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1.
Reduced activity of the interferon-induced double-stranded RNA-dependent protein kinase during a heat shock stress 总被引:7,自引:0,他引:7
M F Dubois J Galabru P Lebon B Safer A G Hovanessian 《The Journal of biological chemistry》1989,264(21):12165-12171
Previous studies have shown that the antiviral response induced by interferon in murine cells could be degraded after a heat shock. Here we have confirmed that a similar effect occurs also in interferon-treated human HeLa cells subjected to a heat shock. In addition, we have investigated the fate of the interferon-induced, double-stranded RNA-dependent protein kinase in heat-shocked cells. This protein kinase is a Mr 68,000 protein (p68 kinase) which, when autophosphorylated, catalyzes phosphorylation of the protein synthesis eukaryotic initiation factor-2, thus mediating inhibition of protein synthesis. After heat shock of interferon-treated HeLa cells, the double-stranded RNA-dependent autophosphorylation of p68 kinase in cytoplasmic extracts is greatly reduced whereas the phosphorylation of other cellular proteins is not affected. In vivo, autophosphorylation of p68 kinase is also reduced in heat-shocked cells whereas there is no apparent effect on the phosphorylation state of other proteins. In such cells, the interferon-mediated antiviral response becomes modified according to the virus challenge, i.e. these cells remain resistant to vesicular stomatitis virus but become partially sensitive to encephalomyocarditis virus (EMCV) infection. The reduction in the activity of p68 kinase is due to its reduced nonionic detergent solubility occurring during the heat shock period. The resultant reduced detergent extractibility of p68 kinase is dependent on the intensity of the thermal stress. In contrast to the effect after a heat shock, arsenite treatment of interferon-treated HeLa cells induces heat shock proteins, but neither modifies the antiviral response nor affects the extractibility of p68 kinase. These results indicate that the degradation of the anti-EMCV response and reduced p68 kinase activity occur in response to heat treatment independently of the induction of heat shock proteins. The role of p68 kinase in the mechanism of the antiviral response against EMCV and vesicular stomatitis virus is discussed. 相似文献
2.
Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment 总被引:1,自引:0,他引:1
J Bag 《European journal of biochemistry》1983,135(2):187-196
When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes. 相似文献
3.
Protein synthesis was drastically inhibited in HeLa cells incubated for 5 min at 42.5 degrees C, but it resumed after 20 min at a rate about 50% that of control cells. After 10 min of heat shock, the binding of Met-tRNAf to 40 S ribosomal subunits was greatly reduced and a polypeptide identified by immunoprecipitation with the alpha subunit of eukaryotic initiation factor-2 (eIF-2) was phosphorylated. Extracts prepared from control and heat-shocked cells were assayed for in vitro protein synthesis. Both extracts were active when supplemented with hemin, but the extract from heat-shocked cells had little initiation activity without this addition. A Mr 90,000 polypeptide and eIF-2 alpha were phosphorylated in this extract, but hemin or an antibody which inhibits the protein kinase designated heme-controlled repressor reduced this phosphorylation. These findings implicated heme-controlled repressor as the kinase at least in part responsible for eIF-2 alpha phosphorylation. Furthermore, the initial inhibition of protein synthesis and eIF-2 alpha phosphorylation after heat shock were reduced by adding hemin to intact HeLa cells. These cells synthesized heat-shock proteins with some delay relative to cells without added hemin. The binding of Met-tRNAf to 40 S ribosomal subunits was inhibited by about 50% in extracts prepared from cells heat-shocked for 40 min, and eIF-2 alpha phosphorylation was increased in these cells. These results suggest that heme-controlled repressor is activated in heat-shocked cells and that eIF-2 alpha phosphorylation limits mRNA translation even after partial recovery of protein synthesis. 相似文献
4.
Chinese hamster ovary (CHO) cells were exposed to a 43 degrees C, 15-min heat shock to study the relationship between protein synthesis and the development of thermotolerance. The 43 degrees C heat shock triggered the synthesis of three protein families having molecular weights of 110,000, 90,000, and 65,000 (HSP). These proteins were synthesized at 37 and 46 degrees C. This heat shock also induced the development of thermotolerance, which was measured by incubating the cells at 46 degrees C 4 h after the 43 degrees C heat treatment. CHO cells were also exposed to 20 micrograms/ml of cycloheximide for 30 min at 37 degrees C, 15 min at 43 degrees C, and 4 h at 37 degrees C. This treatment inhibited the enhanced synthesis of the Mr 110,000, 90,000, and 65,000 proteins. The cycloheximide was then washed out and the cells were incubated at 46 degrees C. HSP synthesis did not recover during the 46 degrees C incubation. This cycloheximide treatment also partially inhibited the development of thermotolerance. These results suggest that for CHO cells to express thermotolerance when exposed to the supralethal temperature of 46 degrees C protein synthesis is necessary. 相似文献
5.
