Low Molecular Mass Heat Shock Research Articles

Functional Role of HSP90 Complexes with Endothelial Nitric-oxide Synthase (eNOS) and Calpain on Nitric Oxide Generation in Endothelial Cells

Although several reports have indicated that eNOS is a highly sensitive calpain substrate, the occurrence of a concomitant Ca(2+)-dependent activation of the synthase and of the protease has never been analyzed in specific direct experiments. In this study, we have explored in vivo how eNOS can undergo Ca(2+)-dependent translocation and activation, protected against degradation by activated calpain. Here we demonstrate that following a brief exposure to Ca(2+)-loading, the cytosolic eNOS-HSP90 complex recruits calpain in a form in which the chaperone and the synthase are almost completely resistant to digestion by the protease. Furthermore, in the presence of the HSP90 inhibitor geldanamycin, a significant decrease in NO production and an extensive degradation of eNOS protein occurs, indicating that dissociation from membranes and association with the chaperone is correlated to the protection of the synthase. Experiments with isolated membrane preparations confirm the primary role of HSP90 in dissociation of eNOS from caveolae. Prolonged exposure of cells to Ca(2+)-loading resulted in an extensive degradation of both eNOS and HSP90, accompanied by a large suppression of NO production. We propose that the protective effect exerted by HSP90 on eNOS degradation mediated by calpain represents a novel and critical mechanism that assures the reversibility of the intracellular trafficking and activation of the synthase.

Acclimatization of In Vitro-Raised Asiatic Hybrid Lily Plants in Subtropical Climate and Associated Changes in Stress Proteins and Antioxidant Enzymes

A protocol for direct differentiation of shoots from leaf segments of Litium cv ‘Orange Pixie’ was developed through in vitro methods. After hardening, tissue-raised plants were transferred in the open field conditions from the very beginning. The acclimatized plants not only grew well but flowered also at 43°C under subtropical climatic conditions and regenerated a new bulb at their base after flowering. The activity of different antioxidant enzymes like superoxide dismutase, peroxidase, catalase and ascorbate peroxidase and their isoenzymes patterns showed differential up and down regulation in control and in different parts of in vitro-raised plants. Characterization of both high and low molecular mass heat-shock proteins (HSPs) was done using HSP70 and HSP 18.1 antibodies against pea (Pisum sativum L), respectively. The level of high molecular mass proteins did not change much and was found to be of constitutive nature, whereas a new small protein of 21 kD was induced only in tissue culture-raised flowering (TF) plants indicating the possible role of this stress protein in acclimatization and flowering of Asiatic hybrid lily plants at 43°C under tropical conditions. The amount of this protein was much higher in petals as compared to stem and leaf.

The Possibility of Novel Antiplatelet Peptides: The Physiological Effects of Low Molecular Weight HSPs on Platelets

Some low molecular mass heat shock proteins (HSPs) appear to act as molecular chaperones, but their exact physiological roles have not been fully elucidated. We reported on a physiological role of HSP20, HSP27 and alphaB-crystallin on platelet function in vitro and ex vivo. HSP20 and alphaB-crystallin inhibited platelet aggregation using human platelets dose-dependently induced by thrombin or botrocetin. On the other hand, HSP27, the other type of low molecular mass HSP, did not affect platelet aggregation. When HSP20 or alphaB-crystallin was injected intravenously as a bolus in hamsters, the development of thrombus after endothelial injury was prevented. Moreover, 9 amino-acid sequences isolated from HSP20 or alphaB-crystallin significantly reduced platelet aggregation induced by TRAP, but not a PAR-4 agonist. These findings strongly suggest that HSP20 or alphaB-crystallin can act intercellularly to regulate platelet functions. Our results may provide the basis for a novel defensive system to thrombus formation in vivo.

