Low Molecular Weight Stress Protein Research Articles

Вплив зaсолення нa склaд білків і вміст проліну в оргaнaх рослин <i>Salix viminalis</i> L.

The salinization effect on protein accumulation and proline content in organs of Salix viminalis L. plants was investigated. The plants had been growing in pots with Stebnyk’s tailing soil (during 30 days). The soil of tailing with renewed biocenosis was used like control, for experiment we used soil with spreading of hlikohalophytes. Under saline stress the accumulation of proteins in stems and roots of S. viminalis plants was noticed. This may indicate a plant adaptation to stress. Only low molecular weight polypeptides, in particular proteins with Mr 30, 23, 22, 20, 17, 15, 12, 10 and 8 kD, were found in the electropherograms of all analyzed organs of S. viminalis plants (leaves, stems, roots). Their content significantly varied depending on the organ of the plant. The spectrums of low molecular weight proteins in organs of S. viminalis plants had qualitative and quantitative differences under normal and stress conditions, especially changes of proteins in experimental organs were more expressive. Low molecular weight proteins with Mr 19—21 kD were found in the roots of S. viminalis plants, both in the control and in the experimental variants, but their quantity was higher under salinity stress. An increased content of 22 kD proteins were detected in the stems of the plant, compared to the control. Also, 17 kD Mr proteins were found in the stems during stress, unlike under normal conditions. Less proteins with a molecular weight of 20—23 kD were synthesized in the plants compared to the control. However, we have found an increased content of Mr 10 kD protein in experimental leaves. The accumulation of proline due to salinity in the shoots and roots of S. viminalis plants was established, compared with the control. This can be explained by the water stress that occurs during salinization. Thus, due to the effect of salt stress in the organs of S. viminalis plants, the accumulation of low molecular weight stress proteins and proline was observed, which may indicate certain peculiarities of plant adaptation to salinity conditions.

MALDI-TOF Mass Spectrometry Enables a Comprehensive and Fast Analysis of Dynamics and Qualities of Stress Responses of Lactobacillus paracasei subsp. paracasei F19.

Lactic acid bacteria (LAB) are widely used as starter cultures in the manufacture of foods. Upon preparation, these cultures undergo various stresses resulting in losses of survival and fitness. In order to find conditions for the subsequent identification of proteomic biomarkers and their exploitation for preconditioning of strains, we subjected Lactobacillus (Lb.) paracasei subsp. paracasei TMW 1.1434 (F19) to different stress qualities (osmotic stress, oxidative stress, temperature stress, pH stress and starvation stress). We analysed the dynamics of its stress responses based on the expression of stress proteins using MALDI-TOF mass spectrometry (MS), which has so far been used for species identification. Exploiting the methodology of accumulating protein expression profiles by MALDI-TOF MS followed by the statistical evaluation with cluster analysis and discriminant analysis of principle components (DAPC), it was possible to monitor the expression of low molecular weight stress proteins, identify a specific time point when the expression of stress proteins reached its maximum, and statistically differentiate types of adaptive responses into groups. Above the specific result for F19 and its stress response, these results demonstrate the discriminatory power of MALDI-TOF MS to characterize even dynamics of stress responses of bacteria and enable a knowledge-based focus on the laborious identification of biomarkers and stress proteins. To our knowledge, the implementation of MALDI-TOF MS protein profiling for the fast and comprehensive analysis of various stress responses is new to the field of bacterial stress responses. Consequently, we generally propose MALDI-TOF MS as an easy and quick method to characterize responses of microbes to different environmental conditions, to focus efforts of more elaborate approaches on time points and dynamics of stress responses.

Phosphorylated Heat Shock Protein 27 Represses Growth of Hepatocellular Carcinoma via Inhibition of Extracellular Signal-regulated Kinase

