, and exposure to the chemical HSF1 inhibitor KNK-437 promoted bortezomib sensitivity in the 253J B-V cells. Finally, shRNA-mediated stable knockdown of Hsp72 in 253J B–V promoted sensitivity to bortezomib in vitro and in tumor xenografts in vivo. Together, our results provide proof-of-concept for using Hsp72 inhibitors to promote bortezomib sensitivity in bladder cancers and suggest that selective targeting of HSPA1B could produce synthetic lethality in tumors that display HSPA1A promoter methylation. 相似文献
2.
3.
4.
5.
Scieglinska D Piglowski W Chekan M Mazurek A Krawczyk Z 《Histochemistry and cell biology》2011,135(4):337-350
In the present study we determined the expression pattern of HSPA1 and HSPA2 proteins in various normal human tissues by tissue-microarray based immunohistochemical analysis. Both proteins belong to the HSPA (HSP70) family of heat shock proteins. The
HSPA2 is encoded by the gene originally defined as testis-specific, while HSPA1 is encoded by the stress-inducible genes (HSPA1A and HSPA1B). Our study revealed that both proteins are expressed only in some tissues from the 24 ones examined. HSPA2 was detected
in adrenal gland, bronchus, cerebellum, cerebrum, colon, esophagus, kidney, skin, small intestine, stomach and testis, but
not in adipose tissue, bladder, breast, cardiac muscle, diaphragm, liver, lung, lymph node, pancreas, prostate, skeletal muscle,
spleen, thyroid. Expression of HSPA1 was detected in adrenal gland, bladder, breast, bronchus, cardiac muscle, esophagus,
kidney, prostate, skin, but not in other tissues examined. Moreover, HSPA2 and HSPA1 proteins were found to be expressed in
a cell-type-specific manner. The most pronounced cell-type expression pattern was found for HSPA2 protein. In the case of
stratified squamous epithelia of the skin and esophagus, as well as in ciliated pseudostratified columnar epithelium lining
respiratory tract, the HSPA2 positive cells were located in the basal layer. In the colon, small intestine and bronchus epithelia
HSPA2 was detected in goblet cells. In adrenal gland cortex HSPA2 expression was limited to cells of zona reticularis. The
presented results clearly show that certain human tissues constitutively express varying levels of HSPA1 and HSPA2 proteins
in a highly differentiated way. Thus, our study can help designing experimental models suitable for cell- and tissue-type-specific
functional differences between HSPA2 and HSPA1 proteins in human tissues. 相似文献
6.
7.
Heat Shock Response and Protein Degradation: Regulation of HSF2 by the Ubiquitin-Proteasome Pathway 总被引:8,自引:3,他引:8
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Anu Mathew Sameer K. Mathur Richard I. Morimoto 《Molecular and cellular biology》1998,18(9):5091-5098
Mammalian cells coexpress a family of heat shock factors (HSFs) whose activities are regulated by diverse stress conditions to coordinate the inducible expression of heat shock genes. Distinct from HSF1, which is expressed ubiquitously and activated by heat shock and other stresses that result in the appearance of nonnative proteins, the stress signal for HSF2 has not been identified. HSF2 activity has been associated with development and differentiation, and the activation properties of HSF2 have been characterized in hemin-treated human K562 erythroleukemia cells. Here, we demonstrate that a stress signal for HSF2 activation occurs when the ubiquitin-proteasome pathway is inhibited. HSF2 DNA-binding activity is induced upon exposure of mammalian cells to the proteasome inhibitors hemin, MG132, and lactacystin, and in the mouse ts85 cell line, which carries a temperature sensitivity mutation in the ubiquitin-activating enzyme (E1) upon shift to the nonpermissive temperature. HSF2 is labile, and its activation requires both continued protein synthesis and reduced degradation. The downstream effect of HSF2 activation by proteasome inhibitors is the induction of the same set of heat shock genes that are induced during heat shock by HSF1, thus revealing that HSF2 affords the cell with a novel heat shock gene-regulatory mechanism to respond to changes in the protein-degradative machinery. 相似文献
8.
Increased thermostability of thermotolerant CHL V79 cells as determined by differential scanning calorimetry 总被引:3,自引:0,他引:3
J R Lepock H E Frey M P Heynen J Nishio B Waters K P Ritchie J Kruuv 《Journal of cellular physiology》1990,142(3):628-634
Heat shock denatures cellular protein and induces both a state of acquired thermotolerance, defined as resistance to a subsequent heat shock, and the synthesis of a category of proteins referred to as heat-shock proteins (HSPs). Thermotolerance may be due to the stabilization of thermolabile proteins that would ordinarily denature during heat shock, either by HSPs or some other factors. We show by differential scanning calorimetry (DSC) that mild heat shock irreversibly denatures a small fraction of Chinese hamster lung V79-WNRE cell protein (i.e., the enthalpy change, which is proportional to denaturation, on scanning to 45 degrees C at 1 degree C/min is approximately 2.3% of the total calorimetric enthalpy). Thermostability, defined by the extent of denaturation during heat shock and determined from DSC scans of whole cells, increases as the V79 cells become thermotolerant. Cellular stabilization appears to be due to an increase in the denaturation temperature of the most thermolabile proteins; there is no increase in the denaturation temperatures of the most thermally resistant proteins, i.e., those denaturing above 65 degrees C. Cellular stabilization is also observed in the presence of glycerol, which is known to increase resistance to heat shock and to stabilize proteins in vitro. A model is presented, based on a direct relationship between the extent of hyperthermic killing and the denaturation or inactivation of a critical target that defines the rate-limiting step in killing, which predicts a transition temperature (Tm) of the critical target for control V79-WNRE cells of 46.0 degrees C and a Tm of 47.3 degrees C for thermotolerant cells. This shift of 1.3 degrees C is consistent with the degree of stabilization detected by DSC. 相似文献
9.