Cloning and Expression Analysis of Salt Responsive Gene from Kandelia candel

Identification of gene expression patterns in mangroves grown under salinity will help to reveal the molecular mechanisms of salt tolerance. Here, 10 cDNAs of genes were isolated from Kandelia candel and identified by representational difference analysis of cDNA (cDNA RDA) under different NaCl concentrations. Of five genes expressed preferentially under salt condition, two were unknown, three were two kinds of low molecular mass heat-shock proteins (sHSPs) and ADP-ribosylation factor, respectively. The expressions of other five genes were repressed under NaCl stress, two encoded cyclophilins, three were tonoplast intrinsic protein, early light-induced protein and 60S ribosomal protein, respectively.

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Some low molecular mass heat shock proteins (HSPs) appear to act as molecular chaperones, but their exact physiological roles have not been fully elucidated. We reported the physiological roles of HSP20, HSP27, and alpha B-crystallin in platelet function in vitro and ex vivo. HSP20 and alpha B-crystallin dose-dependently inhibited the aggregation of human platelets induced by thrombin or botrocetin. On the other hand, HSP27, the other type of low molecular mass HSP, did not affect platelet aggregation. When HSP20 or alpha B-crystallin was injected intravenously as a bolus in hamsters, the development of thrombus after endothelial injury was prevented. Moreover, 9 amino acid sequences isolated from HSP20 or alpha B-crystallin significantly reduced platelet aggregation induced by TRAP, but not a PAR-4 agonist. These findings strongly suggest that HSP20 or alpha B-crystallin can act intercellularly to regulate platelet functions. Our results may provide the basis for a novel defense system against thrombus formation in vivo.

Expression profiles of low molecular mass heat shock proteins by Mycobacterium avium and Mycobacterium intracellulare

Closely related non-tuberculous mycobacterial species, Mycobacterium avium and Mycobacterium intracellulare, were compared for the profiles of their production of low molecular mass heat shock proteins at 45°C, by performing polyacrylamide gel electrophoresis analysis of bacterial cell lysate proteins. All of the M. intracellulare but not M. avium strains potently increased the production of the 18-kDa heat shock protein, when cultured at 45°C. Half of the M. intracellulare strains with high sensitivity to 45°C produced not only the 18-kDa heat shock protein but also the 16-kDa heat shock protein at 45°C. These findings indicate that M. avium and M. intracellulare differentially respond to 45°C heat shock in terms of the production of low molecular mass heat shock proteins.

Effect of Combined Salt and Heat Treatments on Germination and Heat-Shock Protein Synthesis in Lentil Seeds

The germination of lentil seeds was gradually reduced when seeds were exposed to temperature of 30 or 40 °C, either alone or combined with 0.1, 0.2 or 0.3 M NaCl or 34.1 % (m/v) PEG 8000, during 6 -12 h imbibition. [35S]-methionine incorporation in 12 h imbibed lentil axes also decreased with increasing NaCl concentration at 20 and 40 °C, whereas at 30 °C only 0.3 M NaCl treatment partially inhibited protein synthesis. An analysis of newly synthesized proteins by 1-D SDS PAGE, showed that the expression of most polypeptides decreased following increasing stress. Among these, low molecular mass heat-shock proteins declining, higher in 40 °C treated axes than those treated at 30 °C, supports the hypothesis that at this temperature maximal level of expression of these proteins was achieved.

Identification of acquired thermotolerance deficiency within the ditelosomic series of `Chinese Spring' wheat

The relative contribution of individual heat shock proteins to acquired thermotolerance was evaluated through analysis of chromosomal deletions in a ditelosomic series of the hexaploid wheat ( Triticum aestivum L.) cultivar `Chinese Spring'. This study describes the identification of a line within this ditelosomic series that exhibited a reduced level of acquired thermotolerance. Changes in the temperature sensitivity of chlorophyll accumulation were used as an indicator of acquired thermotolerance. The temperature providing maximum chlorophyll accumulation was 30 °C in leaves under continuous light. A 30-min challenge temperature of 48 °C prior to the light exposure was shown to inhibit subsequent chlorophyll accumulation. Preincubation at 40 °C for 4 h before the 30-min 48 °C challenge triggered the acquired thermotolerance system of the plant resulting in chlorophyll accumulation upon exposure to light. Evaluation of the ditelosomic series revealed reductions in acquired thermotolerance levels in the DT7DS line relative to controls. Two-dimensional SDS polyacrylamide gel analysis was used to identify reduction in the level of two low molecular mass heat shock proteins in DT7DS.