《The Journal of cell biology》1981,89(3):666-673
Subjecting 24-h-old cultures of quail myoblasts to incubation at an elevated temperature causes the pattern of protein synthesis to shift from the production of a broad spectrum of different proteins to the enhanced synthesis of a small number of heat-shock proteins. The synthesis of four major heat-induced polypeptides with Mrs of 88,000, 82,000, 64,000 and 25,000 achieve levels comparable to that of the major structural protein, actin. Two-dimensional electrophoretic separation and fluorographic analysis of these polypeptides establish that those with Mrs of 94,000, 88,000, 82,000, and 64,000 and pIs of 5.1, 5.2, 5.2, and 5.4, respectively, are synthesized by heat-shocked as well as by control (albeit not as intense) cultures. However, the synthesis of polypeptides with Mrs of 94,000, 64,000, and 25,000 and pI's of 5.2, 5.8, and 5.4, respectively, is detectable only in myoblasts shifted to a higher temperature. Recovery of heat-shocked myoblasts, to a normal preinduction pattern of polypeptide synthesis, takes approximately 8 h. Similar studies, completed in older, more differentiated myogenic cells, demonstrated that as cells progress through myogenesis their ability to respond to a similar temperature shift is diminished. The synthesis of some myoblastlike heat-shock proteins by fusing of cells or by myotubes requires that they be maintained at an elevated temperature at least twice as long as myoblasts. This observation and the demonstration that heat-shocked myotubes do not synthesize detectable levels of the 25,000-dalton polypeptide found in heat-shocked myoblasts, suggest that the synthetic response of myogenic cells to heat shock is dependent on the differentiative state of these cells. 相似文献
6.
N B LaThangue 《The EMBO journal》1984,3(8):1871-1879
A monoclonal antibody reacts with a polypeptide of 68 000 mol. wt. (p68) that accumulates to high levels during heat shock. The intracellular distribution of this antigen in normal and heat-shocked cells has been studied. It is a major component of non-stressed cells, where it is located predominantly in the cytoplasm, but also occurs in the nucleus. The nuclear accumulation is growth regulated, in that exponentially growing cells have strong nuclear immunofluorescence and confluent cells little. It is concentrated at the leading edge of motile fibroblasts and co-distributes with actin-containing microfilaments. Heat shock causes cytoplasmic and nuclear accumulation and there is new deposition in the periphery of cells. In normal cells the antigen in the nucleus is located in the nuclear lamina and matrix which increases during heat shock. The distribution of this molecule and the structures with which it interacts suggests that it is important in mediating the effects of heat shock. 相似文献
7.
Acquisition of the heat-shock response and thermotolerance during early development of Xenopus laevis 总被引:10,自引:0,他引:10
The ability to synthesize a 68,000- to 70,000-Da protein (hsp) in heat-shocked early Xenopus laevis embryos is dependent on the stage of development. Whereas late blastula and later stage embryos synthesize hsp68-70 after heat shock, cleavage stages are incompetent with respect to hsp synthesis. In vitro translation experiments and RNA blot analyses demonstrate that enhanced synthesis of hsp68-70 is associated with an accumulation of hsp68-70 mRNA. Examination of the effect of heat shock on preexisting actin mRNA reveals that heat shock promotes a reduction in the levels of actin mRNA in cleavage embryos but has no discernible effect on actin mRNA levels in neurula embryos. Finally, the acquisition of the heat-shock response (i.e., synthesis of hsp68-70 and accumulation of hsp70 mRNA) during early Xenopus development is correlated with the acquisition of thermotolerance. 相似文献
8.
