Showing papers on "Heat shock protein published in 1987"
TL;DR: The pattern of accumulation of adenylylated nucleotides during these treatments was inconsistent with a simple role for these nucleotide as alarmones sufficient for triggering the heat shock response, but was consistent with a role in the oxyR-mediated response.
Abstract: Heat and various inhibitory chemicals were tested in Escherichia coli for the ability to cause accumulation of adenylylated nucleotides and to induce proteins of the heat shock (htpR-controlled), the oxidation stress (oxyR-controlled), and the SOS (lexA-controlled) regulons. Under the conditions used, heat and ethanol initiated solely a heat shock response, hydrogen peroxide and 6-amino-7-chloro-5,8-dioxoquinoline (ACDQ) induced primarily an oxidation stress response and secondarily an SOS response, nalidixic acid and puromycin induced primarily an SOS and secondarily a heat shock response, isoleucine restriction induced a poor heat shock response, and CdCl2 strongly induced all three stress responses. ACDQ, CdCl2, and H2O2 each stimulated the synthesis of approximately 35 proteins by factors of 5- to 50-fold, and the heat shock, oxidation stress, and SOS regulons constituted a minor fraction of the overall cellular response. The pattern of accumulation of adenylylated nucleotides during these treatments was inconsistent with a simple role for these nucleotides as alarmones sufficient for triggering the heat shock response, but was consistent with a role in the oxyR-mediated response.
489 citations
TL;DR: The yeast heat-shock factor binds constitutively to DNA but only activates transcription after heat-induced phosphorylation, and the mobility of protein-HSE complexes on polyacrylamide gels is altered following heat shock.
Abstract: When cells are exposed to elevated temperatures, transcription of a small set of genes, the heat-shock genes, is activated. This response is mediated by a short DNA sequence, the heat-shock element (HSE), which is thought to be the binding site for a specific transcription factor. Studies with Drosophila show that this protein binds to HSEs only in heat-shocked cells, suggesting that changes in factor binding are responsible for gene activation. We have investigated the properties of HSE-binding proteins from yeast and HeLa cells. In HeLa cells, binding activity is present only after heat shock. In contrast, control and heat-shocked yeast cells yield the same amount of HSE-binding activity; however, the mobility of protein-HSE complexes on polyacrylamide gels is altered following heat shock. This mobility difference can be significantly reduced by treatment of crude extracts with phosphatase. We propose that the yeast heat-shock factor binds constitutively to DNA but only activates transcription after heat-induced phosphorylation.
429 citations
TL;DR: Results indicate that rat heme oxygenase is a heat shock protein and that hemin induces hemeoxyase through a different mechanism from heat shock.
410 citations
TL;DR: The results indicate that σ32 is directly responsible for regulation of the heat shock response, and measured the amount of ρ32 before and after shift to high temperature and found that it increased transiently during heat shock as a result of changes in σ 32 synthesis and stability.
Abstract: Cells subjected to a heat shock, or a variety of other stresses increase the synthesis of a set of proteins, known as heat shock proteins. This response is apparently universal, occurring in the entire range from bacterial to mammalian cells. In Escherichia coli heat shock protein synthesis transiently increases following a shift from 30 degrees C to 42 degrees C as a result of changes in transcription initiation at heat shock promoters. Heat shock promoters are recognized by RNA polymerase containing a sigma factor of relative molecular mass (Mr) 32,000 (32K) E sigma 32 and not E sigma 70, the major form of RNA polymerase holoenzyme. To determine whether changes in the concentration of sigma 32 regulate this response, we measured the amount of sigma 32 before and after shift to high temperature and found that it increased transiently during heat shock as a result of changes in sigma 32 synthesis and stability. Our results indicate that sigma 32 is directly responsible for regulation of the heat shock response.
389 citations
TL;DR: Drosophila heat shock activator protein, a rare transacting factor which is induced upon heat shock to bind specifically to the heat shock regulatory sequence in vivo, has been purified from shocked cells to more than 95 percent homogeneity by sequence-specific duplex oligonucleotide affinity chromatography.
