Heat shock proteins HSP70 and HSP90 in pea seedlings under clinorotation of different duration

We have previously shown an increase in the Hsp70 and Hsp90 level in pea seedlings (cv. Damir) in response to clinorotation. In this work, Western-blot analysis of the Hsp70 and Hsp90 under clinorotation of different duration--from hours to days--was carried out with pea seedlings (cv. Intensive) with more intensive seed germination and seedling growth. Under clinorotation, the higher Hsp70 and Hsp90 amounts in the germinating seeds seem to be caused by some deceleration of their hydrolysis that correlated with common slowing down of seedling growth. Time-dependent changes in response to clinorotation were determined: some temporary increase in the Hsps level with the maximum for the Hsp70 at 2 h of clinorotation and for the Hsp90--at 6 h and their subsequent returning to the control level.     

Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.     

Conditions for a heat shock response during oogenesis and embryogenesis of the amphibian Pleurodeles waltl

The optimal conditions capable of inducing an increase in HSP70 neosynthesis during development of the urodele amphibian Pleurodeles waltl were determined in this study. These conditions depend on temperature, heat shock duration and recovery duration. In oocytes, a heat shock response was repeatedly obtained at 37°C for 15 min followed by 1 h recovery. These results provided evidence for heat shock response at every stage considered. An increase in HSP70 synthesis was noted throughout oogenesis, but it did not lead to an increase in the amount of soluble HSP70, except for stage VI oocytes. Such results suggest that from stage II to stage IV oocytes, an equilibrium occurs between the HSP70 used and the HSP70 neosynthesized. In contrast, in stage VI oocytes, heat shock led to overproduction of HSP70. During early development, the heat shock response was repeatedly obtained only from the gastrula stage with a 37°C shock and a 15min duration of treatment. Surprisingly, during cleavage stage, the soluble HSP70 total amount increased after heat shock at a time when no HSP70 neosynthesis occurred.     

An Immunohistochemical Study of Localization of the Calcium-Binding Protein Recoverin in Retina of the Newt Pleurodeles waltl

The presence and localization of the calcium-binding protein recoverin, initially found in photoreceptors of the bovine eye, were immunochemically studied in retina of the new Pleurodeles waltl. Polyclonal monospecific antibodies against recoverin were raised and the methods of immunoblotting and indirect immunofluorescence were used. A protein with an apparent molecular mass of 26 kDa was found in the retina extract, which was specifically stained by the antibodies against recoverin. Localization of recoverin was studied on the retina sections: an intense reaction was found in the inner segments and a weak reaction was found in the basal part of the outer segments of photoreceptors and in Landolt's clubs of displaced bipolars. The results we obtained suggest for the first time the presence of recoverin in the retina of a representative of the Urodeles and indicate to interspecific conservativeness of this protein and differences of its localization in the retina photoreceptors in different species. The data obtained open a possibility of using recoverin as a marker protein of photoreceptors and displaced bipolars in studies of retina regeneration in newts.     

HSP70 heat shock proteins and environmental stress in Antarctic marine organisms: A mini-review

The ability to understand and predict the effects of environmental stress on biodiversity is becoming increasingly important in our changing environment. Antarctic marine species are some of the most stenothermal on the planet and many inhabit the waters off the Antarctic Peninsula which is one of the areas where there is rapid regional climate change. Therefore these animals are highly vulnerable to changing environmental temperatures and clearly we need to understand the complexities of their response, not just at the individual species level, but also the implications for the ecosystem as a whole. Heat shock proteins have a long history of use in studies of organism stress responses and have frequently been proposed as potential universal molecular biomarkers, especially for non-model species. In this mini-review, the heat shock response and heat shock proteins (specifically the HSP70 family) are examined in Antarctic marine species alongside their physiological capabilities and limits to answer a series of questions: do these animals have a heat shock response which includes the expression of HSP70 genes? What is the relationship between their heat shock response and physiological capabilities? Can HSP70 genes be used as molecular biomarkers for these species?     

Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas

Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.     

