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.     

Introduction of the carrot HSP17.7 into potato (Solanum tuberosum L.) enhances cellular membrane stability and tuberization in vitro

We have examined the ability of a carrot (Daucus carota L.) heat shock protein gene encoding HSP17.7 (DcHSP17.7) to confer enhanced heat tolerance to potato (Solanum tuberosum L.), a cool-season crop. The DcHSP17.7 gene was fused to a 6XHistidine (His) tag to distinguish the engineered protein from endogenous potato proteins and was introduced into the potato cultivar 'Désirée' under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Western analysis showed that engineered DcHSP17.7 was constitutively, but not abundantly, expressed in transgenic potato lines before heat stress. Leaves from multiple regenerated potato lines that contain the transgene exhibited significantly improved cellular membrane stability at high temperatures, compared with wild-type and vector control plants. Transgenic potato lines also exhibited enhanced tuberization in vitro: under a condition of constant heat stress, at 29 degrees C, nodal sections of the transgenic lines produced larger and heavier microtubers at higher rates, compared to the wild type and vector controls. The dry weight and percentages of microtubers that were longer than 5 mm were up to three times higher in the transgenic lines. Our results suggest that constitutive expression of carrot HSP17.7 can enhance thermotolerance in transgenic potato plants. To our knowledge, this is the first study that shows that the thermotolerance of potato can be enhanced through gene transfer.     

Hsp27 protects mitochondria of thermotolerant cells against apoptotic stimuli

Enhanced cell survival and resistance to apoptosis during thermotolerance correlates with an increased expression of heat shock proteins (Hsps). Here we present additional evidence in support of the hypothesis that the induction of Hsp27 and Hsp72 during acquired thermotolerance in Jurkat T-lymphocytes prevents apoptosis. In thermotolerant cells, Hsp27 was shown to associate with the mitochondrial fraction, and inhibition of Hsp27 induction during thermotolerance in cells transfected with hsp27 antisense potentiated mitochondrial cytochrome c release after exposure to various apoptotic stimuli, despite the presence of elevated levels of Hsp72. Caspase activation and apoptosis were inhibited under these conditions. In vitro studies revealed that recombinant Hsp72 more efficiently blocked cytochrome c-mediated caspase activation than did recombinant Hsp27. A model is presented for the inhibition of apoptosis during thermotolerance in which Hsp27 preferentially blocks mitochondrial cytochrome c release, whereas Hsp72 interferes with apoptosomal caspase activation.     

Involvement of rRNA synthesis in the enhanced survival and recovery of protein synthesis seen in thermotolerance

Although acquired thermotolerance has been linked to the induction of heat shock proteins, the molecular mechanism(s) by which cells become resistant to heat is unknown. The present study shows a strong correlation between the survival of cells following heat shock and the rate of recovery of protein, total RNA, and rRNA synthesis. Increasing exposure of CHO cells to 45 degrees C was found to decrease survival and cause a lengthening delay in these synthetic processes. The same reciprocal correlation was seen in thermotolerant cells. As thermotolerance develops, more cells survive a heat challenge and the delay in synthesis decreases. These data argue that enhanced recovery of protein and RNA synthesis is one factor which plays a key role in thermotolerance. The involvement of rRNA synthesis was further investigated by using actinomycin D at 0.1 microgram m1(-1), a concentration at which rRNA synthesis is selectively inhibited. When the drug was present during the recovery from a challenge heat treatment, the survival of thermotolerant cells was approximately 3-fold lower than expected from the mild toxicity of the drug. As this could not be accounted for by an interaction of the drug with the response of cells to single heat treatments, it is concluded that the drug inhibits the expression of thermotolerance in cells which would otherwise express a full degree of thermotolerance. The time and concentration dependence of this effect indicates that the drug acts though inhibition of rRNA synthesis. Therefore, enhanced recovery of RNA synthesis, presumably rRNA synthesis, is identified as one of the mechanisms responsible for enhanced survival of thermotolerant cells following heat shock.     

Changes in hsp70 alter thermotolerance and heat-shock regulation in Drosophila.

To test the role of the heat shock protein hsp70 in induced thermotolerance and in the regulation of the heat-shock response, we established cell lines with altered expression of the Hsp70 gene. Underexpressing cells were created by transformation with antisense Hsp70 genes, and overexpressing cells by transformation with extra copies of the wild-type gene. Expression at normal temperatures was achieved by placing Hsp70 coding sequences under the control of the metallothionein promoter. Cells that expressed mutant hsp70s were created by transforming cells with deletion and frameshift mutations. The results indicate that hsp70 plays a major role in both thermotolerance and regulation. Surprisingly, they also indicate that these functions can be separated. Overexpression affected thermotolerance more than regulation; underexpression affected regulation more than thermotolerance. A carboxyl-terminal deletion of Hsp70 had a severe dominant-negative effect on thermotolerance but only a minor effect on regulation; an amino-terminal deletion strongly affected regulation but not thermotolerance. A model that explains these observations is presented.     