Abstract: Drosophila heat shock activator protein, a rare transacting factor which is induced upon heat shock to bind specifically to the heat shock regulatory sequence in vivo, has been purified from shocked cells to more than 95 percent homogeneity by sequence-specific duplex oligonucleotide affinity chromatography. The purified protein has a relative molecular mass of 110 kilodaltons, binds to the regulatory sequence with great affinity and specificity, and strongly stimulates transcription of the Drosophila hsp70 gene. Studies with this regulatory protein should lead to an understanding of the biochemical pathway underlying the heat shock phenomenon.
336 citations
TL;DR: It is shown that within 30 seconds of heat shock of intact cells the sequence-specific binding activity in whole cell extracts increases significantly, reaching a plateau by 5min after the start of the shock; removal of the heat stimulus returns the activity to basal levels.
Abstract: Drosophila tissue culture cells stimulated by heat shock contain high levels of heat shock activator protein, which binds specifically to the heat-shock control DNA element. In contrast, nonshocked cells have low basal levels of binding activity. Here, we show that within 30 seconds of heat shock of intact cells the sequence-specific binding activity in whole cell extracts increases significantly, reaching a plateau by 5 min after the start of the shock; removal of the heat stimulus returns the activity to basal levels. Known chemical inducers of heat-shock genes elicit a similar pattern of specific binding activity. Moreover, this pattern is observed in the presence of protein synthesis inhibitors, even if the stimulus-withdrawal is repeated sequentially through five cycles. Our results are inconsistent with models which propose proteolysis as the chief means of mediating heat-shock transcriptional control. Rather, they suggest that heat shock activator pre-exists in normal cells in a nonbinding form, which is converted upon cell stimulus to a high affinity, sequence-specific binding form, most probably by a post-translational modification. This conversion may be crucial for the transcriptional activation of heat shock genes.
334 citations
TL;DR: The relative affinities of the protein for a range of variant HSE sequences correlates with the ability of these sequences to support heat‐inducible transcription in vivo, suggesting that this polypeptide is involved in the activation of heat‐shock promoters.
Abstract: The promoters of heat shock genes are activated when cells are stressed. Activation is dependent on a specific DNA sequence, the heat-shock element (HSE). We describe the purification to homogeneity of an HSE-binding protein from yeast (Saccharomyces cerevisiae), using sequential chromatography of whole cell extracts on heparin-agarose, calf thymus DNA-Sepharose and an affinity column consisting of a repetitive synthetic HSE sequence coupled to Sepharose. The protein runs as a closely spaced doublet of approximately 150 kd on SDS-polyacrylamide gels; mild proteolysis generates a stable 70-kd fragment which retains DNA binding activity. The relative affinities of the protein for a range of variant HSE sequences correlates with the ability of these sequences to support heat-inducible transcription in vivo, suggesting that this polypeptide is involved in the activation of heat-shock promoters. However, the protein was purified from unshocked yeast, and may therefore represent an unactivated form of heat-shock transcription factor. Study of the purified protein should help to define the mechanistic basis of the heat-shock response.
314 citations
TL;DR: It is shown that temperature- mediated dissociation of hsp90 from the receptor is a hormone-dependent event in the same manner as temperature-mediated transformation to the DNA-binding state and ammonium sulfate causes both dissociation and conversion of the receptor to theDNA-binding form in a manner that does not require the presence of steroid.
258 citations
TL;DR: HSP70 RNA is more stable in cells treated with protein synthesis inhibitors, suggesting that a heat shock-sensitive labile protein regulates its turnover, and heat shock is not required for the efficient translation of HSP70 mRNA.
Abstract: We have examined the posttranscriptional regulation of hsp70 gene expression in two human cell lines, HeLa and 293 cells, which constitutively express high levels of HSP70. HSP70 mRNA translates with high efficiency in both control and heat-shocked cells. Therefore, heat shock is not required for the efficient translation of HSP70 mRNA. Rather, the main effect of heat shock on translation is to suppress the translatability of non-heat shock mRNAs. Heat shock, however, has a marked effect on the stability of HSP70 mRNA; in non-heat-shocked cells the half-life of HSP70 mRNA is approximately 50 min, and its stability increases at least 10-fold upon heat shock. Moreover, HSP70 mRNA is more stable in cells treated with protein synthesis inhibitors, suggesting that a heat shock-sensitive labile protein regulates its turnover. An additional effect on posttranscriptional regulation of hsp70 expression can be found in adenovirus-infected cells, in which HSP70 mRNA levels decline precipititously late during infection although hsp70 transcription continues unabated.