Molecular cloning and analysis of DNA complementary to three mouse Mr = 68,000 heat shock protein mRNAs 总被引:16,自引:0,他引:16
The construction and isolation of three recombinant DNAs complementary to different mouse L-cell Mr = 68,000 heat shock protein (hsp68) mRNAs is described. cDNA libraries derived from heat-shocked mouse L-cell poly(A)+ RNA by the vector-linked primer strategy of cDNA synthesis and cloning of Okayama and Berg (Okayama, H., and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170) were screened first with a Drosophila hsp70 heterologous probe and subsequently with a cDNA probe isolated from the first screening. Positive clones were assigned to one of three sets based on their restriction map, and the largest member of each group was chosen for further analysis. All three cDNAs hybrid-select mRNA for the mouse major heat shock protein (hsp68) as assayed by in vitro translation and hybridize preferentially to two heat shock-induced hsp68 mRNAs on Northern blots. The coding regions of the cDNAs are almost identical and closely resemble other HSP70 genes but the 3' untranslated regions diverge considerably. Differences in the lengths of the untranslated regions are responsible for the two different sized induced hsp68 mRNAs in mouse L-cells. The physical maps of these cDNA clones and the limited number of mouse genomic DNA fragments detected on Southern blots suggest that there are at least three closely related heat shock-inducible members of the mouse HSP70 gene family. None of the cloned cDNAs are derived from the two related cognate genes known to be present in the mouse genome. 相似文献
9.
To investigate whether sublethal heat shock protects Perkinsus marinus (Dermo)-infected oysters Crassostrea virginica from lethal heat stress, and the effects of P. marinus infection on sublethal heat shock response, oysters were first experimentally challenged with P. marinus. Then, when infections in oysters progressed to moderate levels (parasite burden = 10(4) to 10(5) cells g(-1) wet tissue weight), oysters were treated with a sublethal heat shock at 40 degrees C for 1 h (heat shock + Dermo challenge). Other treatment groups included heat-shocked, unchallenged (non-P. marinus challenged) oysters and non-heat-shocked, P. marinus-challenged and -unchallenged oysters. Thermal tolerance was compared among these treatments by administering a lethal heat treatment at 44 degrees C for 1 h, 7 d after sublethal heat shock. Sublethal heat shock enhanced survival to lethal heat treatment in both P. marinus-challenged and -unchallenged oysters. Although levels of hsp70 isoforms (hsp69 and hsp72) did not vary significantly by heat shock or infection with P. marinus, responses due to these treatments were apparent when comparing hsp70 levels within infected and uninfected oysters. Infection enhanced expression of hsp69, regardless of whether oysters were heat shocked or not. In uninfected oysters, hsp72 increased due to heat shock 2 and 7 d post heat shock. Overall, this study demonstrates that heat shock can improve survival in oysters, even in oysters infected with P. marinus. Expression of hsp70 varied among isoforms after sublethal and lethal heat shocks and in infected and uninfected oysters. The heat shock response was not negatively affected by P. marinus infection. 相似文献
10.
We have measured the effect of heat shock on three mycoplasmas (Acholeplasma laidlawii K2 and JA1 and Mycoplasma capricolum Kid) and demonstrated the induction of mycoplasma heat shock proteins under these conditions. Increased synthesis of at least 5 heat shock proteins in A. laidlawii K2, 11 heat shock proteins in A. laidlawii JA1, and 7 heat shock proteins in M. capricolum was observed by electrophoretic analysis of proteins from heat-shocked cells in sodium dodecyl sulfate-polyacrylamide gels. In all three strains, major heat shock proteins (66 to 68 and 26 to 29 kilodaltons [kDa]) were found. The 66- to 68-kDa protein cross-reacted with antibody to Escherichia coli DnaK protein, suggesting that this heat shock protein has been conserved in spite of major reductions in genetic complexity during mycoplasma evolution. A. laidlawii also contained a 60-kDa protein that cross-reacted with eubacterial GroEL protein and a 40-kDa protein that cross-reacted with E. coli RecA protein. Unlike with coliphages, the mycoplasma virus L2 progeny yield was not increased when virus was plated on heat-shocked A. laidlawii host cells. However, UV-irradiated L2 virus could be host cell reactivated by both A. laidlawii SOS repair and heat shock systems. 相似文献
11.
12.
Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves. 总被引:24,自引:3,他引:21
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Biochemical and electron microscopic analyses of heat-shocked suspension cultures of Peruvian tomato (Lycopersicon peruvianum) revealed that a considerable part of the dominant small heat shock proteins (hsps) with an Mr of approximately 17,000 are structural proteins of newly forming granular aggregates in the cytoplasm (heat shock granules), whose formation strictly depends on heat shock conditions (37 to 40 degrees C) and the presence or simultaneous synthesis of hsps. However, under certain conditions, e.g., in preinduced cultures maintained at 25 degrees C, hsps also accumulate as soluble proteins without concomitant assembly of heat shock granules. Similar heat shock-induced cytoplasmic aggregates were also observed in other cell cultures and heat-shocked tomato leaves and corn coleoptiles. 相似文献
13.
