Tissue-specific variations in the induction of Hsp70 and Hsp64 by heat shock in insects

The patterns of heat-induced synthesis (37 degrees C to 45 degrees C) of heat shock proteins (Hsps) in different tissues of grasshoppers and cockroaches from natural populations and in laboratory-reared gram-pest (Heliothis armigera) were examined by 35S-methionine labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography. Whereas 45 degrees C was lethal in most cases, optimal induction of Hsp synthesis was seen between 37 degrees C and 42 degrees C. The ongoing protein synthesis was not much affected at these temperatures, except in the tissues of adult H. armigera exposed to 42 degrees C. The profiles of the Hsps induced in the tissues of the insects, however, were different. From the relative abundance of the synthesis of 70-kDa (Hsp70) and 64-kDa (Hsp64) polypeptides, three categories of heat shock response were identified: (1) induction of abundant Hsp70 but little Hsp64 (malpighian tubules, male accessory glands, and ovaries of adult grasshoppers), (2) abundant Hsp64 but little Hsp70 (testes of adult grasshoppers, testes and malpighian tubules of adult cockroaches, and testes, malpighian tubules, and fat bodies of H. armigera larvae), and (3) induction of both Hsp70 and Hsp64 in more or less equal abundance (ovaries of adult cockroaches, salivary glands of H. armigera larvae, and malpighian tubules, male accessory glands, testes, and ovaries of adult H. armigera). Cockroaches collected from storerooms showed detectable synthesis of Hsp64 and/or Hsp70 only after heat shock, but those collected from drains showed detectable synthesis of both Hsp70 and Hsp64 in different tissues without heat stress. Western blotting showed that the 64-kDa polypeptide in these insects is a member of the Hsp60 family. Grasshopper testes, which synthesized negligible Hsp70 but abundant Hsp64 after heat shock, developed thermotolerance. Thus, heat shock response is modulated by developmental and environmental factors in different tissues of insects.     

Isoproterenol potentiates exercise-induction of Hsp70 in cardiac and skeletal muscle

The response to exercise stress is characterized by an increase in circulating catecholamines and rapid synthesis of the inducible member of the 70 kDa family of heat shock proteins (Hsp70). Cell culture studies indicate that Hsp70 expression is influenced by beta-adrenergic receptor intermediates including cyclic AMP (cAMP) and cAMP dependent protein kinase (PKA). Thus, in the present investigation, the effect of a beta-adrenergic agonist, isoproterenol (ISO; 10 mg/kg) and a beta-adrenergic antagonist, nadolol (NAD; 25 mg/kg), on the in vivo expression of Hsp70 in rodent cardiac and skeletal muscle following moderate (MOD; 17 m/min) and exhaustive (EXH; 30 m/min) exercise was examined. While ISO alone did not induce Hsp70 synthesis, ISO treatment potentiated Hsp70 expression following MOD in the white vastus and heart (395+/-29 and 483+/-29% greater than control respectively, P < 0.05). Furthermore, this effect was reversed with combined beta-adrenergic agonist and antagonist treatment (ISO+NAD) indicating that the isoproterenol induced increase in post-exercise Hsp70 expression was mediated via beta-adrenergic receptor activity. However, there were no differences in Hsp70 levels among treatment groups following EXH. The failure of NAD to attenuate Hsp70 accumulation following EXH suggests that beta-adrenergic receptor activity is not the main signal in the induction of Hsp70 following exercise. Hsp70 induction was dependent on exercise intensity and ISO administration prior to MOD resulted in Hsp70 levels similar to those observed following EXH. The results from the present investigation indicate that beta-adrenergic receptor stimulation does not induce Hsp70 synthesis per se, but may be one factor involved in the complex regulation of the stress response to exercise in vivo.     

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.     