252 citations
TL;DR: It is demonstrated that all three proteins are related isoforms, with two of the isoforms containing phosphate, and a relatively simple and rapid purification of the proteins employing both ion-exchange and gel filtration chromatography.
251 citations
TL;DR: The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines and suggest that the p 53-hsc70 protein complex could have functional significance in these transformed cells.
Abstract: A rabbit antiserum was prepared against the C-terminal peptide of 21 amino acids from the human heat shock protein hsp70. These antibodies were shown to be specific for this highly inducible heat shock protein (72 kilodaltons [kDa] in rat cells), and for a moderately inducible, constitutively expressed heat shock protein, hsc70 (74 kDa). In six independently derived rat cell lines transformed by a murine cDNA-genomic hybrid clone of p53 plus an activated Ha-ras gene, elevated levels of p53 were detected by immunoprecipitation by using murine-specific anti-p53 monoclonal antibodies. In all cases, the hsc70, but not the hsp70, protein was coimmunoprecipitated with the murine p53 protein. Similarly, antiserum to heat shock protein coimmunoprecipitated p53. Western blot (immunoblot) analysis demonstrated that the hsc70 and p53 proteins did not share detectable antigenic epitopes. The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines. A p53 cDNA clone, p11-4, failed to produce clonable cell lines from foci of primary rat cells transfected with p11-4 plus Ha-ras. A mutant p53 cDNA clone derived from p11-4, SVKH215, yielded a 2- to 35-fold increase in the number of foci produced after transfection of rat cells with SVKH215 plus Ha-ras. When cloned, 87.5% of these foci produced transformed cell lines. SVKH215 encodes a mutant p53 protein that binds preferentially to the heat shock proteins of 70 kDa compared with binding by the parental p11-4 p53 gene product. These data suggest that the p53-hsc70 protein complex could have functional significance in these transformed cells.
TL;DR: It is shown that sigma 32 is an unstable protein, degraded with a half-life of approximately 4 min, and that both the induction and decline in the synthesis of heat shock proteins can be controlled by changes in the rate of synthesis of s Sigma 32.
Abstract: The Escherichia coli rpoH (htpR) gene product, sigma 32, is required for the normal expression of heat shock genes and for the heat shock response. We present experiments indicating a direct role for sigma 32 in controlling the heat shock response. Both the induction and decline in the synthesis of heat shock proteins can be controlled by changes in the rate of synthesis of sigma 32. Specifically, we show that: (1) sigma 32 is an unstable protein, degraded with a half-life of approximately 4 min; (2) increasing the rate of synthesis of sigma 32, by inducing expression from a Plac or Ptac-rpoH fusion, is sufficient to increase the rate of synthesis of heat shock proteins; (3) during the shut-off phase of the heat shock response synthesis of sigma 32 is repressed post-transcriptionally, and the dnaK756 mutation, which causes a defect in the shut-off phase, prevents the post-transcriptional repression of synthesis of sigma 32. These results serve as a basis for understanding the role of DnaK in the heat shock response, the regulation of sigma 32 synthesis, and the role of sigma 32 in controlling transcription of heat shock genes.
TL;DR: The observation that cells of the FM3A ts85 mutant, which have a thermolabile ubiquitin activating enzyme (E1), do not accelerate protein degradation in response to a 43 degrees C heat shock, whereas wild‐typeFM3A mouse cells do indicates that the ubiquit in system is involved in the degradation of heat‐denatured proteins.
