Expression of the Heat Shock Response in a Tomato Interspecific Hybrid Is Not Intermediate between the Two Parental Responses

While it is apparent that the heat shock response is ubiquitous, variabilities in the nature of the heat shock response between closely related species have not been well characterized. The heat shock response of three genotypes of tomato, Lycopersicon esculentum, Lycopersicon pennellii, and the interspecific sexual hybrid was characterized. The two parental genotypes differed in the nature of the heat shock proteins synthesized; the speciesspecific heat shock proteins were identified following in vivo labeling of leaf tissue with [³⁵S]methionine and cysteine. The duration of, and recovery from, heat shock varied between the two species: L. esculentum tissue recovered more rapidly and protein synthesis persisted longer during a heat shock than in the wild species, L. pennellii. Both species induced heat shock protein synthesis at 35°C and synthesis was maximal at 37°C. The response of the F1 to heat shock was intermediate to the parental responses for duration of, and recovery from, heat shock. In other aspects, the response of the F1 to heat shock was not intermediate to the parental responses: the F1 induced only half of the L. esculentum specific heat shock proteins, and all of the L. pennellii specific heat shock proteins. A discussion of the inheritance of the regulation of the heat shock response is presented.     

Heat shock mRNA accumulation during recovery from cold shock in Drosophila melanogaster

Heat shock protein synthesis is induced in response to a variety of chemical and physical stresses. Among these are heating above normal growing temperatures, treatment with heavy metals, amino acid analogues, steroid hormones and a variety of other chemicals (CRC Crit. Rev. Biochem. 18, 239–280). We have shown previously that heat shock proteins are also synthesized during recovery from prolonged 0°C treatment in Drosophila larval salivary glands. In this paper we describe the cold treatments which induce heat shock protein synthesis in more detail, and show that heat shock mRNA does not accumulate during the cold treatment, but rather during the recovery period when the larvae are returned to 25°C. The implications of these results for the regulation of heat shock mRNA levels, and for the role of heat shock proteins in recovery from cold shock are discussed.     

Induced Levels of Heat Shock Proteins in a dnaK Mutant of Lactococcus lactis

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and proteases, including the DnaK-GrpE-DnaJ and the GroELS chaperone complexes. In order to investigate the importance of the DnaK chaperone complex for growth and heat shock response regulation in Lactococcus lactis, we have constructed two dnaK mutants with C-terminal deletions in dnaK. The minor deletion of 65 amino acids in the dnaKΔ2 mutant resulted in a slight temperature-sensitive phenotype. BK6, containing the larger deletion of 174 amino acids (dnaKΔ1), removing the major part of the inferred substrate binding site of the DnaK protein, exhibited a pronounced temperature-sensitive phenotype and showed altered regulation of the heat shock response. The expression of the heat shock proteins was increased at the normal growth temperature, measured as both protein synthesis rates and mRNA levels, indicating that DnaK could be involved in the regulation of the heat shock response in L. lactis. For Bacillus subtilis, it has been found (A. Mogk, G. Homuth, C. Scholz, L. Kim, F. X. Schmid, and W. Schumann, EMBO J. 16:4579–4590, 1997) that the activity of the heat shock repressor HrcA is dependent on the chaperone function of the GroELS complex and that a dnaK insertion mutant has no effect on the expression of the heat shock proteins. The present data from L. lactis suggest that the DnaK protein could be involved in the maturation of the homologous HrcA protein in this bacterium.     

Heat shock response of Dictyostelium

In response to a shift from 22 to 30°C the relative rate of synthesis of a small number of proteins is dramatically increased in Dictyostelium discoideum. The cells neither grow nor develop at this temperature but die slowly with a half-life of 18 hr. The major protein synthesized in response to a heat shock to 30°C in either growing cells or developing cells has an apparent molecular weight of 70,000 (70K). An increase in the relative rate of synthesis of 70K can be seen as early as 20 min following heat shock. Synthesis of 70K remains high for 4 hr at 30°C and then decreases. Similar kinetics of 70K synthesis occur during recovery at 22°C following a 1-hr heat shock. RNA synthesis during the first half-hour of heat shock is essential for the high rate of 70K measured 2 hr later. By isoelectric focusing the 70K protein can be separated into two spots, one of which overlaps one of the major heat shock proteins of Drosophila melanogaster. The relative rate of synthesis of several other proteins (82K, 60K, 43K) increases less dramatically in Dictyostelium during heat shock at 30°C. A heat shock to 34°C results in rapid synthesis of these proteins but not of 70K. The relative rates of synthesis of most other proteins made at 22°C decreases, most notably that of actin. Synthesis of heat shock proteins at 30°C does not significantly affect viability at 30°C but dramatically prolongs the period of time the cells can survive at 34°C. Thus, 30°C appears to be a stasis condition for Dictyostelium which elicits a response essential for protection from lethal temperatures. The similarity of the heat shock response in Dictyostelium to that in Drosophila and vertebrate cells suggests that certain aspects of the response may be universal in eukaryotes.     