10.
11.
The molecular basis of heat shock response (HSR), a cellular defense mechanism against various stresses, is not well understood. In this, the first comprehensive analysis of gene expression changes in response to heat shock and MG132 (a proteasome inhibitor), both of which are known to induce heat shock proteins (Hsps), we compared the responses of normal mouse fibrosarcoma cell line, RIF-1, and its thermotolerant variant cell line, TR-RIF-1 (TR), to the two stresses. The cellular responses we examined included Hsp expressions, cell viability, total protein synthesis patterns, and accumulation of poly-ubiquitinated proteins. We also compared the mRNA expression profiles and kinetics, in the two cell lines exposed to the two stresses, using microarray analysis. In contrast to RIF-1 cells, TR cells resist heat shock caused changes in cell viability and whole-cell protein synthesis. The patterns of total cellular protein synthesis and accumulation of poly-ubiquitinated proteins in the two cell lines were distinct, depending on the stress and the cell line. Microarray analysis revealed that the gene expression pattern of TR cells was faster and more transient than that of RIF-1 cells, in response to heat shock, while both RIF-1 and TR cells showed similar kinetics of mRNA expression in response to MG132. We also found that 2,208 genes were up-regulated more than 2 fold and could sort them into three groups: 1) genes regulated by both heat shock and MG132, (e.g. chaperones); 2) those regulated only by heat shock (e.g. DNA binding proteins including histones); and 3) those regulated only by MG132 (e.g. innate immunity and defense related molecules). This study shows that heat shock and MG132 share some aspects of HSR signaling pathway, at the same time, inducing distinct stress response signaling pathways, triggered by distinct abnormal proteins. 相似文献
12.
13.
Increased ubiquitin-dependent degradation can replace the essential requirement for heat shock protein induction
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Serine palmitoyltransferase, the first enzyme in ceramide biosynthesis, is required for resistance to heat shock. We show that increased heat shock sensitivity in the absence of serine palmitoyltransferase activity correlates with a lack of induction of the major heat shock proteins (Hsps) at high temperature. Normal heat shock resistance can be restored, without restoration of ceramide synthesis or induction of Hsps, by overexpression of ubiquitin. This function of ubiquitin requires the proteasome. These data imply that the essential function of Hsp induction is the removal of misfolded or aggregated proteins, not their refolding. This suggests that cells stressed by heat shock do not die because of the loss of protein activity due to their denaturation, but because of the inherent toxicity of the denatured and/or aggregated proteins. 相似文献
14.
Mathew A Mathur SK Jolly C Fox SG Kim S Morimoto RI 《Molecular and cellular biology》2001,21(21):7163-7171
15.
Kettner S Kalthoff F Graf P Priller E Kricek F Lindley I Schweighoffer T 《Molecular and cellular biology》2007,27(21):7718-7726
The activation of dendritic cells is marked by changes both on their cell surfaces and in their functions. We define EWI-2/CD316 as an early activation marker of dendritic cells upregulated by Toll-like receptor ligands clearly before CD86 and CD83. By expression cloning, human heat shock protein A8 (HSPA8), a member of the hsp70 family, was identified as the ligand for EWI-2. Soluble EWI-2 bound both to cells expressing HSPA8 and also to immobilized HSPA8 protein. Although heat shock proteins are evolutionarily well conserved, other members of this class, including human hsp60 and mycobacterial hsp65, did not bind to EWI-2. The ligation of EWI-2 enhanced the CCL21/SLC-dependent migration of activated mature dendritic cells but attenuated their antigen-specific stimulatory capacities. Important functions of recently activated dendritic cells are thus critically modulated by the newly discovered HSPA8-EWI-2 interaction. 相似文献
16.