Abstract: Exposure of cultured rat hepatoma (HTC) cells to a 43 degrees C heat shock transiently accelerates the degradation of the long-lived fraction of cellular proteins. The rapid phase of proteolysis which lasts approximately 2 h after temperature step-up is followed by a slower phase of proteolysis. During the first 2 h after temperature step-up there is a wave of ubiquitin conjugation to cellular proteins which is accompanied by a fall in ubiquitin and ubiquitinated histone 2A (uH2A) levels. Upon continued incubation at 43 degrees C the levels of ubiquitin conjugates fall with a corresponding increase of ubiquitin and uH2A to initial levels. The burst of protein degradation and ubiquitin conjugation after temperature step-up is not affected by the inhibition of heat shock protein synthesis. Cells of the FM3A ts85 mutant, which have a thermolabile ubiquitin activating enzyme (E1), do not accelerate protein degradation in response to a 43 degrees C heat shock, whereas wild-type FM3A mouse cells do. This observation indicates that the ubiquitin system is involved in the degradation of heat-denatured proteins. Sequential temperature jump experiments show that the extent of proteolysis at temperatures up to 43 degrees C is related to the final temperature and not to the number of steps taken to attain it. Temperature step-up to 45 degrees C causes the inhibition of intracellular proteolysis. We propose the following explanation of the above observations. Heat shock causes the conformational change or denaturation of a subset of proteins stable at normal temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation, suggesting that they are caused by a loss rather than an alteration of DnaK function.
Abstract: DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp70s) of Drosophila melanogaster and humans. Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E. coli genome by homologous recombination in a recB recC sbcB strain. Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37 degrees C) and could not form colonies at a high temperature (42 degrees C); furthermore, they also formed long filaments at 42 degrees C. The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42 degrees C for 2 h were no longer capable of forming colonies at 30 degrees C. The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42 degrees C. These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent. In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain. The well-characterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function. The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation.(ABSTRACT TRUNCATED AT 250 WORDS) Images
Journal Article•
TL;DR: It is important that studies investigating the role of hsps in stress and other cellular responses such as growth and differentiation define the specific gene (including its regulatory sequences) that encodes the protein being investigated, in order to avoid apparently contradictory and confusing reports ofHsps expression.
Abstract: Heat shock proteins appear to be causatively involved in the acquisition of thermotolerance in prokaryotes but not in eukaryotes. Further, the enhanced synthesis of hsps may be necessary for some cellular responses to stress but not others. In prokaryotic cells the development of thermotolerance, as measured by cell survival, is dependent upon protein synthesis. However, in eukaryotes, enhanced hsp synthesis following an inducing stress and prior to a subsequent heat shock is neither necessary nor sufficient for the development of thermotolerance as measured by colony-forming assays. The enhanced expression of hsps may be required for some mammalian cellular stress responses, such as the ability to reform both actin microfilament bundles and nucleolar morphology. These latter two thermotolerant responses have not been correlated with colony-forming ability. Future work should address the relationships between these various physiological responses to stress and determine if hsps function in some repair mode with regard to colony formation responses. Evidence is accumulating that hsps or their cognates may function in growth and differentiation in some manner as yet to be fully explained. Recent studies indicate that genes controlling cell division in E. coli may be linked to those of several stress regulons, and it would not be surprising to find a similar relationship in eukaryotes. At this time, it is important that studies investigating the role of hsps in stress and other cellular responses such as growth and differentiation define the specific gene (including its regulatory sequences) that encodes the protein being investigated, in order to avoid apparently contradictory and confusing reports of hsps expression.
TL;DR: A factor found in nuclear extracts of human cells bound to the heat shock element of a human heat shock protein 70 gene was significantly increased after heat shock, suggesting that an initial event in the response of ahuman cell to heat is the activation of a preexisting regulatory factor.
Abstract: A factor found in nuclear extracts of human cells bound to the heat shock element of a human heat shock protein 70 gene. The level of this factor was significantly increased after heat shock. This induction was rapid and was not blocked by cycloheximide, suggesting that an initial event in the response of a human cell to heat is the activation of a preexisting regulatory factor.