Modulation of heat shock protein 90 affects TGF-β-induced collagen synthesis in human dermal fibroblast cells

Heat shock protein 90 is a chaperone molecule that aids in proper folding of target proteins. Recently, heat shock protein 90 was found to play a role in would healing through regulation of fibroblast functions. The aim of the present study was to investigate the role of heat shock protein 90 in collagen synthesis in human dermal fibroblasts. The effects of transforming growth factor-β, 17-N-allylamino-17-demethoxygeldanamycin, and transfection of heat shock protein 90 were evaluated by real-time PCR, western blot, and immunofluorescence assays. The Smad 2/3 and Akt pathways were evaluated to identify the signaling pathways involved in collagen synthesis. Heat shock protein 90 and collagen levels were compared in keloid and control tissues by immunohistochemical analysis. The expression of collagen was significantly increased after treatment with transforming growth factor-β, while 17-N-allylamino-17-demethoxygeldanamycin inhibited transforming growth factor-β-induced collagen synthesis. Overexpression of heat shock protein 90 itself with or without transforming growth factor-β increased collagen synthesis. These effects were dependent on Smad 2/3 pathway signaling. Finally, expression of heat shock protein 90 was increased in keloid tissue compared with control tissues. Taken together, these results demonstrate that modulation of heat shock protein 90 influences transforming growth factor-β-induced collagen synthesis via regulation of Smad 2/3 phosphorylation.     

Increase in synthesis and stability of σ 32 on treatment with inhibitors of DNA gyrase in Escherichia coli

We report here that in Escherichia coli, the anti-bacterial agent nalidixic acid induces transient stabilization and increased synthesis of σ³², accompanied by the induction of heat shock proteins (Dnak and GroEL proteins). The induction of heat shock proteins, increased synthesis of σ³², and stabilization of σ³² observed on treatment of wild-type cells with nalidixic acid were not observed in a nalA26 mutant, a strain that is resistant to nalidixic acid as the result of a mutation in the gyrA gene. Not only oxolinic acid, but also novobiocin, whose targets are the A and B subunits of DNA gyrase, respectively, also induced stabilization and increased synthesis of σ³². Thus, inhibition of the activity of DNA gyrase may cause stabilization and increased synthesis of σ³², resulting in turn in induction of heat shock proteins.     

Protein Synthesis in Maize during Anaerobic and Heat Stress

Protein accumulation and protein synthesis were investigated during anaerobic stress and heat shock in maize seedlings (Zea mays L.). Antibodies against alcohol dehydrogenase (ADH) and cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) were used to investigate the expression of the genes encoding these proteins during stress treatment. ADH1 protein accumulation is shown to increase about 10-fold in the root after 24 hours of anaerobic treatment. The Gpc gene products are separable into two size classes: the slow mobility GAPC1 and GAPC2 (GAPC1/2), and the faster GAPC3 and GAPC4 (GAPC3/4). The GAPC1/2 antigen did not increase at all, whereas the GAPC3/4 antigen increased less than fourfold. The proteins synthesized in the root during aerobic and anaerobic conditions were compared, and GAPC3/4 was identified as an anaerobic polypeptide. In vitro translations were used to estimate the levels of different mRNAs in roots following anaerobiosis, recovery from anaerobiosis, and heat shock. This was compared with the in vivo protein synthesis rates in roots labeled under identical conditions. In vivo labeling indicates that GAPC and ADH are not heat shock proteins. Although both GAPC3/4- and ADH1-translatable mRNA levels increase about 10-fold during anaerobiosis, in vivo labeling of these proteins (relative to total protein synthesis) is further enhanced, leading to a selective translation effect for ADH1 of threefold, and for GAPC3/4 of sixfold. In contrast, anoxia causes no change in GAPC1/2-translatable mRNA levels or in vivo labeling. As an additional comparison, β-glucosidase mRNA levels are found to be constant during anoxia, but in vivo synthesis decreases.     

Synthesis of heat shock proteins in Cf124 cells: Effect of virus infection

The transient synthesis of a class of proteins known as heat shock or stress response proteins was induced when Cf124 cells were incubated at high temperature. When cells were infected with Chilo iridescent virus and simultaneously heat shocked, heat shock protein (hsp) synthesis was delayed, and the shut-off of hsp synthesis was suppressed. In previously heat shocked cells, inhibition of hsp synthesis was dependent upon the multiplicity of infection, however, when infection preceded heat shock, the synthesis of hsp started immediately after heat shock. In all cases, hsp synthesis was dependent upon newly synthesized messenger RNA.     