Heat shock protein 27, one of the low molecular weight stress proteins, is recognized as a molecular chaperone; however, other functions have not yet been well established. Phosphorylated heat shock protein 27 levels inversely correlate with the progression of human hepatocellular carcinoma. This study shows that phosphorylated heat shock protein 27 interferes with cell growth of the hepatocellular carcinoma-derived HuH7 cells in the presence of the proinflammatory cytokine, tumor necrosis factor-alpha, via inhibition of the sustained activation of the extracellular signal-regulated kinase signal pathway. The activities of Raf/extracellular signal-regulated kinase and subsequent activator protein-1 transactivation and the induction levels of cyclin D1 were lower in HuH7 cells transfected with phosphorylated heat shock protein 27 than those with unphosphorylated heat shock protein 27. Moreover, phosphorylated heat shock protein 27 up-regulated the levels of p38 mitogen-activated protein kinase and mitogen-activated protein kinase phosphatase-1, an inhibitory protein of extracellular signal-regulated kinase. These results indicate that phosphorylated heat shock protein 27 might suppress the extracellular signal-regulated kinase activity in the hepatocellular carcinoma cells via two separate pathways in an inflammatory state. The extracellular signal-regulated kinase activity is inversely correlated with phosphorylated heat shock protein 27 at serine 15 and also in human hepatocellular carcinoma tissues in vivo. Because the extracellular signal-regulated kinase signal pathway is a major proliferation signal of hepatocellular carcinoma, activator protein-1 activation is an early event in hepatocarcinogenesis. These findings strongly suggest that the control of the phosphorylated heat shock protein 27 levels could be a new therapeutic strategy especially to counter the recurrence of hepatocellular carcinoma.

Increased expression of high but not low molecular weight heat shock proteins in resectable lung carcinoma

Strong expression of high-molecular-weight (HMW) heat-shock proteins (HSP) by lung carcinoma has been documented using immunohistochemistry. Far less is known about the expression of low-molecular-weight (LMW) HSP in lung cancer. We compared the quantitative expression of HMW (HSP-60, HSP-70) and LMW (HSP-27, ubiquitin) HSP in tumor and non-tumor lung tissue obtained from 47 patients undergoing surgical resection of lung carcinoma. HSP levels were determined in cell lysates from tissue samples by ELISA using streptavidin-biotin technology. Results were normalized to total protein content measured by spectrophotometry. Compared to disease-free lung tissue, tumor tissue samples showed higher levels of both HSP-60 (median value: 227 pg versus 96 pg per mg protein ( P<0.001 by Wilcoxon Rank test for paired data) and HSP-70 (median value: 525 ng versus 401 ng per mg protein ( P=0.01 by Wilcoxon Rank test for paired data). Tumor and tumor-free tissues show similar levels of ubiquitin and HSP-27. Neither the survival rate nor the histologic type and extent of cancer are correlated with the observed differences in HSP-60 and HSP-70 expression ( P>0.1 by one way analysis of variance for repeated measures with one between subject factor). Our data confirm, on a quantitative basis, the increased expression of HSP-60 and HSP-70 in non-small-cell lung carcinoma. However, no prognostic value was found to be associated with this over-expression. In contrast, LMW stress proteins such as ubiquitin and HSP-27, although implicated in cellular processes potentially related to malignant transformation, show no increased expression in lung carcinoma.

Anthracycline-induced erythroid differentiation of K562 cells is inhibited by p28, a novel mammalian glutathione-binding stress protein

When exposed to the anthracycline doxorubicin, K562 cells undergo differentiation which is characterized by arrested cell division, an increased mean cell diameter, and the production of hemoglobin. The influence of expression of p28, a low-molecular weight stress protein, on the differentiation of K562 cells was examined. Expression of p28 was modulated by transfection of K562 cells with expression vectors containing the murine p28 cDNA in either the sense or antisense orientation, or without the p28 cDNA. In K562 cells where p28 expression was either unaltered or downregulated, exposure to 40 nM Doxorubicin resulted in an arrest of cell division, the production of hemoglobin, and an increased cell diameter consistent with cells undergoing differentiation. K562 cells that overexpressed p28 continued to divide, had fewer hemoglobin-producing cells, had a smaller mean cell diameter and had a 5.5-fold increase in cell survival. Consistent with an inhibition of doxorubicin-induced erythroid differentiation, p28 may act by changes in redox regulation via the glutathione-binding activity of p28 and suggests a general role for p28 in cellular differentiation. Furthermore, p28 expression may be useful in predicting resistance to chemo- or radiation therapy in the treatment of leukemia and lymphoma.

Ischemic Preconditioning: a Potential Role for Constitutive Low Molecular Weight Stress Protein Translocation and Phosphorylation?