Differential expression of small heat shock protein genes Hsp23 and Hsp40, and heat shock gene Hsr-omega in fruit flies (Drosophila melanogaster) along a microclimatic gradient

We examined the role of small Hsp genes (Hsp23 and Hsp40) and heat shock gene Hsr-omega in the thermoadaptation of Drosophila melanogaster inhabiting a highly heterogeneous microsite (Nahal Oren canyon, Carmel massif, Israel). We tested whether interslope differences in Drosophila thermoadaptation, revealed in our previous studies, are associated with the differential expression of these genes. Our results demonstrate an increased expression of the Hsp40 gene in thermotolerant lines subjected to mild heat shock treatment (P < 10(-6), analysis of variance test). A high positive correlation was found between the levels of Hsp40 expression and scores of basal (R = 0.74; P < 0.001, based on the Spearman rank correlation test) and induced thermotolerance (R = 0.78; P < 0.0001), implying a significant contribution of Hsp40 gene in thermoadaptation.     

In vivo chaperone activity of heat shock protein 70 and thermotolerance

Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.     

Thermolability of mouse oocytes is due to the lack of expression and/or inducibility of Hsp70

Eukaryotic and prokaryotic cells have been shown to respond to physical and chemical stress by the induction of proteins called heat shock proteins. Heat shock protein 70 (Hsp70), is the most ubiquitous of these proteins. Although heat shock proteins are generally thought to protect cells from physiologically stressful stimuli, it cannot be assumed that this is so, because several cases exist in which thermotolerance is acquired without the production of heat shock proteins, and in several other cases the hyperproduction of these heat shock proteins does not produce thermotolerance. In this study we show that unfertilized mouse oocytes are sensitive to elevated temperatures, and that the synthesis of Hsp70 cannot be induced in these oocytes. Furthermore, our data demonstrate that the expression of Hsp70 in mouse oocytes is sufficient for the acquisition of thermotolerance. Mouse oocytes were injected with mRNA for Hsp70, and the viability of these oocytes was determined after heating. The number of viable oocytes was significantly higher in the group injected with Hsp70 mRNA and then heated compared with oocytes injected with Hsp70 antisense mRNA and sham-injected controls treated in an identical manner. No significant differences in the number of viable oocytes were found between the group that had been injected with Hsp70 mRNA, heated, and then allowed to recover for 3 hr and the group maintained at 37 degrees C throughout.(ABSTRACT TRUNCATED AT 250 WORDS)     

Loss of Hsp70 in Drosophila is pleiotropic, with effects on thermotolerance, recovery from heat shock and neurodegeneration

The heat-shock response is a programmed change in gene expression carried out by cells in response to environmental stress, such as heat. This response is universal and is characterized by the synthesis of a small group of conserved protein chaperones. In Drosophila melanogaster the Hsp70 chaperone dominates the profile of protein synthesis during the heat-shock response. We recently generated precise deletion alleles of the Hsp70 genes of D. melanogaster and have used those alleles to characterize the phenotypes of Hsp70-deficient flies. Flies with Hsp70 deletions have reduced thermotolerance. We find that Hsp70 is essential to survive a severe heat shock, but is not required to survive a milder heat shock, indicating that a significant degree of thermotolerance remains in the absence of Hsp70. However, flies without Hsp70 have a lengthened heat-shock response and an extended developmental delay after a non-lethal heat shock, indicating Hsp70 has an important role in recovery from stress, even at lower temperatures. Lack of Hsp70 also confers enhanced sensitivity to a temperature-sensitive lethal mutation and to the neurodegenerative effects produced by expression of a human polyglutamine disease protein.     

Inhibition of Hsp90 function delays and impairs recovery from heat shock

The induction of the heat shock response as well as its termination is autoregulated by heat shock protein activities. In this study we have investigated whether Hsp90 functional protein levels influence the characteristics and duration of the heat shock response. Treatment of cells with several benzoquinone ansamycin inhibitors of Hsp90 (geldanamycin, herbimycin A) activated a heat shock response in the absence of heat shock, as reported previously. Pretreatment of cells with the Hsp90 inhibitors significantly delayed the rate of restoration of normal protein synthesis following a brief heat shock. Concurrently, the rate of Hsp synthesis and accumulation was substantially increased and prolonged. The cessation of heat shock protein synthesis did not occur until the levels of Hsp70 were substantially elevated relative to its standard threshold for autoregulation. The elevated levels of HSPS 22-28 (the small HSPS) and Hsp70 are not able to promote thermotolerance when Hsp90 activity is repressed by ansamycins; rather a suppression of thermotolerance is observed. These results suggest that a multicomponent protein chaperone complex involving both Hsp90 and Hsp70 signals the cessation of heat shock protein synthesis, the restoration of normal translation, and likely the establishment of thermotolerance. Impaired function of either component is sufficient to alter the heat shock response.     