Translational thermotolerance provided by small heat shock proteins is limited to cap-dependent initiation and inhibited by 2-aminopurine

Heat shock results in inhibition of general protein synthesis. In thermotolerant cells, protein synthesis is still rapidly inhibited by heat stress, but protein synthesis recovers faster than in naive heat-shocked cells, a phenomenon known as translational thermotolerance. Here we investigate the effect of overexpressing a single heat shock protein on cap-dependent and cap-independent initiation of translation during recovery from a heat shock. When overexpressing alphaB-crystallin or Hsp27, cap-dependent initiation of translation was protected but no effect was seen on cap-independent initiation of translation. When Hsp70 was overexpressed however, both cap-dependent and -independent translation were protected. This finding indicates a difference in the mechanism of protection mediated by small or large heat shock proteins. Phosphorylation of alphaB-crystallin and Hsp27 is known to significantly decrease their chaperone activity; therefore, we tested phosphorylation mutants of these proteins in this system. AlphaB-crystallin needs to be in its non-phosphorylated state to give protection, whereas phosphorylated Hsp27 is more potent in protection than the unphosphorylatable form. This indicates that chaperone activity is not a prerequisite for protection of translation by small heat shock proteins after heat shock. Furthermore, we show that in the presence of 2-aminopurine, an inhibitor of kinases, among which is double-stranded RNA-activated kinase, the protective effect of overexpressing alphaB-crystallin is abolished. The synthesis of the endogenous Hsps induced by the heat shock to test for thermotolerance is also blocked by 2-aminopurine. Most likely the protective effect of alphaB-crystallin requires synthesis of the endogenous heat shock proteins. Translational thermotolerance would then be a co-operative effect of different heat shock proteins.     

Characterization of the Heat Shock Response in Lactococcus lactis subsp. lactis

The heat shock response in Lactococcus lactis subsp. lactis was characterized with respect to synthesis of a unique set of proteins induced by thermal stress. A shift in temperature from 30 to 42°C was sufficient to arrest the growth of L. lactis subsp. lactis, but growth resumed after a shift back to 30°C. Heat shock at 50°C reduced the viable cell population by 10³; however, pretreatment of the cells at 42°C made them more thermoresistant to exposure at 50°C. The enhanced synthesis of approximately 13 proteins was observed in cells labeled with ³⁵S upon heat shock at 42°C. Of these heat shock-induced proteins, two appeared to be homologs of GroEL and DnaK, based on their molecular weights and reactivity with antiserum against the corresponding Escherichia coli proteins. Therefore, we conclude that L. lactis subsp. lactis displays a heat shock response similar to that observed in other mesophilic bacteria.     

Genotype-specific heat shock proteins in two maize inbreds

Summary Leaf blade tissue of maize inbred lines B73 and Mo17 was analyzed for intraspecific genetic variability in the heat shock response. The maize inbreds were characterized for acquired thermal tolerance and patterns of heat shock protein synthesis. The leakage conductivity assay of membrane stability during stress indicated that Mol7 possesses greater potential than B73 to acquire thermal tolerance. Poly(A)⁺ RNA, extracted from leaf blades, was translated in vitro in the presence of ³⁵S-methionine and the translation products separated by twodimensional gel electrophoresis. Major genotypic differences were observed in the translation products. Mo 17 synthesized twelve unique heat shock proteins in the 15–18 kD range, but B73 synthesized only three unique heat shock proteins in the same range. DNA polymorphisms were observed between the maize lines using ³²P labeled heat shock protein gene probes.Abbreviations HKT Heat-killing time - HS Heat shock - HSP Heat shock protein - HMW High molecular weight - LMW Low molecular weight Contribution of the College of Agricultural Sciences, Texas Tech University, Journal No. T-4-333     

Effects of cadmium on gene expression in cadmium-tolerant and cadmium-sensitiveDatura innoxia cells

The effect of Cd on gene expression in suspension cultures of twoDatura innoxia cell lines with differing Cd tolerance was studied.In vivo labeling experiments using [³H] leucine showed that Cd induced the synthesis of a similar range of proteins in both cell lines at a concentration which will kill the sensitive but not the tolerant cells. Corresponding changes in levels of translatable mRNA were also observed. The induction of the synthesis of proteins by Cd was transient since Cd-tolerant cells growing continuously in 250 M CdCl₂ contained a similar set ofin vitro translation products to cells growing in the absence of Cd. Although Cd had a similar effect on gene expression in both cell lines, Cd-tolerant cells possess two abundant mRNAs which are constitutively produced. These mRNAs encode proteins of low molecular weight (about 11 kDa) and are either absent or present at a low level in Cd-sensitive cells. The functions of these proteins are not known but they may be involved in the tolerance mechanism. Two-dimensional gel electrophoresis ofin vitro translation products showed that many of the Cd-induced proteins are also induced by heat shock. A 42°C heat shock resulted in agreater range and more intense induction of translatable mRNAs than 4 h exposure to 250 M CdCl₂. However a subset of mRNAs were induced specifically by Cd while other mRNAs were heat shock-specific. There was no difference in the ability of the two cell lines to tolerate heat shock. This was also reflected by the same pattern of major proteins induced by heat shock in the two cell lines.