Nuclear and nucleolar localization of the 72,000-dalton heat shock protein in heat-shocked mammalian cells 总被引:75,自引:0,他引:75
The intracellular location of the major induced mammalian heat shock (or stress) protein (Mr = 72,000) has been determined by both biochemical and immunological methods. This protein, shown here to be comprised of at least three structurally related isoforms, is produced at high levels within 30 min to 1 h following heat treatment of cells. Biochemical fractionation of cells grown under heat shock showed that following its synthesis a portion of the 72,000-Da protein (and its isoforms) becomes associated with the nucleus while some remains in the cytoplasm. Indirect immunofluorescence studies using antiserum directed against the major isoforms of the 72,000-Da protein were carried out in normal and heat-shocked cells as well as in cells grown under stress by exposure to either an amino acid analogue or to sodium arsenite. Diffuse cytoplasmic and nuclear staining was observed in cells grown at 37 degrees C. In cells grown under heat shock conditions, both the cytoplasmic staining and the nuclear staining were found to increase with the nuclear staining consisting of both granular and patch-like structures, the latter being coincident with phase-dense nucleoli. In the case of cells exposed to amino acid analogues or to sodium arsenite, only cytoplasmic and to a lesser extent nuclear staining was observed, i.e. no localized nucleolar fluorescence was observed. Following return of heat shock-treated cells to normal growth temperatures, both the synthesis of the 72,000-Dalton stress protein and its nucleolar staining were found to diminish. 相似文献
14.
《Research in virology》1991,142(1):25-31
Three major Mayaro virus proteins of 62, 50 and 34 kDa were detected in Aedes albopictus cells after 48 h postinfection at 28°C. When the infected cells were shifted from 28 to 37°C for 90 min (heat shock conditions), the synthesis of two major heat shock proteins (HSP) 82 and 70 kDa was induced concomitantly with strong inhibition of virus and normal protein synthesis. Total cellular RNA was isolated from mock and infected cells incubated at 28°C or under heat shock. Northern blot analysis with HSP genomic probes from Drosophila sp showed that (1) the probe for HSP 82 hybridized with an RNA of 2.6 kb present only in heat-shocked cells, (2) the HSP 70 probe hybridized with RNA species of 2.5 kb, present only in RNA from heat-shocked cells. These results showed that Mayaro virus was not able to alter the reprogrammation of gene expression induced by heat shock in A. albopictus cells. 相似文献
15.
We examined the synthesis of shock proteins in cultured fetal mouse myocytes. The preparation is free from fibroblasts, and the cells are vital and morphologically intact with respect to beat frequency and electron microscopy. Cultured myocytes from fetal mouse heart respond to heat shock and cadmium chloride, H2O2, allylamine, cyclosporine, and azathioprine exposure with the synthesis of shock proteins. Heat shock induces the de novo synthesis of two proteins of 71 and 68 kDa; cadmium chloride induces, in addition, a protein of 30 kDa. The other substances tested provoke the synthesis only of the 30-kDa polypeptide. The formation of heat shock proteins is concentration-dependent: Cyclosporine provokes the de novo synthesis of the 30-kDa polypeptide at concentrations above 10 ng/ml, whereas azathioprine causes the same effect at concentrations above 50 micrograms/ml. Hence cyclosporine might be cardiotoxic already at concentrations below the pharmacological dosages while azathioprine influences the myocytes only at concentrations much higher than the therapeutic level. Our results indicate that heat shock protein expression in cultured myocytes may be a useful tool to monitor cardiotoxicity. 相似文献
16.
B Allan M Linseman L A MacDonald J S Lam A M Kropinski 《Journal of bacteriology》1988,170(8):3668-3674
The general properties of the heat shock response in Pseudomonas aeruginosa were characterized. The transfer of cells from 30 to 45 degrees C repressed the synthesis of many cellular proteins and led to the enhanced production of 17 proteins. With antibodies raised against the Escherichia coli proteins, two polypeptides of P. aeruginosa with apparent molecular weights of 76,000 and 61,000 (76K and 61K proteins) were shown to be analogous to the DnaK and GroEL heat shock proteins of E. coli due to their immunologic cross-reactivity. The major sigma factor (sigma 87) of P. aeruginosa was shown to be a heat shock protein that was immunologically related to the sigma 70 of E. coli by using polyclonal antisera. A hybridoma was produced, and the monoclonal antibody MP-S-1 was specific for the sigma 87 and did not cross-react with sigma 70 of E. coli. A smaller 40K protein was immunoprecipitated with RNA polymerase antisera from cells that had been heat shocked. The 40K protein was also associated with RNA polymerase which had been purified from heat-shocked cells and may be the heat shock sigma factor of P. aeruginosa. Exposure to ethanol resulted in the production of seven new proteins, three of which appeared to be heat shock proteins. 相似文献
17.