Pharmacological analysis of integrin function: inhibition of vascular stenosis and identification of a novel antiplatelet substance

The long-lasting success of PTCA, a common treatment for patients with coronary artery disease, is hampered by acute reocclusion due to thrombus and by late restenosis due to smooth muscle cell (SMC) migration and proliferation. Recently potent antiplatelet agents such as inhibitors of platelet glycoprotein (GP) IIb/IIIa (alpha IIb/beta 3) have become available and are highly effective in humans. We investigated the inhibitory effects of these compounds on vascular stenosis. Administration of an RGD-containing peptide (non-selective alpha IIb/beta 3 antagonist) results in reduction of neointima, but selective alpha IIb/beta 3 antagonists have no effect. This effect is due both to an early event, which could be due to the inhibition of secretion of PDGF from activated platelets with blocked alpha IIb/beta 3, and to a late event, by interference with SMC alpha v/beta 3. Moreover, platelet adhesion via GPIb/V/IX is a trigger for thrombus formation. Inhibition of platelet adhesion results in the prevention of thrombus formation and the suppression of neointima. However, this effect was supported by the reduction of SMC proliferation. Therefore, these dual inhibitions markedly reduced vascular stenosis. Finally some low molecular mass heat shock proteins (HSPs) appear to act as molecular chaperones, but their physiological roles have not been fully elucidated. We have investigated the physiological role of p20, a kind of HSP, on platelet function. p20 inhibited platelet aggregation. Our findings may provide the basis for a novel defensive system against thrombus formation.

AL–2 - Pharmacological analysis of integrin function: Inhibition of vascular stenosis and identification of a novel antiplatelet substance.

The long-lasting success of PTCA, a common treatment for patients with coronary artery disease, is hampered by acute reocclusion due to thrombus and by late restenosis due to smooth muscle cell (SMC) migration and proliferation. Recently potent antiplatelet agents such as inhibitors of platelet glycoprotein (GP) IIb/IIIa (alpha IIb/beta 3) have become available and are highly effective in humans. We investigated the inhibitory effects of these compounds on vascular stenosis. Administration of an RGD-containing peptide (non-selective alpha IIb/beta 3 antagonist) results in reduction of neointima, but selective alpha IIb/beta 3 antagonists have no effect. This effect is due both to an early event, which could be due to the inhibition of secretion of PDGF from activated platelets with blocked alpha IIb/beta 3, and to a late event, by interference with SMC alpha v/beta 3. Moreover, platelet adhesion via GPIb/V/IX is a trigger for thrombus formation. Inhibition of platelet adhesion results in the prevention of thrombus formation and the suppression of neointima. However, this effect was supported by the reduction of SMC proliferation. Therefore, these dual inhibitions markedly reduced vascular stenosis. Finally some low molecular mass heat shock proteins (HSPs) appear to act as molecular chaperones, but their physiological roles have not been fully elucidated. We have investigated the physiological role of p20, a kind of HSP, on platelet function. p20 inhibited platelet aggregation. Our findings may provide the basis for a novel defensive system against thrombus formation.

A heat shock-related protein, p20, plays an inhibitory role in platelet activation

Some low molecular mass heat shock proteins (HSPs) appear to act as molecular chaperones, but their exact physiological roles have not been fully elucidated. We reported previously that a 20-kDa protein (p20), which is classified as a low molecular mass HSP, is present at high levels in skeletal and smooth muscles. In the present study, we investigated a physiological role of p20 on platelet function in vitro and ex vivo. p20 inhibited platelet aggregation using human platelets dose-dependently induced by botrocetin. On the other hand, HSP27, the other type of low molecular mass HSP, did not affect platelet aggregation. When p20 (300 μg/kg) was injected intravenously as a bolus in hamsters, platelet aggregation ex vivo induced by botrocetin was also significantly inhibited. In order to further investigate the inhibitory effect by p20 on platelet activation, we performed platelet aggregation induced by thrombin or ADP using human platelets. p20 markedly prevented platelet aggregation induced by thrombin, but not ADP. These findings suggest that p20 can act intercellularly to regulate platelet functions. Our results may provide the basis for a novel defensive system to thrombus formation.