P. Eaton, W. I. Awad, J. I. A. Miller, D. J. Hearse and M. J. Shattock. Ischemic Preconditioning: a Potential Role for Constitutive Low Molecular Weight Stress Protein Translocation and Phosphorylation?Journal of Molecular and Cellular Cardiology (2000) 32, 961–971. We have investigated whether translocation of constitutive low molecular weight stress proteins (α B-crystallin and HSP27) to the myofilament/cytoskeletal compartment occurs during ischemic preconditioning and assessed if this is causally associated with cardioprotection. Triton-insoluble preparations from fresh or aerobically perfused rat hearts (n=4/group) contained relatively little α B-crystallin (96±43 and 43±36 units respectively) or HSP27 (177±32 and 101±26 units respectively). Three preconditioning cycles of (5 min ischemia+5 min reperfusion) increased the Triton-insoluble crystallin to 864±61 units (P<0.05) and HSP27 to 1353±53 units (P<0.05). Two hours of aerobic perfusion following the preconditioning protocol resulted the return of α B-crystallin and HSP27 to near control levels (189±14 units and 252±24 units, respectively). Stress protein translocation, comparable to that achieved by the IPC protocol was induced by aerobic perfusion with hypercarbic (pH 6.8) perfusion. Thus, three cycles of 5 min hypercarbia+5 min normocarbia increased α B-crystallin to 628 ± 30 units (P<0.05) and HSP27 to 1353±53 units. In parallel functional studies, the recovery of LVDP after 35 min ischemia and 60 min of reperfusion was 43±7% in the ischemic control group, 61±3% (P<0.05) in the preconditioned group and 42±6% in the hypercarbic group. Thus, translocation of α B-crystallin and/or is not of-itself sufficient to induce cardioprotection. Using a phospho-specific antibody, we have demonstrated that preconditioning not only translocates α B-crystallin but also increases its phosphorylation at Ser-59 by 9.7-fold compared to aerobic controls (1616±402 v 166±28 units respectively). In contrast, hypercarbia while eliciting a comparable translocation, failed to alter the phosphorylation state of α B-crystallin. Preconditioning-induced phosphorylation was significantly attenuated by 50 μ m genistein (by 61%), 10 μ m SB203580 (by 91%) and 10 μ m bisindolylmaleimide (by 68%), but not by 10 μ m PD98059 (by 4%). Our findings are consistent with the possibility that ischemic preconditioning may be mediated by phosphorylation and translocation of constitutive low molecular weight stress proteins, particularly α B-crystallin.

UVB irradiation induces changes in cellular localization and phosphorylation of mouse HSP27.

We investigated the induction, cellular localization and phosphorylation of a low-molecular weight stress protein (heat shock protein 27, HSP27) by UVB (290-320 nm, max. 312 nm) irradiation stress using immunoblot and indirect immunofluorescence analysis in in vivo and in vitro experiments. The HSP27 was constitutively expressed and distributed in the cytoplasmic fraction of Pam 212 cells (mouse keratinocyte line) or dorsal skin. The increase in the cytoplasm HSP27 level induced by UVB irradiation was less than two-fold that in nonirradiated controls. On the other hand, the translocation of HSP27 from cytoplasm to the nucleus or perinuclear area was time- and dose-dependently induced by UVB irradiation. After UVB irradiation, three isoforms having different isoelectric points were detected in nucleic HSP27 by two-dimensional immunoblotting. The most basic isoform was the unphosphorylated type and the two acidic isoforms were phosphorylated, suggesting that HSP27 is phosphorylated in response to UVB irradiation and accumulates in or around the nucleus as a phosphorylated isoform. These results suggest that the translocation and phosphorylation of HSP27 are induced in response to UVB-irradiation stress.

Ontogeny of low molecular weight stress protein p26 during early development of the brine shrimp, Artemia franciscana.