Characterization of progesterone receptor binding to the 90- and 70-kDa heat shock proteins

In this study a model system for expression of the chicken progesterone receptor in cultured cells was developed using a quail fibroblast cell line, QT6. The chicken progesterone receptor form A expressed in QT6 cells was evaluated and determined to have a number of similarities to receptor isolated from chicken oviduct. These include hormone binding, sedimentation profile, phosphorylation pattern, heat shock protein (hsp) 70 and hsp90 associations and the ability to stimulate a reporter gene construct. Therefore, the receptor expressed in this system functioned adequately for further evaluation of the particular region (or regions) involved in hsp70 and hsp90 binding. Several receptor deletion mutants were tested for hsp70/hsp90 binding; only the d369-659 mutant, which has the entire steroid-binding domain deleted, was unable to bind hsp90 and hsp70. Three separate regions of the steroid-binding domain were found to partially restore hsp90 and hsp70 binding to the d369-659 mutant protein. However, hsp binding was not abolished when these or other regions of the steroid binding domain were deleted individually. These findings indicate that hsp90 and hsp70 both bind to the steroid-binding domain of the receptor through interactions at multiple locations or through some structural quality that is distributed throughout this region of the protein.     

Evidence that the 90-kDa heat shock protein (HSP90) exists in cytosol in heteromeric complexes containing HSP70 and three other proteins with Mr of 63,000, 56,000, and 50,000

Monoclonal antibody (mAb) 8D3 and 3G3 are unique antibodies capable of precipitating both free 90-kDa heat shock protein (HSP90) and HSP90-protein complexes. Immunoprecipitation of [35S]methionine-labeled Hepa 1c1c7 cytosolic extracts were performed using mAb 8D3 or 3G3. The resulting immunoprecipitates can be dissociated from the mAb with a 500 mM NaCl wash. These washes were subjected to both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Five major protein spots were detected in addition to HSP90 with the following relative molecular weights: 68,000, 63,000, 56,000, 50,000, and 188,000. On Western blots mAb 3G3 was capable of specifically binding to HSP90. Each of these proteins was localized on two-dimensional gels. Using one-dimensional gel electrophoresis and immunochemical localization on Western blots, the p68 spot was identified as HSP70, and the p56 spot was found to cross-react with polyclonal antibody JP-1 raised against a 59-kDa protein. This 59-kDa protein has been found previously to be associated with several steroid hormone receptors in rabbit uterine cytosol. Immunoprecipitation of [32P]orthophosphate-labeled Hepa 1c1c7 cytosol with mAb 8D3 or 3G3 revealed two major phosphorylated proteins with relative molecular weights of 90,000 and 50,000. The identities of p63 and p188 are currently unknown. This is the first report examining the major proteins that are complexed with HSP90 in mammalian cells.     

The role of heat shock proteins HSP90 in the response of immune cells to centimeter microwaves

The effects of low-level electromagnetic waves (8.15-18 GHz, 1 microW/cm2, 1 h) on the production of heat shock proteins, several cytokines, and nitric oxide in isolated mouse macrophages and lymphocytes were examined both under normal conditions and after the treatment of the cells with geldanamycin (GA), a depressor of activity of the heat shock protein 90 (Hsp90). The irradiation of cells without GA induced the production of Hsp70, nitric oxide (NO), interleukin-1beta (IL-1beta), interleukin-10 (IL-10), and the tumor necrosis factor -alpha (TNF-alpha). No changes in the production of Hsp90 in irradiated cells were observed, but intracellular locations of Hsp25 and Hsp70 altered. The preliminary treatment of cells with GA did not remove the effects of microwaves: in these conditions, the synthesis of all cytokines tested, nitric oxide, as well as total and membrane amount of Hsp70, and the amount of Hsp25 in the cytoplasm and cytoskeleton increased. Moreover, the exposure of cells incubated with GA resulted in the reduction of Hsp90-alpha production.     

The neuroprotective potential of heat shock protein 70 (HSP70)

In response to many metabolic disturbances and injuries, including stroke, neurodegenerative disease, epilepsy and trauma, the cell mounts a stress response with induction of a variety of proteins, most notably the 70-kDa heat shock protein (HSP70). Whether stress proteins are neuroprotective has been hotly debated, as these proteins might be merely an epiphenomenon unrelated to cell survival. Only recently, with the availability of transgenic animals and gene transfer, has it become possible to overexpress the gene encoding HSP70 to test directly the hypothesis that stress proteins protect cells from injury. A few groups have now shown that overproduction of HSP70 leads to protection in several different models of nervous system injury. This review will cover these studies, along with the potential mechanisms by which HSP70 might mediate cellular protection.     

