Simultaneous exposure of Xenopus A6 kidney epithelial cells to concurrent mild sodium arsenite and heat stress results in enhanced hsp30 and hsp70 gene expression and the acquisition of thermotolerance

In this study, we examined the effect of concurrent low concentrations of sodium arsenite and mild heat shock temperatures on hsp30 and hsp70 gene expression in Xenopus A6 kidney epithelial cells. RNA blot hybridization and immunoblot analysis revealed that exposure of A6 cells to 1–10 µM sodium arsenite at a mild heat shock temperature of 30 °C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 µM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26 °C with larger responses at 28 and 30 °C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 µM) and heat shock (30 °C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge (37 °C). Acquired thermotolerance was not observed with cells treated with the two mild stresses individually.     

Roles of Escherichia coli heat shock proteins DnaK, DnaJ and GrpE in mini-F plasmid replication

Summary A subset of Escherichia coli heat shock proteins, DnaK, DnaJ and GrpE were shown to be required for replication of mini-F plasmid. Strains of E. coli K12 carrying a missense mutation or deletion in the dnaK, dnaJ, or grpE gene were virtually unable to be transformed by mini-F DNA at the temperature (30° C) that permits cell growth. When excess amounts of the replication initiator protein (repE gene product) of mini-F were provided by means of a multicopy plasmid carrying repE, these mutant bacteria became capable of supporting mini-F replication under the same conditions. However, the copy number of a high copy number mini-F plasmid was reduced in these mutant bacteria as compared with the wild type in the presence of excess RepE protein. Furthermore, mini-F plasmid mutants that produce altered initiator protein and exhibit a very high copy number were able to replicate in strains deficient in any of the above heat shock proteins. These results indicate that the subset of heat shock proteins (DnaK, DnaJ and GrpE) play essential roles that help the functioning of the RepE initiator protein in mini-F DNA replication.     

The heat shock response in meso- and thermoacidophilic chemolithotrophic bacteria

Abstract The heat shock response was studied in a chemolithotrophic thermoacidophilic archaebacterium Sulfolobus acidocaldarius (shifted from 70° to 85°C) and a mesoacidop0ilic microorganism Thiobacillus ferrooxidans (from 30° to 41°C). When transferred from their normal growth temperature to the stress temperature, cells showed a decrease in the incorporation of Na₂¹⁴CO₃ into proteins, and at the same time, the synthesis of a specific subset of heat shock proteins was observed. Ethanol (4%) at 30°C, also caused a response similar to the heat shock upon T. ferrooxidans cells, whereas Sulfolobus cells at 70°C did not incorporate radioactive CO₂ in the presence of ethanol, apparently being damaged by the organic solvent.     

Dobutamine mediates cytoprotection by induction of heat shock protein 70 in vitro

Aims

Dobutamine is cytoprotective when applied before a subsequent stress. However, the underlying molecular mechanism is unknown. Dobutamine also inhibits nuclear factor (NF)-κB in human T lymphocytes. Other inhibitors of NF-κB induce a so-called heat shock response. We hypothesized that dobutamine mediates protection from apoptotic cell death by the induction of a heat shock response.

Main methods

Jurkat T lymphoma cells were preincubated with dobutamine (0.1, 0.5 mM) before the induction of apoptosis (staurosporine, 2 μM). DNA-binding of heat shock factor (HSF)-1 was analyzed by electrophoretic mobility shift assay, mRNA-expression of heat shock protein (hsp)70 and hsp90 by Northern Blot, activity of caspase-3 by fluorogenic caspase activity assay and cleavage of pro-caspase-3 by Western Blot. Apoptosis was assessed by flow cytometry after annexin V-fluorescein isothiocyanate staining. Hsp70 and hsp90 were inhibited using N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolaetam and 17-allylamino-17-demethoxygeldana-mycin, respectively. All data are given as median and 25/75% percentile.