Changes in heat-shock protein synthesis and thermotolerance of Arabodopsis thaliana seedlings resulting from Hsp90 inhibition by geldanamycin

The influence of geldanamycin (GA), a specific inhibitor of heat-shock protein Hsp90, on the synthesis of Hsp70 and Hsp90 and thermotolerance of Arabidopsis thaliana seedlings has been studied. Incubation of seedlings with GA under normal conditions induced synthesis of these stress proteins. Treatment of seeds with the Hsp90 inhibitor resulted in elevated constitutive levels of Hsp70 and Hsp90 in seedlings, as well as increased induction of their synthesis under heat shock. The GA effect increased with its concentration. Hsp up-regulation promoted thermotolerance of seedlings. The findings suggest autoregulation of heatshock protein synthesis and regulation of plant tolerance by Hsp90.     

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.     

Bag1-Hsp70 mediates a physiological stress signalling pathway that regulates Raf-1/ERK and cell growth

Survival after stress requires the precise orchestration of cell-signalling events to ensure that biosynthetic processes are alerted and cell survival pathways are initiated. Here we show that Bag1, a co-chaperone for heat-shock protein 70 (Hsp70), coordinates signals for cell growth in response to cell stress, by downregulating the activity of Raf-1 kinase. Raf-1 and Hsp70 compete for binding to Bag1, such that Bag1 binds to and activates Raf-1, subsequently activating the downstream extracellular signal-related kinases (ERKs). When levels of Hsp70 are elevated after heat shock, or in cells conditionally overexpressing Hsp70, Bag1-Raf-1 is displaced by Bag1-Hsp70, and DNA synthesis is arrested. Mutants Bag1C204A and Bag1E208A, which cannot bind Hsp70, constitutively activate Raf-1/ERK kinases but are unaffected by Hsp70; consequently neither Bag1-Raf-1 nor DNA synthesis is negatively affected during heat shock. Likewise, mutants Hsp70F245S, Hsp70R262W and Hsp70L282R, which retain chaperone activity but do not bind to Bag1, fail to repress Bag1 activation of Raf-1/ERK kinase. We propose that Bag1 functions in the heat-shock response to coordinate cell growth signals and mitogenesis, and that Hsp70 functions as a sensor in stress signalling.     

Molecular identification and expression of heat shock cognate 70 (hsc70) and heat shock protein 70 (hsp70) genes in the Pacific oyster Crassostrea gigas

The 70-kDa heat shock protein (Hsp) family is composed of both environmentally inducible (Hsp) and constitutively expressed (Hsc) family members. We sequenced 2 genes encoding an Hsp70 and an Hsc70 in the Pacific oyster Crassostrea gigas. The Cghsc70 gene contained introns, whereas the Cghsp70 gene did not. Moreover, the corresponding amino acid sequences of the 2 genes presented all the characteristic motifs of the Hsp70 family. We also investigated the expression of Hsp70 in tissues of oysters experimentally exposed to metal. A recombinant Hsc72 was used as an antigen to produce a polyclonal antibody to quantify soluble Hsp70 by enzyme-linked immunosorbent assay in protein samples extracted from oysters. Our results showed that metals (copper and cadmium) induced a decrease in cytosolic Hsp70 level in gills and digestive gland of oysters experimentally exposed to metal. These data suggest that metals may inhibit stress protein synthesis.     

The level and phosphorylation of Hsp70 in the rat liver cytosol after adrenalectomy and hyperthermia

Hepatic heat shock protein Hsp70 synthesis and in vitro phosphorylation were studied in the liver cytosol of intact, adrenalectomized and dexamethasone-administered adrenalectomized rats after 41 degrees C whole body hyperthermic stress. Hsp70 was detected by immunoblotting with N27F3-4 monoclonal antibody recognizing both constitutive and inducible forms of the protein. A comparison between basal and heat stress-induced levels of the protein in the liver cytosol of the three groups of animals suggested that glucocorticoid hormones stimulate the basal synthesis of Hsp70 and inhibit its induction by stress. In both unstressed and hyperthermia-exposed animals, hepatic Hsp70 was detected as a phosphoprotein. The extent of its in vitro phosphorylation was found to be significantly reduced by heat stress or adrenalectomy, but dexamethasone failed to restore it to the original level.     