TL;DR: It is suggested that, at least in the case of radiation and treatment with H2O2, the 32-kDa protein might be induced in response to cellular oxidative stress, supported by the observation that depletion of endogenous cellular glutathione prior to solar near ultraviolet irradiation lowers the fluence threshold for induction of the 32 -kDa stress protein.
TL;DR: The isolation of an E. coli homologue of hsp83 illustrates the remarkable conservation of heat shock proteins in evolution and will facilitate genetic and biochemical experiments aimed at determining the function of hSp83.
Abstract: We have isolated a gene from Escherichia coli homologous to the gene encoding the Mr 83,000 Drosophila heat shock protein (hsp83). In E. coli the protein homologous to hsp83 is a heat shock protein called C62.5. The predicted amino acid sequence of C62.5 is 41% and 42% identical to the Drosophila and human hsp83 proteins, respectively. Selected regions of the protein have conservation as high as 90%. The gene encoding C62.5 (named htpG) is located between the dnaZ and adk genes at 11.1 minutes on the E. coli chromosome. The htpG gene appears to be a newly identified locus. The isolation of an E. coli homologue of hsp83 illustrates the remarkable conservation of heat shock proteins in evolution and will facilitate genetic and biochemical experiments aimed at determining the function of hsp83.
TL;DR: Since hypoxia and/or nutrient deprivation can occur during tumor development, a GRP-induced state in the tumor may confer resistance to doxorubicin treatment.
Abstract: The glucose-regulated protein (GRP) system in mammalian cells is induced by glucose deprivation, anoxia, the calcium ionophore A23187, and 2-deoxyglucose. In Chinese hamster ovary cells the major GRPs are approximately equal to 76, 97, and 170 kDa. Removal of each of these four GRP-inducing stresses leads to the coordinate repression of GRPs and induction of the major heat shock proteins at 70 and 89 kDa. The application of each of these four GRP-inducing conditions leads to a significant induction of resistance to the drug doxorubicin. Removal of each GRP-inducing condition results in the rapid disappearance of this resistance in a manner that correlates with the repression of the GRPs. The retention of doxorubicin by GRP-induced cells does not explain the induced drug resistance. When the RIF in vitro/in vivo tumor system is probed with an antibody against the 76-kDa GRP, a significant increase in this GRP is observed in cells obtained from the central regions of tumors. Since hypoxia and/or nutrient deprivation can occur during tumor development, a GRP-induced state in the tumor may confer resistance to doxorubicin treatment.
TL;DR: The genome of the yeast Saccharomyces cerevisiae contains a family of genes related to the HSP70 genes (encoding the 70-kDa heat shock protein) of other eukaryotes, and mutations constructed in vitro and substituted into the yeast genome in place of the wild-type alleles indicate that at least three distinct functions are carried out by genes of the H SP70 family.
Abstract: The genome of the yeast Saccharomyces cerevisiae contains a family of genes related to the HSP70 genes (encoding the 70-kDa heat shock protein) of other eukaryotes. Mutations in two of these yeast genes (SSC1 and SSD1), whose expression is increased a few fold after temperature upshift, were constructed in vitro and substituted into the yeast genome in place of the wild-type alleles. No phenotypic effects of the mutation in SSD1 were detected. However, a functional SSC1 gene is essential for vegetative growth. This result, in conjunction with experiments involving mutations in other members of this multigene family, indicates that at least three distinct functions are carried out by genes of the HSP70 family.
TL;DR: It is shown that the levels of hsc73 mRNA are approximately 5‐fold higher in rapidly growing tissue‐culture cells than in cells whose growth has been arrested by serum starvation, and the gene appears to be subject to more than one form of regulation, mediated by different promoter elements that are intermingled.