Embryogenesis in the brine shrimp, Artemia sp., occurs by one of two pathways: (i) the direct, uninterrupted development of nauplius larvae within the female or (ii) the production of embryos that arrest development at the gastrula stage and enter diapause. Diapause embryos are released from females into the aqueous environment where they remain in diapause until activated by appropriate environmental cues and resume development. These encysted embryos possess at least one low molecular weight stress protein, which we refer to as p26 and which has been implicated previously in the stress response of activated embryos. We investigated the appearance of p26 in developing diapause embryos in utero and looked for its presence in embryos developing directly into nauplii. We found p26 to be specific to diapause-destined embryos; it was not detected in direct-developing embryos. We conclude that p26 is not required for the basic developmental program that produces the nauplius. In diapause-destined embryos, p26 was first detectable after 3 days of development, at which time the embryos were late gastrulae. This protein continues to increase in amount until the encysted embryos are released, approximately 5 days after fertilization. At the time of release almost all p26 is located in the low speed supernatant fraction, but as released embryos continue diapause, p26 transfers to the pelleted nuclear fraction in increasing amounts. Our working hypothesis views p26 as a molecular chaperone preventing protein denaturation and aggregation under conditions associated with metabolic arrest and other stressful states, which these encysted embryos encounter.

Immunohistochemical localization of the low molecular weight stress protein HSP27 following focal cerebral ischemia in the rat

We immunohistochemically investigated the induction and localization of a low-molecular weight stress protein, HSP27, in the rat brain following 1 hr of middle cerebral artery occlusion in comparison with those of HSP70. The brains were perfusion-fixed after 4 h, 1 day, 3 days, 7 days, and 14 days of reperfusion. Frozen sections were then prepared and used for immunohistochemistry. In normal brains, we observed no immunoreactivities to HSP70 and HSP27. HSP70 was localized predominantly in neurons in areas peripheral to the ischemic center after 1 day and 3 days, and in endothelial cells and perivascular cells within the ischemic center after 1 day. In contrast, HSP27 was induced in microglia in the ischemic center after 4 h, and then in reactive astrocytes distributed widely in the ipsilateral hemisphere and in part of the contralateral hemisphere after 1 through 14 days. In the center of ischemia where infarction developed, only nonspecific staining was seen. Thus, the expression patterns of HSP70 and HSP27 were quite different with regard to cell type, distribution, and time course following focal cerebral ischemia. HSP70 may be a sensitive marker of acute neuronal stress in the penumbral areas, whereas HSP27, which was most prominently induced in reactive astrocytes in periischemic and remote areas, may be a component of glial reaction to injury.

Variation in the Expression and/or Phosphorylation of the Human Low Molecular Weight Stress Protein during in Vitro Cell Differentiation

Members of the low molecular weight heat shock protein (hsp) family show regulated expression in both Drosophila and mice during development and differentiation. Here we have examined whether similar regulation of the single low molecular weight hsp (hsp 28) of humans exhibits differences in either its expression and/or phosphorylation during the course of in vitro differentiation of hematopoietic cells. In the promyelocytic leukemic cell line, HL-60, we show that early after commitment of the cells to a macrophage-like phenotype (via exposure to phorbol ester myristate, PMA) there occurs an accompanying increased phosphorylation of hsp 28. Over time and as the cells become terminally differentiated the levels of hsp 28 increase significantly. In contrast, cells stimulated to adopt a granulocyte-like phenotype (e.g. exposed to either dimethyl sulfoxide or retinoic acid) show no changes in either the phosphorylation or expression of hsp 28. Moreover, once differentiated the granulocyte-like cells no longer appear capable of phosphorylating hsp 28. Human K562 cells, in response to hemin, rapidly increase their expression and phosphorylation of hsp 28 during the course of their differentiation into erythroid-like cells. Addition of PMA to the K562 cells induces differentiation into a megakaryocyte-like phenotype but is not accompanied by changes in hsp 28 phosphorylation/expression. In the case of the HL-60 cells, differentiation toward the macrophage like lineage is accompanied by an increased adherence of the cells to their substratum and an apparent association of hsp 28 with the actin cytoskeleton.

Induction of 27-kDa heat shock protein following cerebral ischemia in a rat model of ischemic tolerance