Key findings

Pre-incubation with dobutamine inhibited staurosporine-induced annexin V-fluorescence (28 [20–32] % vs. 12 [9–15] % for dobutamine 0.1 mM and 7 [5–12] % for dobutamine 0.5 mM, p < 0.001), cleavage of pro-caspase-3 as well as caspase-3-like activity (0.46 [0.40–0.48] vs. 0.32 [0.27–0.39] for Dobutamine 0.1 mM and 0.20 [0.19–0.23] for Dobutamine 0.5 mM, p < 0.01). Dobutamine induced DNA-binding of HSF-1 and mRNA-expression of hsp70 and hsp90. While inhibition of Hsp90 had no effect, inhibition of Hsp70 increased the number of annexin V-positive cells (33 [32–36] % vs. 18 [16–24] %) and caspase-3-like activity (0.21 [0.19–0.23] vs. 0.16 [0.13–0.17], p < 0.05).

Significance

Dobutamine protects from apoptotic cell death via the induction of Hsp70.     

HSP12, a new small heat shock gene of Saccharomyces cerevisiae: Analysis of structure, regulation and function

Summary We have isolated a new small heat shock gene, HSP12, from Saccharomyces cerevisiae. It encodes a polypeptide of predicted Mr 12 kDa, with structural similarity to other small heat shock proteins. HSP12 gene expression is induced several hundred-fold by heat shock and on entry into stationary phase. HSP12 mRNA is undetectable during exponential growth in rich medium, but low levels are present when cells are grown in minimal medium. Analysis of HSP12 expression in mutants affected in cAMP-dependent protein phosphorylation suggests that the gene is regulated by cAMP as well as heat shock. A disruption of the HSP12 coding region results in the loss of an abundant 14.4 kDa protein present in heat shocked and stationary phase cells. It also leads to the induction of the heat shock response under conditions normally associated with low-level HSP12 expression. The HSP12 disruption has no observable effect on growth at various temperatures, nor on the ability to acquire thermotolerance.     

An Escherichia coli mutant refractory to nitrosoguanidine mutagenesis

Summary A newly-isolated Escherichia coli mutant suffers only about 10% as many mutations as normal strains on exposure to nitrosoguanidine¹. The responsible mutation, inm-1, maps at approximately minute 79 in the current E. coli genetic map. The mutant is normal for overall growth, nitrosoguanidine lethality, spontaneous mutagenesis, ultraviolet light lethality and mutagenesis, ethyl methanesulfonate lethality and mutagenesis, and the adaptive repair induced by alkylating agents. The existence of this mutation proves that nitrosoguanidine mutagenesis is not merely the result of reactions between the chemical and DNA, but requires specific cellular function(s), and underscores the peculiarity of nitrosoguanidine as a mutagen.     

Heat-shock response in a yeast tps1 mutant deficient in trehalose synthesis

Exponential cells of the Saccharomyces cerevisiae tps1 mutant underwent a rapid loss of viability upon a non-lethal heat exposure (from 28 to 42°C). However, a further more severe heat stress (52.5°C 5 min) induced an increase in the fraction of viable cells. This mutant can not synthesize trehalose either at 28° C or at 42°C due to the lack of a functional trehalose-6P synthase complex. In control experiments carried out with the wild-type W303-1 B, heat-stressed exponential phase cultures grown on YPgal at 28°C acquired thermotolerance to a higher extent than identical cultures grown on YPD, although in both cultures the level of stored trehalose was negligible. These data suggest that the bulk of trehalose accumulated in yeast upon mild heat treaments is not sufficient to account for the acquisition of thermotolerance.     

Roles of Hsp104 and trehalose in solubilisation of mutant huntingtin in heat shocked Saccharomyces cerevisiae cells

Inhibition of huntingtin aggregation, either in the nucleus and/or in the cytosol, has been identified as a major strategy to ameliorate the symptoms of Huntington's disease. Chaperones and other protein stabilisers would thus be key players in ensuring the correct folding of the amyloidogenic protein and its expression in the soluble form. By transient activation of the global heat stress response in Saccharomyces cerevisiaeBY4742, we show that heterologous expression of mutant huntingtin (103Q-htt) could be modulated so that the protein was partitioned off in the soluble fraction of the cytosol. This led to lower levels of reactive oxygen species and improved cell viability. Previous reports had speculated on the relationship between trehalose and the heat shock response in ensuring enhanced cell survival but no direct evidence of such an interaction was available. Using mutants of an isogenic strain which do not express the major trehalose synthetic or metabolising enzymes or the chaperone, heat shock protein 104 (Hsp104), we were able to identify the functions of Hsp104 and the osmoprotectant trehalose in solubilising mutant huntingtin. We propose that the beneficial effect of the protein refolding machinery in solubilising the aggregation-prone protein is exerted by maintaining a tight balance between the trehalose synthetic enzyme, trehalose-6-phosphate synthase 1 and Hsp104. This ensures that the level of the osmoprotectant, trehalose, does not exceed the limit beyond which it is reported to inhibit protein refolding.     