Abstract: The isolation and complete DNA sequence of a rat genomic clone encoding hsc73, the major hsp70-like protein found in growing cells is described. Unlike the heat-inducible genes characterized so far, the hsc73 gene is interrupted by introns, and there are also numerous intronless hsc73 pseudogenes in the rat genome. We show that the levels of hsc73 mRNA are approximately 5-fold higher in rapidly growing tissue-culture cells than in cells whose growth has been arrested by serum starvation. The abundance of hsc73 mRNA is not significantly increased by heat shock of either fed or starved cells. The hsc73 promoter contains putative binding sites for transcription factor Sp1, two CCAAT boxes and, surprisingly, two matches to the consensus heat-shock regulatory element. When fused to the CAT gene and transfected into COS or HeLa cells, the promoter is constitutively active, showing only a small induction by heat shock. Deletion of some constitutive elements makes it more strongly heat-inducible. The gene thus appears to be subject to more than one form of regulation, mediated by different promoter elements that are intermingled.
TL;DR: Treatment of a wild strain of E. coli with various toxic agents revealed no correlation between the development of thermotolerance and the induction of any subset of the heat shock proteins, and Thermotolerance appears to develop by processes other than the htpR-dependent induction of heatshock proteins.
Abstract: The addition of isopropyl thio-~-D-galactoside (IPTG) to Escherichia coli cells containing multiple copies of the heat shock regulatory gene htpR (rpoI-l) under the control of an IPTG-inducible promoter (P-tac) induced 15 of the 17 polypeptides of the heat shock (HTP) regulon. The time course and magnitude of the induction closely resembled that caused by a shift to 42°C. Nevertheless the two means of inducing the heat shock regulon differed in outcome. Cultures grown at 28°C and induced by incubation at 42°C for 15 min gave significant protection against a challenge temperature of 50°C, but no protection was afforded by a 15-min IPTG treatment at 28°C. It could be shown that there was no interference by IPTG with the development of thermotolerance at 42°C. Also, treatment of a wild strain of E. coil with various toxic agents revealed no correlation between the development of thermotolerance and the induction of any subset of the heat shock proteins. Thermotolerance appears to develop by processes other than the htpR-dependent induction of heat shock proteins.
Journal Article•
TL;DR: Examination of the expression of a series of mouse mutant, as well as wild-type, p53 proteins in SV40-transformed monkey COS cells shows that these proteins are identical to the hsp 72/73 heat shock proteins.
Abstract: We have examined the expression of a series of mouse mutant, as well as wild-type, p53 proteins in SV40-transformed monkey COS cells. Wild-type mouse p53 binds predominantly to SV40 large T antigen in these cells. However, several of the mutants co-precipitate exclusively with proteins of approximately 68 Kd relative molecular mass. We show by immunological and proteolytic mapping techniques that these proteins are identical to the hsp 72/73 heat shock proteins. p53 mutants in complex with hsp 72/73 have an altered subcellular location compared to the wild-type protein and the hsp 72/73 binding p53 mutants fail to exhibit an epitope recognized by monoclonal antibody PAb 246. The existence of at least two antigenically distinct subclasses of hsp 72/73 complexed to mutant p53 is shown.
TL;DR: A comparison of the translated product of the human cDNA to the published yeast 90-kDa heat-shock protein reveals more than 60% homology at both the nucleotide and amino acid levels.
TL;DR: Results are consistent with the observation that these inducers of the heat shock response, as well as cycloheximide, repress protein synthesis and suggest that the increase in the level of c-fos mRNA is caused by an inhibition of protein synthesis.
Abstract: Several known inducers of the heat shock response (heat stress, arsenite, and heavy metals) were shown to cause a significant elevation of c-fos mRNA in HeLa cells. Heat stress resulted in a time- and temperature-dependent prolonged elevation in the level of c-fos mRNA, which was accompanied by increased translation of c-fos protein and its appearance in the nucleus. Elevated expression of c-fos during heat stress was paralleled by induction of hsp 70 mRNA, while levels of c-myc and metallothionein mRNAs declined. Treatment of HeLa cells with arsenite or heavy metals also resulted in increased levels of hsp 70, as well as c-fos mRNA. Although elevated expression of c-fos was prevented by inhibitors of RNA synthesis, analysis of relative rates of gene transcription showed that during heat stress there was a negligible change in c-fos transcription. Therefore, the enhanced expression of c-fos during the heat shock response is likely to occur primarily through posttranscriptional processes. Cycloheximide was also shown to significantly increase the c-fos mRNA level in HeLa cells. There results are consistent with the observation that these inducers of the heat shock response, as well as cycloheximide, repress protein synthesis and suggest that the increase in the level of c-fos mRNA is caused by an inhibition of protein synthesis. This supports the hypothesis that c-fos mRNA is preferentially stabilized under conditions which induce the heat shock response, perhaps by decreased synthesis of a short-lived protein which regulates c-fos mRNA turnover.