Preconditioning the brain with sublethal cerebral ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). The purpose of this study is to investigate the role of low-molecular weight stress proteins, 27-kDa heat shock protein (HSP27) and αB crystallin, in ischemic tolerance. We measured the content of these proteins with enzyme immunoassay in the rat hippocampus and cerebral cortex following 6 min of ischemia with and without preconditioning with 3 min of ischemia and 3 days of reperfusion. We also visualized the localization of HSP27 immunohistochemically in comparison with that of HSP70. A 3-min period of ischemia caused a 2.4-fold increase in HSP27 content in the hippocampus after 3 days. Immunohistochemical localization of HSP27 was found in glial cells in all subregions of the hippocampus, whereas HSP70 immunostaining was seen only in CA1 pyramidal neurons. HSP27 content in the hippocampus decreased 2 h after 6 min of ischemia. HSP27 content progressively increased in the unpreconditioned hippocampus after 1 and 3 days, but returned to preischemic levels in the preconditioned hippocampus. HSP27 and HSP70 immunostaining was seen in CA1 pyramidal neurons after 1 day both with and without preconditioning. After 3 and 7 days, an intense HSP27 staining was observed in reactive glial cells in the CA1 without preconditioning, whereas the staining decreased in the preconditioned hippocampus. HSP70 staining was seen only in neurons at these time points. We observed no significant changes in HSP27 content in the cerebral cortex although neurons in the third and fifth layers were immunostained after 1 and 3 days. We observed no alterations in αB crystallin content after ischemia both in the hippocampus and the cortex. The present study demonstrated that cerebral ischemia induces HSP27 expression but not αB crystallin. Both HSP27 and HSP70 induction had a good temporal correlation with the induction of ischemic tolerance. However, different sites of action were suggested because the localization and cell types of HSP27 induction were quite different from those of HSP70 induction. The result suggests that it is unlikely that HSP27 is directly involved in the protection afforded by ischemic preconditioning.

Phosphorylation of the stress protein HSP27 is an early event in murine myelomonocytic leukemic cell differentiation induced by leukemia inhibitory factor/D-factor

Leukemia inhibitory factor/D-factor, a potent differentiation-inducing glycoprotein for murine myelomonocytic leukemic M1 cells, rapidly stimulated the phosphorylation of a 27 kDa protein with an isoelectric point of 5.6 in a LIF-sensitive M1-T22 cell line but not in a LIF-resistant M1-D(−) cell line. The increase in phosphorylation was detectable 5 min after LIF treatment and was maximal at 10 min. Heat shock treatment at 44.5 °C for 30 min also induced the phosphorylation of the same 27 kDa protein. Although this 27 kDa protein did not become labeled with [ 35S]-methionine, metabolic labeling experiments using [ 35S]-cysteine or [ 3H]-leucine clearly demonstrated that the synthesis of this protein was enhanced after heat shock. These results suggest that the phosphorylated 27 kDa protein is a low molecular weight stress protein and that the protein may play a role at an early stage in the LIF signaling pathway probably linked to macrophagic differentiation.

Decreased heat- and tumor necrosis factor-mediated hsp28 phosphorylation in thermotolerant Hela cells

Heat shock or tumor necrosis factor rapidly stimulated the phosphorylation of the mammalian low molecular weight stress protein hsp28. We have found that both phenomena are greatly decreased in cells which are made tolerant to heat. This observation correlated with a better survival of thermotolerant cells exposed to either heat or TNF treatment. The results suggest that the phosphorylation of hsp28 may be linked to the resistance of the cells to the deleterious effects induced by either heat or a mediator of inflammation such as TNF.

Both near ultraviolet radiation and the oxidizing agent hydrogen peroxide induce a 32-kDa stress protein in normal human skin fibroblasts.

We have analyzed the pattern of protein synthesis in solar near ultraviolet (334 nm, 365 nm) and near visible (405 nm) irradiated normal human skin fibroblasts. Two hours after irradiation we find that one major stress protein of approximately 32 kDa is induced in irradiated cells. This protein is not induced by ultraviolet radiation at wavelengths shorter than 334 nm and is not inducible by heat shock treatment of these cells. Although sodium arsenite, diamide, and menadione all induced a 32-kDa protein, they also induced the major heat shock proteins. In contrast, the oxidizing agent, hydrogen peroxide, induced the low molecular weight stress protein without causing induction of the major heat shock proteins. A comparison of the 32-kDa proteins induced by sodium arsenite, H2O2, and solar near ultraviolet radiation using chemical peptide mapping shows that they are closely related. These results imply that the pathways for induction of the heat shock response and the 32-kDa protein are not identical and suggest that, at least in the case of radiation and treatment with H2O2, the 32-kDa protein might be induced in response to cellular oxidative stress. This conclusion is supported by the observation that depletion of endogenous cellular glutathione prior to solar near ultraviolet irradiation lowers the fluence threshold for induction of the 32-kDa stress protein.