Characterization of two heat shock proteins (Hsp70/Hsc70) from grass carp (Ctenopharyngodon idella): evidence for their differential gene expression, protein synthesis and secretion in LPS-challenged peripheral blood lymphocytes

Two cDNAs, encoding the stress-inducible 70-kDa heat shock protein (Hsp70) and the constitutively expressed 70-kDa heat shock cognate protein (Hsc70), were isolated from grass carp. The Hsp70 and Hsc70 cDNAs were 2250 bp and 2449 bp in length and contained 1932 bp and 1953 bp open reading frames, respectively. Tissue distribution results showed that Hsp70/Hsc70 was highly expressed in gill, kidney, head kidney and peripheral blood lymphocytes (PBLs). Using grass carp PBLs as a cell model, effects of lipopolysaccharide (LPS) on the mRNA and protein levels of Hsp70/Hsc70 were examined. In this case, LPS increased the mRNA expression of Hsp70 in a time- and dose-dependent manner, but had no effect on Hsc70 mRNA expression. In agreement with this, LPS elevated the intracellular Hsp70 markedly, but not the Hsc70 protein levels in parallel experiments. Furthermore, Hsp70 protein was also detected in culture medium. Moreover, inhibition of LPS on Hsp70 release in a time-dependent manner was observed, indicating that there may be a dynamic balance between Hsp70 stores and Hsp70 release in grass carp PBLs following exposure to LPS. Taken together, these results not only shed new insights into the different regulations of LPS on Hsp70/Hsc70 gene expression, protein synthesis and release, but also provide a basis for further study on the functional role of Hsp70 in fish immune response.     

Neuronal differentiation in PC12 cells is accompanied by diminished inducibility of Hsp 70 and Hsp60 in response to heat and ethanol

The stress response of PC12 cells was characterized by evaluating the production of heat shock proteins of the 70 kDa (Hsp70), 60 kDa (Hsp60) and 90 kDa (Hsp90) families by western blot analysis. Induction of Hsp synthesis was elicited by brief exposure to elevated temperatures or by addition of ethanol to the cultures. Normal PC12 cells responded to stress with rapid up-regulation of Hsp70 and Hsp60 production. However, fully differentiated PC12 cells (induced by nerve growth factor, NGF) failed to produce Hsp70 or Hsp60 in response to heat or ethanol treatment. The disappearance of the heat shock response of the cells was directly related to the extent of neuronal differentiation. The cellular levels of the constitutive proteins, Hsc70 and Hsp90, were not altered by differentiation of the cells. Production of Hsps was restored in the differentiated cells by removal of NGF which coincided with the loss of neurite expression and retraction of processes.     

Genetic heterogeneity of heat shock protein synthesis as a factor determining the resistance to stressors in mammalia

The relationship between the levels of 70 kDa family heat shock protein (Hsp) synthesis and lymphocyte sensitivity to stressors was investigated. Lymphocyte cultivation in mitogen deprived culture medium and/or the cell treatment with alkylating agents have been used as a stress challenge. Model experiments with two inbred murine strains genetically contrasting by the sensitivity to alkylating agents demonstrated that the basic level of Hsp synthesis depends on genotype. The quantity Hsp70 mRNA, as well as intracellular level of the proteins, in BALB/c was significantly higher than those in C57BL/6 mice. The mice, which were characterized by higher Hsp levels, demonstrated higher resistance to alkylating agent action. The induction of surplus amount of Hsp by heat shock increased the cell resistance to an alkylating agent melphalan. Lymphocyte isolated from high Hsp producers BALB/c mice were more resistant to apoptotic signals induced by mitogen deprivation.     

Histidine 89 is an essential residue for Hsp70 in the phosphate transfer reaction

Autophosphorylation of Hsp70 is detected in the process of substrate refolding in the presence of adenosine triphosphate (ATP) in the reaction mixture. But to date, the role and mechanism of Hsp70 autophosphorylation have not been elucidated. In this study we determined the site of histidine phosphorylation of Hsp70 as an intermediate in the process of phosphate transfer reaction by site-directed mutagenesis. We selected two possible sites (ie, His89 and His227) of intermediate histidine phosphorylation based on our hypothesis of the transfer of gamma-phosphoryl groups and replacement by glycine and serine. Although an acid labile autophosphorylation intermediate of Hsp70 and its cytidine diphosphate-dependent dephosphorylation were detected in wild-type Hsp70, they were markedly suppressed in the H89S mutation of Hsp70, but not on the H227S mutation. The ATPase activity and ATP synthesis activity of Hsp70 were almost completely suppressed in the H89S and H89G mutations. The role of His89 in the phosphate transfer reaction of Hsp70 is discussed.     

Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1.

The Hsp70 family of molecular chaperones has an essential role in the synthesis, folding and translocation of the nascent peptide chain. While the general features of these activities are well documented, less is understood about the regulation of these activities. The ATPase rate is stimulated by non-native proteins, furthermore, interaction with ATP leads to the release of protein substrate concurrent with a conformational change in Hsp70. One interpretation of these data is that the two domains of Hsp70 interact. In the process of mapping the carboxyl-terminal boundary of the substrate binding domain for human Hsp70, we identified a regulatory motif, EEVD, which is conserved at the extreme carboxyl terminus among nearly all cloned cytosolic eukaryotic Hsp70s. Deletion or mutation of EEVD affects the ATPase activity, the ability to interact with substrates, and interferes with the ability of the mutant Hsp70 to interact with HDJ-1 in the refolding of denatured firefly luciferase. Examination of the biophysical properties of the mutant Hsp70s reveals a change in the overall shape and conformation of the protein consistent with reduced interactions between the two domains. These data suggest that the EEVD motif is involved in the intramolecular regulation of Hsp70 function and intermolecular interactions with HDJ-1.     

Transforming growth factor-beta 1 rapidly induces Hsp70 and Hsp90 molecular chaperones in cultured chicken embryo cells.

In this report we show that: (1) molecular chaperones in the heat shock protein (hsp) family are a new class of cellular proteins induced by Transforming Growth Factor-beta 1 (TGF beta), a cytokine present in serum, (2) rapid induction of Hsc70 precedes a general increase in protein synthesis and may be a preparatory event, (3) TGF beta is a potent regulator of overall protein synthesis in chicken embryo cells (CEC), and (4) isoforms of Hsp90 with different biochemical properties exist, raising the possibility that they may have different functions. TGF beta can substitute for serum in stimulating synthesis of members of the Hsp90 and Hsp70 families of stress proteins, whereas other cytokines, including PDGF, FGF, and EGF, were not effective nor did they enhance the stimulatory effect of TGF beta on the hsp's. Analysis of the induction of hsp's using one- and two-dimensional polyacrylamide gel electrophoresis indicated that members of the Hsp70 family of molecular chaperones were induced rapidly by TGF beta, reaching maximum rates of accumulation by 5 hours of treatment. Total protein synthesis increased more slowly, undergoing an approximately twofold increase in 24 hours. Using a modified protocol for two-dimensional gel electrophoresis, the Hsp90 protein family was separated into four isoelectric forms, two of which were phosphorylated (Hsp90-2 and -4). These phosphorylated isoforms turned over faster than the unphosphorylated forms of Hsp90. All four isoforms were heat inducible, but only Hsp90-2 and -3 were induced rapidly by TGF beta, again within 5 hours of treatment. The effects of serum on these protein families were similar to those of TGF beta, suggesting that this cytokine may be the serum component primarily responsible for up-regulating members of the Hsp90 and Hsp70 families. We hypothesize that cells rapidly increase their chaperoning capacity for newly synthesized polypeptides in preparation for an increase in the rate of synthesis of proteins up-regulated by TGF beta.     

Diurnal regulation of Hsp70s in leaf tissue

Steady-state mRNA levels for three Hsp70s were found to be regulated by a distinctive light/dark mechanism in spinach leaves. Messenger RNAs for the chloroplast stromal and two cytosolic forms displayed a diurnal expression pattern under isothermal conditions that appeared to be independent of circadian control. While protein blot data showed relatively constant Hsp70 protein levels, the higher Hsp70 mRNA levels in the light paralleled the diurnal cycle of total cell protein synthesis. Fractionation studies showed that the major cytosolic Hsp70 cognate group was associated with polysomes. Therefore, the variation of Hsp70 mRNAs is consistent with the diurnal metabolic activity of plant photosynthetic cells in which the demand of protein biogenesis for chaperone function and tissue temperature are highest during the day.