TL;DR: In situ hybridization reveals striking regional differences in the expression of constitutive and inducible heat shock genes in the rabbit brain, with dramatic induction of heat shock mRNA after hyperthermia.
TL;DR: A diverse array of low molecular weight heat shock proteins was synthesized in each of these species and exhibited considerable heterogeneity in isoelectric point, molecular weight, stainability, and radiolabel incorporation.
Abstract: Heat shock of living tissue induces the synthesis of a unique group of proteins, the heat shock proteins. In plants, the major group of heat shock proteins has a molecular mass of 15 to 25 kilodaltons. Accumulation of these proteins to stainable levels has been reported in only a few species. To examine accumulation of the low molecular weight heat shock proteins in a broader range of species, two-dimensional electrophoresis was used to resolve total protein from the following species: soybean (Glycine max L. Merr., var Wayne), pea (Pisum sativum L., var Early Alaska), sunflower (Helianthus annuus L.), wheat (Triticum aestivum L.), rice (Oryza sativa L., cv IR-36), maize (Zea mays L.), pearl millet (Pennisetum americanum L. Leeke, line 23DB), and Panicum miliaceum L. When identified by both silver staining and incorporation of radiolabel, a diverse array of low molecular weight heat shock proteins was synthesized in each of these species. These proteins accumulated to significant levels after three hours of heat shock but exhibited considerable heterogeneity in isoelectric point, molecular weight, stainability, and radiolabel incorporation. Although most appeared to be synthesized only during heat shock, some were detectable at low levels in control tissue. Compared to the monocots, a higher proportion of low molecular weight heat shock proteins was detectable in control tissues from dicots.
TL;DR: Spinach (Spinacia oleracea L. cv Bloomsdale) seedlings cultured in vitro were used to study changes in protein synthesis during cold acclimation and heat shock altered the pattern of protein synthesis and induced the synthesis of several heat shock proteins (HSPs).
Abstract: Spinach (Spinacia oleracea L. cv Bloomsdale) seedlings cultured in vitro were used to study changes in protein synthesis during cold acclimation. Seedlings grown for 3 weeks postsowing on an inorganic-nutrient-agar medium were able to increase their freezing tolerance when grown at 5°C. During cold acclimation at 5°C and deacclimation at 25°C, the kinetics of freezing tolerance induction and loss were similar to that of soil-grown plants. Freezing tolerance increased after 1 day of cold acclimation and reached a maximum within 7 days. Upon deacclimation at 25°C, freezing tolerance declined within 1 day and was largely lost by the 7th day. Leaf proteins of intact plants grown at 5 and 25°C were in vivo radiolabeled, without wounding or injury, to high specific activities with [35S]methionine. Leaf proteins were radiolabeled at 0, 1, 2, 3, 4, 7, and 14 days of cold acclimation and at 1, 3, and 7 days of deacclimation. Up to 500 labeled proteins were separated by two-dimensional gel electrophoresis and visualized by fluorography. A rapid and stable change in the protein synthesis pattern was observed when seedlings were transferred to the low temperature environment. Cold-acclimated leaves contained 22 polypeptides not found in nonacclimated leaves. Exposure to 5°C induced the synthesis of three high molecular weight cold acclimation proteins (CAPs) (Mr of about 160,000, 117,000, and 85,000) and greatly increased the synthesis of a fourth high molecular weight protein (Mr 79,000). These proteins were synthesized during day 1 and throughout the 14 day exposure to 5°C. During deacclimation, the synthesis of CAPs 160, 117, and 85 was greatly reduced by the first day of exposure to 25°C. However, CAP 79 was synthesized throughout the 7 day deacclimation treatment. Thus, the induction at low temperature and termination at warm temperature of the synthesis of CAPs 160, 117, and 85 was highly correlated with the induction and loss of freezing tolerance. Cold acclimation did not result in a general posttranslational modification of leaf proteins. Most of the observed changes in the two-dimensional gel patterns could be attributed to the de novo synthesis of proteins induced by low temperature. In spinach leaf tissue, heat shock altered the pattern of protein synthesis and induced the synthesis of several heat shock proteins (HSPs). One polypeptide synthesized in cold-acclimated leaves had a molecular weight and net charge (Mr 79,000, pI 4.8) similar to that of a HSP (Mr 83,000, pI 4.8). However, heat shock did not increase the freezing tolerance, and cold acclimation did not increase heat tolerance over that of nonacclimated plants, but heat-shocked leaf tissue was more tolerant to high temperatures than nonacclimated or cold-acclimated leaf tissue. When protein extracts from heat-shocked and cold-acclimated leaves were mixed and separated in the same two-dimensional gel, the CAP and HSP were shown to be two separate polypeptides with slightly different isoelectric points and molecular weights.