Cyclic AMP and the heat shock response in Chinese hamster ovary cells

Heat shock leads to transient increases in cAMP levels in HA-1-CHO cells. Such pulses are correlated temporally with the induction of heat resistance (thermotolerance) and with heat shock protein synthesis. Although the kinetics of cAMP increase after heating suggest a role in thermotolerance induction, raising cAMP levels directly using dBcAMP did not produce full thermotolerance. The resistance induced by dBcAMP may thus be either a component of or different to heat-shock triggered resistance. Cells which had been made thermotolerant by heat shock did not produce a pulse in cAMP level on heating. The cAMP producing system thus seemed desensitized to heat in thermotolerant cells.     

Effects of dexamethasone,heat shock,and serum responses on the inhibition of hsc70 synthesis by antisense RNA in NIH 3T3 cells

A dexamethasone (Dex)-inducible antisense RNA expression vector was constructed that contains the 5′-untranslated region and one third of the coding sequence for the bovine hsc70 protein. This vector was used to transfect NIH 3T3 cells from which clonal cell lines expressing hsc70 antisense RNA were developed. Quantitative Northern blot analysis with strand-specific probes was used to demonstrate the Dex-inducible accumulation of hsc70 antisense RNA in proliferating cell cultures and the inhibition of hsc70 RNA levels. Surprisingly, antisense RNA was either much less effective in reducing the amounts of hsc70 RNA in Dex-treated cultures than in untreated controls or cells compensated by producing more hsc70 RNA in response to increasing amounts of antisense RNA. Hsc70 protein synthesis did not decrease in either Dex-treated or untreated cultures: it actually increased, again suggesting the activation of a compensatory response. In Dex-treated cultures subjected to heat shock, hsc70 antisense RNA blocked the induction of hsp70, indicating that newly synthesized RNA was targeted effectively before it became translationally active. To test this hypothesis further, Dex-treated cultures were made quiescent by serum deprivation and then restimulated with serum, which causes a burst of RNA and protein synthesis. Consistent with this hypothesis, increased synthesis of hsc70 was blocked in serum-stimulated cultures expressing antisense RNA. © 1995 Wiley-Liss, Inc.     

Heat shock protein 27 stimulates recovery of RNA and protein synthesis following a heat shock

Constitutive expression of human hsp27 resulted in a 100-fold increase in survival to a single lethal heat shock in CHO cells without effecting the development of thermotolerance. A possible mechanism for the thermoprotective function of hsp27 may be increased recovery of protein synthesis and RNA synthesis following a heat shock. A lethal heat shock (44°C, 30 min) results in a 90% reduction in the rate of protein synthesis in non-tolerant cells. Control transfected cells recovered protein synthesis to a pre-heat shock rate 10 h after the heat shock; while cell lines that constitutively express human hsp27 recovered 6 h after the heat shock. Thermotolerant cells had a 50% reduction in protein synthesis, which recovered within 7 h following the heat shock. The same lethal heat shock (44°C, 30 min) reduced RNA synthesis by 60% in the transfected cell lines, with the controls recovering in 7 h; while the hsp27 expressing cell lines recovered within 5 h. Thermotolerant cells had a 40% reduction in RNA synthesis and were able to recover within 4 h. The enhanced ability of hsp27 to facilitate recovery of protein synthesis and RNA synthesis following a heat shock may provide the cell with a survival advantage. J. Cell. Biochem. 66:153–164, 1997. © 1997 Wiley-Liss Inc.     

Characterization of a small heat shock protein, Mx Hsp16.6, of Myxococcus xanthus

A number of heat shock proteins in Myxococcus xanthus were previously identified by two-dimensional (2D) gel electrophoresis. One of these protein was termed Mx Hsp16.6, and the gene encoding Mx Hsp16.6 was isolated. Mx Hsp16.6 consists of 147 amino acid residues and has an estimated molecular weight of 16,642, in accordance with the apparent molecular mass in the 2D gel. An alpha-crystallin domain, typically conserved in small heat shock proteins, was found in Mx Hsp16.6. Mx Hsp16.6 was not detected during normal vegetative growth but was immediately induced after heat shock. Expression of the hsp16.6 gene was not induced by other stresses, such as starvation, oxidation, and high osmolarity. Mx Hsp16.6 was mostly localized in particles formed after heat shock and precipitated by low-speed centrifugation. Furthermore, Mx Hsp16.6 was detected in highly electron-dense particles in heat-shocked cells by immunoelectron microscopy, suggesting that it forms large complexes with heat-denatured proteins. An insertion mutation in the hsp16.6 gene resulted in lower viability during heat shock and lower acquired thermotolerance. Therefore, it is likely that Mx Hsp16.6 plays critical roles in the heat shock response in M. xanthus.