A heat shock protein 90 inhibitor that modulates the immunophilins and regulates hormone receptors without inducing the heat shock response

When a cell encounters external stressors, such as lack of nutrients, elevated temperatures, changes in pH or other stressful environments, a key set of evolutionarily conserved proteins, the heat shock proteins (hsps), become overexpressed. Hsps are classified into six major families with the hsp90 family being the best understood; an increase in cell stress leads to increased levels of hsp90, which leads to cellular protection. A hallmark of hsp90 inhibitors is that they induce a cell rescue mechanism, the heat shock response. We define the unique molecular profile of a compound (SM145) that regulates hormone receptor protein levels through hsp90 inhibition without inducing the heat shock response. Modulation of the binding event between heat shock protein 90 and the immunophilins/homologs using SM145, leads to a decrease in hormone receptor protein levels. Unlike N-terminal hsp90 inhibitors, this hsp90 inhibitor does not induce a heat shock response. This work is proof of principle that controlling hormone receptor expression can occur by inhibiting hsp90 without inducing pro-survival protein heat shock protein 70 (hsp70) or other proteins associated with the heat shock response. Innovatively, we show that blocking the heat shock response, in addition to hsp90, is key to regulating hsp90-associated pathways.     

Ribosomal protein modification in Escherichia coli

Summary A mutant of Escherichia coli K12 has been isolated which shows an alteration in the ribosomal protein S18. Genetic analyses have revealed that the mutation causing this alteration maps at 99.3 min of the E. coli genetic map, between dnaC and deo. This indicated that the mutation has occurred in a gene different from the structural gene for this protein which has been located at 94 min. From the N-terminal amino acid sequence analysis it is concluded that the mutation has resulted in loss of the N-terminal acetyl group of this protein. The gene which is affected in this mutant is termed rimI that most likely specifies an enzyme acetylating the N-terminal alanine of protein S18. The mutation does not affect the acetylation of two other ribosomal proteins, S5 and L12, both of which are known to be acetylated in wild-type E. coli K12.     

Developmental study of thermotolerance and the heat shock response inLucilia cuprina (Weidemann)

The ability to withstand thermal stress in a laboratory population of the blowflyLucilia cuprina (measured as per cent adult survival following varying periods of exposure to elevated temperature up to a maximum of 48°C) was in the order pupa > larva > adult. Pre-exposure to a mild heat shock (37°C) induced tolerance to temperatures which were otherwise lethal. An analysis of heat shock-induced protein synthesis during development at similar elevated temperatures presented patterns corresponding to the above observations on thermotolerance. The induced level of synthesis of major heat shock proteins (viz., 79, 69, 28, 20 and 19 kDa) were greater in larval tissues than in most of the adult tissues except gonads. The response varied between young (2 days) and old (30 days) adults in a tissue-specific manner. In general, heat shock protein 69 kDa was most abundant in all the tissues studied. Control as well as heat shocked Malpighian tubules of adults uniquely showed two major [³⁵S]methionine labelled bands corresponding to approximately 62 and 64 kDa. Immunoblots showed the 62 kDa protein to cross react with an antibody againstHelioihis HSP60. Although the synthesis of the 62 kDa polypeptide was prominent only in Malpighian tubules of adult blowflies, nearly equal levels of this HSP60 family polypeptide were present in all tissues (control as well heat shocked) except the larval salivary glands.     