TL;DR: Ubiquitin was radiolabeled by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using erythrocyte-mediated microinjection and degraded denatured hemoglobin more rapidly than native hemoglobin and ubiquitin-globin conjugates formed within them, ensuring stabilization of proteins after heat-shock.
Abstract: Ubiquitin was radiolabeled by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using erythrocyte-mediated microinjection. The injected cells were then incubated at 45 degrees C for 5 min (reversible heat-shock) or for 30 min (lethal heat-shock). After either treatment, there were dramatic changes in the levels of ubiquitin conjugates. Under normal culture conditions, approximately 10% of the injected ubiquitin is linked to histones, 40% is found in conjugates with molecular weights greater than 25,000, and the rest is unconjugated. After heat-shock, the free ubiquitin pool and the level of histone-ubiquitin conjugates decreased rapidly, and high molecular weight conjugates predominated. Formation of large conjugates did not require protein synthesis; when analyzed by two-dimensional electrophoresis, the major conjugates did not co-migrate with heat-shock proteins before or after thermal stress. Concomitant with the loss of free ubiquitin, the degradation of endogenous proteins, injected hemoglobin, BSA, and ubiquitin was reduced in heat-shocked HeLa cells. After reversible heat-shock, the decrease in proteolysis was small, and both the rate of proteolysis and the size of the free ubiquitin pool returned to control levels upon incubation at 37 degrees C. In contrast, neither proteolysis nor free ubiquitin pools returned to control levels after lethal heat-shock. However, lethally heat-shocked cells degraded denatured hemoglobin more rapidly than native hemoglobin and ubiquitin-globin conjugates formed within them. Therefore, stabilization of proteins after heat-shock cannot be due to the loss of ubiquitin conjugation or inability to degrade proteins that form conjugates with ubiquitin.
TL;DR: The results suggest that a decrease in the level of cAMP-dependent protein phosphorylation results in the heat shock response, including elevated synthesis of three heat shock proteins, acquisition of thermotolerance, and transient arrest of the cell cycle.
Abstract: When Saccharomyces cerevisiae cells grown at 23 degrees C were transferred to 36 degrees C, they initiated synthesis of heat shock proteins, acquired thermotolerance to a lethal heat treatment given after the temperature shift, and arrested their growth transiently at the G1 phase of the cell division cycle. The bcy1 mutant which resulted in production of cyclic AMP (cAMP)-independent protein kinase did not synthesize the three heat shock proteins hsp72A, hsp72B, and hsp41 after the temperature shift. The bcy1 cells failed to acquire thermotolerance to the lethal heat treatment and were not arrested at the G1 phase after the temperature shift. In contrast, the cyr1-2 mutant, which produced a low level of cAMP, constitutively produced three heat shock proteins and four other proteins without the temperature shift and was resistant to the lethal heat treatment. The results suggest that a decrease in the level of cAMP-dependent protein phosphorylation results in the heat shock response, including elevated synthesis of three heat shock proteins, acquisition of thermotolerance, and transient arrest of the cell cycle.