The heat shock protein HSP70 and heat shock cognate protein HSC70 contribute to antimony tolerance in the protozoan parasite leishmania

Antimony-containing drugs are still the drugs of choice in the treatment of infections caused by the parasite Leishmania. Resistance to antimony is now common in some parts of the world, and several mechanisms of resistance have been described. By transfecting cosmid banks and selecting with potassium antimonyl tartrate (SbIII), we have isolated a cosmid associated with resistance. This cosmid contains 2 copies of the heat shock protein 70 (HSP70) and 1 copy of the heat shock cognate protein 70 (HSC70). Several data linked HSP70 to antimony response and resistance. First, several Leishmania species, both as promastigotes and amastigotes, increased the expression of their HSP70 proteins when grown in the presence of 1 or 2 times the Effect Concentration 50% of SbIII. In several mutants selected for resistance to either SbIII or to the related metal arsenite, the HSP70 proteins were found to be overexpressed. This increase was also observed in revertant cells grown for several passages in the absence of SbIII, suggesting that this increased production of HSP70 is stable. Transfection of HSP70 or HSC70 in Leishmania cells does not confer resistance directly, though these transfectants were better able to tolerate a shock with SbIII. Our results are consistent with HSP70 and HSC70 being a first line of defense against SbIII until more specific and efficient resistance mechanisms take over.     

Identification of the vitellogenin proteins in the newt Pleurodeles waltl (urodele amphibian)

Vitellogenin derived from the blood of estrogen-treated Pleurodeles waltl was identified by immunochemical and electrophoretic analyses, using an antiserum against plasma vitellogenin isolated by dimethylformamide precipitation. Pleurodeles vitellogenin migrates as four bands on native PAGE, designated alpha-, beta-, gamma- and delta- VTG, with apparent mol. wts of 250,000, 270,000, 280,000 and 520,000 respectively. In the plasma, from estrogen-treated males like from ovariectomized estrogen-treated females, an additional band (mu-VTG) was found by native PAGE, never observed in estrogen-treated female plasma. It has a mol. wt of about 380,000 and shows complete immunological cross-reactivity with the vitellogenin antiserum. At least two polypeptides, termed VTG-I and VTG-II (mol. wt = 180,000 and 210,000) were identified by SDS-PAGE. Rocket immunoelectrophoresis displays three distinct precipitate lines indicating major immunological differences between the plasma vitellogenins.     

Heat shock proteins HSP70 and GP96: structural insights

Several heat shock proteins (HSPs) act as potent adjuvants for eliciting anti-tumor immunity. HSP-based tumor vaccine strategies have been highly successful in animal models and are undergoing testing in clinical trials. It is generally accepted that HSPs, functioning as chaperones for tumor antigens, elicit tumor-specific adaptive immune responses. HSPs also appear to induce innate immune responses in an antigen-independent fashion. Innate responses generated by HSPs may contribute to anti-tumor immunity. Immunologically active chaperones with anti-tumor activity are referred to as “immunochaperones”. Here, we review the studies that address the role of structural domains or regions of the immunochaperones HSP70 and GP96 that may be involved in the induction of adaptive or innate immune responses. This article forms part of the Symposium in Writing “Thermal stress-related modulation of tumor cell physiology and immune responses”, edited by Elfriede Noessner.     

Secretion of the heat shock proteins HSP70 and HSC70 by baby hamster kidney (BHK‐21) cells

The HSPs (heat‐shock proteins) of the 70‐kDa family, the constitutively expressed HSC70 (cognate 70‐kDa heat‐shock protein) and the stress‐inducible HSP70 (stress‐inducible 70‐kDa heat‐shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK‐21) cells released HSP70 and HSC70 in a serum‐free medium and that this process was the result of an active secretion of HSPs rather than the non‐specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non‐classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl‐β‐cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK‐21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK‐21 cells in a serum‐free medium through a non‐classical pathway in which lipid rafts play an important role.     

Putative 65 kDa protein of beet yellows closterovirus is a homologue of HSP70 heat shock proteins

A portion of the RNA genome of beet yellows closterovirus (BYV) has been sequenced encompassing a complete long open reading frame (ORF) potentially encoding a 65 kDa protein. The sequence of this putative protein was strikingly similar to those of HSP70-related heat shock proteins. The counterparts of all the eight segments strongly conserved in HSP70s could be confidently identified in the BYV 65 kDa protein. It is suggested that some of these segments might be the ATP-binding site(s) and that, similarly to the heat shock proteins, the 65 kDa is probably ATP-binding. Generally, however, the divergence between the 65 kDa sequence and the sequences of the HSP70s was much more pronounced than that between any two members of the latter family, allowing a clearer delineation of clusters of conserved residues that might be crucial for protein function. It is suggested that these observations will be helpful in functional dissection of the proteins of the HSP70 family. Analysis of the sequence of a portion of the ORF found upstream from the 65 kDa ORF showed that the C-terminal domain of the encoded protein could be an RNA-dependent RNA polymerase closely related to those of tricornaviruses, a family of RNA plant viruses with three component genomes.