A cDNA Encoding a Low Molecular Mass Heat-Shock Protein from Opium Poppy

A cDNA Encoding a Low Molecular Mass Heat-Shock Protein from Opium Poppy

The 28-kDa protein whose phosphorylation is induced by protein kinase C activators in MCF-7 cells belongs to the family of low molecular mass heat shock proteins and is the estrogen-regulated 24-kDa protein

We have previously reported the presence of a 28-kDa protein in human mammary adenocarcinoma MCF-7 cells, whose phosphorylation by phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and permeant diacylglycerol 1,2-dioctanoyl-sn-glycerol was correlated to growth arrest induced by the protein kinase C (PKC) activators. We now investigate the possible identity of this protein with the estrogen-regulated "24-kDa" protein shown as related to the mammalian heat shock protein 27 (Fuqua, S. A. W., Blum-Salingaros, M., and McGuire, W. L. (1989) Cancer Res 49, 4126-4129). 32P-Labeled 28-kDa protein from TPA-treated MCF-7 cells was immunoprecipitated with a 24-kDa-specific monoclonal antibody. Immunoblots from cell extracts fractionated by two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis demonstrated that TPA induced the conversion of a 28-kDa isoform "a" (pI 6.7) to a more acidic isoform "b" (pI 6.2). Two-dimensional gel analysis of [3H]leucine-labeled MCF-7 cell extracts demonstrated that conversely to TPA, which induced only phosphorylation of 28-kDa protein, heat shock induced both synthesis (increase of isoform a) and phosphorylation (conversion of isoforms a to b) of the protein. 32P labeling of MCF-7 cells allowed demonstration of the presence of an extra phosphoisoform "c" (pI 5.9) upon TPA as well as heat shock treatment. When cells were pretreated with the bisindolylmaleimide GF109203X, a selective inhibitor of PKC, the heat shock-induced phosphorylation was unchanged, while the TPA effect was almost abolished, suggesting that the heat shock-activated protein kinase was very likely different from PKC. However, peptide mapping of the 28-kDa phosphoprotein suggested identical sites of phosphorylation upon TPA and heat shock stimulation. Partial amino acid sequencing of the 28-kDa protein revealed identity with both the 24-kDa protein and the mammalian HSP27. The fact that estrogens and PKC, respectively, regulate expression and phosphorylation of this 24/28-kDa protein strongly argues for its key role in MCF-7 cell proliferation and differentiation.

A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein.

The 25-kD inhibitor of actin polymerization (25-kD IAP), isolated from turkey smooth muscle (Miron, T., M. Wilchek, and B. Geiger, 1988. Eur. J. Biochem. 178:543-553), is shown here to be a low molecular mass heat shock protein (HSP). Direct sequence analysis of the purified protein, as well as cloning and sequencing of the respective cDNA, disclosed a high degree of homology (67% identity, 80% similarity) to the human 27-kD HSP. Southern blot of chicken genomic DNA disclosed one band, suggesting the presence of a single gene, and Northern blot analysis revealed abundant transcript of approximately 1 kb in gizzard and heart tissues and lower amounts in total 18-d chick embryo RNA and in cultured fibroblasts. Exposure of the latter cells to 45 degrees C resulted in over 15-fold increase in the apparent level of the 25-kD IAP protein, confirming that its expression is regulated by heat shock. Immunofluorescent microscopic localization indicated that after heat treatment, the levels of the 25-kD IAP were markedly increased and the protein was apparently associated with cytoplasmic granules. Heat shock also had a transient, yet prominent, effect on the microfilament system in cultured fibroblasts: stress fibers disintegrated within 10-15 min after incubation at 45 degrees C, yet upon further incubation at the elevated temperature, conspicuous actin bundles were apparently reformed.