Ribosomal protein modification in Escherichia coli

Summary Among mutants of E. coli selected for temperaturesensitive growth, four were found to possess alterations in ribosomal proteins L7/L12. Of these, three apparently lack protein L7, the acetylated form of protein L12. Genetic analyses have revealed that the mutation responsible for this alteration maps at a locus around 34 min of the current E. coli genetic map, which is clearly different from the location for the structural gene for protein L7/L12 which is situated at 89 min. Hence, the gene affected in these mutants was termed rimL. Tryptic and thermolysin fingerprints of the protein L12 purified from the rimL mutants showed a profile indistinguishable from that of wild-type protein. It was found that the acetylase activity specific for protein L12 was negligible, when assayed in vitro, in the high-speed supernatant prepared from mutant cells. These results indicated that the three mutants contain mutations in the gene rimL that codes for an acetylating enzyme specific for ribosomal protein L12.Previous paper in this series is Isono and Isono (1980)     

Phenotypic instability in a tif-1 mutant of Escherichia coli

Summary A recent study showed that in E. coli T44 () carrying the tif-1 mutation, elevated temperature and adenine can interfere with the translation process. The present study shows that the expression of tif phenotypes (thermoinduction and filamentation) is suppressed by factors which affect ribosomal function. Ethanol suppresses thermoinduction and, in some spc ^r mutants, both thermoinduction and filamentation are suppressed. An unknown factor(s) in yeast extract suppresses both thermoinduction and filamentation. In thermoresistant revertant (ts⁺), the expression of the ts⁺ phenotype is suppressed by yeast extract, ethanol, guanosine+cytidine and by the addition of a spc ^r mutation. This indicates that this phenotype could be due to suppressor mutations, and the interaction between factors affecting ribosomal function and the ts⁺ phenotype suggests that the suppression of tif in the ts⁺ strains could operate on the ribosomal level. In vitro studies show that in extracts from either spc ^r or ts⁺ strains, or in the presence of ethanol, translational restriction is relieved, suggesting that the suppression of tif phenotypes could involve the translation process.     

Stress-induced thermotolerance of ventilatory motor pattern generation in the locust, Locusta migratoria

Ventilation is a crucial motor activity that provides organisms with an adequate circulation of respiratory gases. For animals that exist in harsh environments, an important goal is to protect ventilation under extreme conditions. Heat shock, anoxia, and cold shock are environmental stresses that have previously been shown to trigger protective responses. We used the locust to examine stress-induced thermotolerance by monitoring the ability of the central nervous system to generate ventilatory motor patterns during a subsequent heat exposure. Preparations from pre-stressed animals had an increased incidence of motor pattern recovery following heat-induced failure, however, prior stress did not alter the characteristics of the ventilatory motor pattern. During constant heat exposure at sub-lethal temperatures, we observed a protective effect of heat shock pre-treatment. Serotonin application had similar effects on motor patterns when compared to prior heat shock. These studies are consistent with previous studies that indicate prior exposure to extreme temperatures and hypoxia can protect neural operation against high temperature stress. They further suggest that the protective mechanism is a time-dependent process best revealed during prolonged exposure to extreme temperatures and is mediated by a neuromodulator such as serotonin.     

In vitro roles of Escbericbia coli DnaJ and DnaK heat shock proteins in the replication of oriC plasmids

Summary Heat shock proteins have been shown to be involved in many cellular processes in procaryotic and eucaryotic cells. Using an in vitro DNA replication assay, we show that DNA synthesis initiated at the chromosomal origin of replication of Escherichia coli (oriC) is considerably reduced in enzyme extracts isolated from cells bearing mutations in the dnaK and dnaJ genes, which code for heat shock proteins. Furthermore, unlike DNA synthesis in wild-type extracts, residual DNA synthesis in dnaK and dnaJ extracts is thermosensitive. Although thermosensitivity can be complemented by the addition of DnaK and DnaJ proteins, restoration of near wild-type replication levels requires supplementary quantities of purified DnaA protein. This key DNA synthesis initiator protein is shown to be adsorbed to DnaK affinity columns. These results suggest that at least one of the heat shock proteins, DnaK, exerts an effect on the initiation of DNA synthesis at the level of DnaA protein activity. However, our observation of normal oriC plasmid transformation ratios and concentrations in heat shock mutants at permissive temperatures would suggest that heat shock proteins play a role in DNA replication mainly at high temperatures or under other stressful growth conditions.     

A new radiation sensitive mutant of Escherichia coli K-12

Summary Streptomycin treatment of competent cultures of Bacillus subtilis kills the non competent cells. This method was used to look for mutagenic effects of Streptomyces coelicolor DNA. This DNA was found to induce a class of met⁺ clones harbouring aromatic adventitious mutations.