Distinct activation mechanisms of protein kinase B by growth-factor stimulation and heat-shock treatment 总被引:1,自引:0,他引:1
Protein kinase B (PKB) alpha, having the pleckstrin homology (PH) and catalytic domains in its amino- and carboxyl-terminal regions, respectively, is activated in the signaling pathway of growth factors as a downstream target of phosphatidylinositol 3-kinase and becomes an active form in heat-shocked cells in a manner independent of the lipid kinase. Therefore, the activation mechanisms of PKBalpha were compared in platelet-derived growth factor (PDGF)-stimulated and heat-shocked cells by monitoring the protein kinase activity and phosphorylation of the mutant molecules expressed in COS-7 cells. In heat-shocked cells, PKBalpha was activated to a certain level without phosphorylation on Thr-308 in the activation loop and on Thr-450 and Ser-473 in the carboxyl-terminal end region, which is critical for growth-factor-induced activation of PKBalpha. Metabolic labeling with (32)P-orthophosphate in the transfected cells revealed that there is no major phosphorylation site other than the three residues in PKBalpha. PKBalpha activated by heat shock was more stable than the enzyme stimulated by PDGF in the cells, and PKBalpha recovered from heat-shocked cells was resistant to the protein phosphatase treatment, whereas the enzyme obtained from the growth-factor-stimulated cells was inactivated by dephosphorylation. Heat shock also enhanced the association of the PH-domain fragment to the full-length PKBalpha in the transfected cells. On the other hand, the PH-domain fragment of PKBalpha, which moves from the cytosol to the plasma membrane upon PDGF stimulation by the interaction with the phosphatidylinositol 3-kinase products, did not translocate but stayed in the cytosol in heat-shocked NIH 3T3 cells. Furthermore, PKBalpha was associated with the nuclear region in heat-shocked cells, which is not observed in growth-factor-stimulated cells. These results indicate that heat shock induces the conformational change of PKBalpha that accompanies the protein complex formation and perinuculear/nuclear localization of the enzyme, to generate an active form by a mechanism distinct from that in the growth-factor-signaling pathway. 相似文献
18.
Regulation of protein synthesis in heat-shocked Drosophila cells. Soluble factors control translation in vitro 总被引:1,自引:0,他引:1
We have developed an in vitro translation system from heat-shocked and normal Drosophila cultured cells. The lysates retain regulation of translation typical of the whole cells from which they were prepared, both when programmed by endogenous mRNA and when RNA-dependent. These systems have been used to investigate the mechanism of shutdown of normal protein synthesis and selection of heat shock mRNAs for translation in heat shock in Drosophila. Supplementation of intact RNA-dependent lysates with separated ribosome or supernatant fractions from normal or heat-shocked translation systems showed the normal supernatant fraction could "rescue" normal protein synthesis in a heat shock lysate. Normal ribosomes had no rescuing activity and neither heat shock fraction affected translation in normal lysates. Reconstitution of the system from separated ribosomes and supernatant in normal and mixed combinations showed heat shock and normal ribosomes were both competent to support normal protein synthesis with normal supernatant. Heat shock supernatant did not support normal protein synthesis with ribosomes from either source. We conclude that the factors regulating translation in heat-shocked Drosophila cells are soluble factors in the lysate and that the soluble factors present in the normal lysate are dominant. 相似文献
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20.
Heat shocks did not increase the resistance of Listeria monocytogenes to an ultrasonication treatment under pressure (Mano-Sonication; MS). While heat-shocked cells (180 min, 45 degrees C) became sixfold more heat resistant than native cells (D62 = 1.8 min vs D62 = 0.24 min), the resistance of native and heat-shocked cells to MS (200 kPa, 117 microns) was the same (DMS = 1.6 min). The inactivation rate of non-heat-shocked cells of L monocytogenes by a combined heat/ultrasonication treatment under pressure (Mano-Thermo-Sonication; MTS) was an additive effect. On the contrary, on heat-shocked cells, the inactivation rate of MTS was greater than that of heat added to the inactivation rate of MS (synergistic effect) in the range 62-68 degrees C. 相似文献