Studies of repair enzyme activities in a uv-sensitive cell line (V79/UC) derived from Chinese hamster V79 cells have revealed levels of total DNA polymerase that are about 50% of the levels in the parental cell line. There are a number of DNA polymerase inhibitors available which allow us to distinguish between the major forms of DNA polymerase (alpha, beta, gamma, and delta) identified in mammalian cells. Enzyme assays with these inhibitors indicate that the aphidicolin-sensitive DNA polymerase is defective in the V79/UC cell line. This could be either polymerase alpha or delta, or both. The V79/UC cells do not express resistance to aphidicolin in standard toxicity studies. However, when aphidicolin is added postirradiation in survival assays designed to measure the extent of inhibitable repair, V79/UC cells do not respond with the further decrease in survival seen in the parental line. Further evidence of a polymerase-dependent repair defect is evident from alkaline elution data. In this case the V79/UC cells show the appearance of single-strand breaks following uv irradiation in the absence of any added inhibitor. Cells of the V79/M12G parental line, on the other hand, show the appearance of single-strand breaks only when aphidicolin is present. 相似文献
17.
18.
Andrija Finka Vishal Sood Manfredo Quadroni Paolo De Los Rios Pierre Goloubinoff 《Cell stress & chaperones》2015,20(4):605-620
Classic semiquantitative proteomic methods have shown that all organisms respond to a mild heat shock by an apparent massive accumulation of a small set of proteins, named heat-shock proteins (HSPs) and a concomitant slowing down in the synthesis of the other proteins. Yet unexplained, the increased levels of HSP messenger RNAs (mRNAs) may exceed 100 times the ensuing relative levels of HSP proteins. We used here high-throughput quantitative proteomics and targeted mRNA quantification to estimate in human cell cultures the mass and copy numbers of the most abundant proteins that become significantly accumulated, depleted, or unchanged during and following 4 h at 41 °C, which we define as mild heat shock. This treatment caused a minor across-the-board mass loss in many housekeeping proteins, which was matched by a mass gain in a few HSPs, predominantly cytosolic HSPCs (HSP90s) and HSPA8 (HSC70). As the mRNAs of the heat-depleted proteins were not significantly degraded and less ribosomes were recruited by excess new HSP mRNAs, the mild depletion of the many housekeeping proteins during heat shock was attributed to their slower replenishment. This differential protein expression pattern was reproduced by isothermal treatments with Hsp90 inhibitors. Unexpectedly, heat-treated cells accumulated 55 times more new molecules of HSPA8 (HSC70) than of the acknowledged heat-inducible isoform HSPA1A (HSP70), implying that when expressed as net copy number differences, rather than as mere “fold change” ratios, new biologically relevant information can be extracted from quantitative proteomic data. Raw data are available via ProteomeXchange with identifier PXD001666.
Electronic supplementary material
The online version of this article (doi:10.1007/s12192-015-0583-2) contains supplementary material, which is available to authorized users. 相似文献19.
Protein misfolding is implicated in neurodegenerative diseases and occurs in aging. However, the contribution of the misfolded ensembles to toxicity remains largely unknown. Here we introduce 2 primate cell models of destabilized proteins devoid of specific cellular functions and interactors, as bona fide misfolded proteins, allowing us to isolate the gain-of-function of non-native structures. Both GFP-degron and a mutant chloramphenicol-acetyltransferase fused to GFP (GFP-Δ9CAT) form perinuclear aggregates, are degraded by the proteasome, and colocalize with and induce the chaperone Hsp70 (HSPA1A/B) in COS-7 cells. We find that misfolded proteins neither significantly compromise chaperone-mediated folding capacity nor induce cell death. However, they do induce growth arrest in cells that are unable to degrade them and promote stress-induced death upon proteasome inhibition by MG-132 and heat shock. Finally, we show that overexpression of all heat-shock factor-1 (HSF1) and Hsp70 proteins, as well as wild-type and deacetylase-deficient (H363Y) SIRT1, rescue survival upon stress, implying a noncatalytic action of SIRT1 in response to protein misfolding. Our study establishes a novel model and extends our knowledge on the mechanism of the function-independent proteotoxicity of misfolded proteins in dividing cells. 相似文献
20.
Recent studies have demonstrated that inhibition of the proteasome, an enzyme responsible for the majority of intracellular proteolysis, may contribute to the toxicity associated with oxidative stress. In the present study we demonstrate that exposure to oxidative injury (paraquat, H(2)O(2), FeSO(4)) induces a rapid increase in reactive oxygen species (ROS), loss of mitochondrial membrane potential, inhibition of proteasome activity, and induction of cell death in neural SH-SY5Y cells. Application of proteasome inhibitors (MG115, epoxomycin) mimicked the effects of oxidative stressors on mitochondrial membrane potential and cell viability, and increased vulnerability to oxidative injury. Neural SH-SY5Y cells stably transfected with human HDJ-1, a member of the heat shock protein family, were more resistant to the cytotoxicity associated with oxidative stressors. Cells expressing increased levels of HDJ-1 displayed similar degrees of ROS formation following oxidative stressors, but demonstrated a greater preservation of mitochondrial function and proteasomal activity following oxidative injury. Cells transfected with HDJ-1 were also more resistant to the toxicity associated with proteasome inhibitor application. These data support a possible role for proteasome inhibition in the toxicity of oxidative stress, and suggest heat shock proteins may confer resistance to oxidative stress, by preserving proteasome function and attenuating the toxicity of proteasome inhibition. 相似文献