PubMed Health⌕ Search

Biomedical subjects

M Gaestel

Publications and source records attributed to M Gaestel.

At least 37 records · Page 2Linked to original sources
PubMed1999

The dynamics of Hsp25 quaternary structure. Structure and function of different oligomeric species.

Small heat shock proteins (sHsps), including alpha-crystallin, represent a conserved and ubiquitous family of proteins. They form large oligomers, ranging in size from 140 to more than 800 kDa, which seem to be important for the interaction with non-native proteins as molecular chaperones. Here we analyzed the stability and oligomeric structure of murine Hsp25 and its correlation with function. Upon unfolding, the tertiary and quaternary structure of Hsp25 is rapidly lost, whereas the secondary structure remains remarkably stable. Unfolding is completely reversible, leading to native hexadecameric structures. These oligomers are in a concentration-dependent equilibrium with tetramers and dimers, indicating that tetramers assembled from dimers represent the basic building blocks of Hsp25 oligomers. At high temperatures, the Hsp25 complexes increase in molecular mass, consistent with the appearance of "heat shock granules" in vivo after heat treatment. This high molecular mass "heat shock form" of Hsp25 is in a slow equilibrium with hexadecameric Hsp25. Thus, it does not represent an off-pathway reaction. Interestingly, the heat shock form exhibits unchanged chaperone activity even after incubation at 80 degrees C. We conclude that Hsp25 is a dynamic tetramer of tetramers with a unique ability to refold and reassemble into its active quaternary structure after denaturation. So-called heat shock granules, which have been reported to appear in response to stress, seem to represent a novel functional species of Hsp25.

HSP27 Heat-Shock Proteins↗
PubMed1999

MAPKAP kinase 2 phosphorylates serum response factor in vitro and in vivo.

Several growth factor- and calcium-regulated kinases such as pp90(rsk) or CaM kinase IV can phosphorylate the transcription factor serum response factor (SRF) at serine 103 (Ser-103). However, it is unknown whether stress-regulated kinases can also phosphorylate SRF. We show that treatment of cells with anisomycin, arsenite, sodium fluoride, or tetrafluoroaluminate induces phosphorylation of SRF at Ser-103 in both HeLa and NIH3T3 cells. This phosphorylation is dependent on the kinase p38/SAPK2 and correlates with the activation of MAPKAP kinase 2 (MK2). MK2 phosphorylates SRF in vitro at Ser-103 with similar efficiency as the small heat shock protein Hsp25 and significantly better than CREB. Comparison of wild type murine fibroblasts with those derived from MK2-deficient mice (Mk(-/-)) reveals MK2 as the major SRF kinase induced by arsenite. These results demonstrate that SRF is targeted by several signal transduction pathways within cells and establishes SRF as a nuclear target for MAPKAP kinase 2.

3T3 Cells↗
PubMed1999

MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis.

MAPKAP kinase 2 (MK2) is one of several kinases that are regulated through direct phosphorylation by p38 MAP kinase. By introducing a targeted mutation into the mouse MK2 gene, we have determined the physiological function of MK2 in vivo. Mice that lack MK2 show increased stress resistance and survive LPS-induced endotoxic shock. This is due to a reduction of approximately 90% in the production of tumor necrosis factor-alpha (TNF-alpha) and not to a change in signalling from the TNF receptor. The level and stability of TNF-alpha mRNA is not reduced and TNF-alpha secretion is not affected. We conclude that MK2 is an essential component in the inflammatory response which regulates biosynthesis of TNF-alpha at a post-transcriptional level.

Animals↗
PubMed1999

Preoperative treatment of rectal cancer with radiation, chemotherapy and hyperthermia: analysis of treatment efficacy and heat-shock response.

Preoperative treatment of locally advanced rectal cancer with radiation, chemotherapy and hyperthermia is analyzed with regard to heat-shock response. In 23 patients with locally advanced rectal cancer (uT3/uT4), hyperthermia was administered in combination with radiotherapy and chemotherapy. In parallel, the effect of the treatment on levels of the heat-shock proteins HSP27 and inducible HSP70 in tumors and surrounding tissues was investigated by Western blotting. The patients' sera were also examined for autoantibodies against HSPs. HSP27 and inducible HSP70 were detected in most rectal tumors and surrounding tissues before and after treatment. HSP27 and inducible HSP70 levels had changed in 10 tumors after treatment. However, prior to treatment, there existed an unexpected diversity in HSP levels in the tumors and surrounding tissue. Hyperthermia doses in cumulative minutes for which 90% of the tumor is above the reference temperature (cum min T90 > or = 15 min) led to increased survival and response compared to that of a control group of patients treated without or with low-dose hyperthermia (cum min T90 < 15 min). However, there was no correlation to different expression of the HSPs. Hyperthermia as used in this setting does not lead to any sustained expression of HSPs in either the tumor or the surrounding tissue.

Antibodies↗
PubMed1998

Thiol/disulfide exchange between small heat shock protein 25 and glutathione.

Murine small heat shock protein 25 (Hsp25) carries a single Cys-residue at position 141 of its amino acid sequence. In glutathione redox buffers, Hsp25 equilibrates between reduced protein (PSH), mixed disulfide (PSSG) and protein dimer (PSSP) forms. At highly oxidative conditions, native Hsp25 predominantly forms PSSP while denatured Hsp25 forms PSSG. Conversion of PSSP to PSSG correlates with urea and temperature denaturation of tertiary and/or quaternary structure of Hsp25. At pH 7.5, 25 degreesC, the second-order rate constant for the formation of PSSP in the reaction of native PSH with GSSG is 20.1+/-1.4 M-1 min-1. This is approximately 3-fold lower than the reaction velocity of GSSG with a typical, unhindered thiol of pKa 8.6. At redox equilibrium, the fractions of PSSP, PSSG, and PSH depend on the concentration of GSH and less on the ratio [GSH]/[GSSG] (R). At a constant R, the fractions of PSSG and PSH species depend similarly on GSH concentration, being approximately equal in glutathione redox buffers with low R. It is concluded that in oligomeric complexes, Hsp25 subunits in vitro form stable dimers, in which the reacting -SH groups are in a proximity to form intersubunit disulfide bonds. Within a reaction of one of these -SH groups with GSSG, steric hindrances and electrostatic repulsion complicate penetration of another reduced or oxidized glutathione molecule to the reaction site.

Buffers↗
PubMed1998

Evidence for a hsp25-specific mechanism involved in transcriptional activation by heat shock.

Transcriptional stimulation of heat shock genes is generally due to the activation of heat shock transcription factor (HSF) 1. We demonstrate that in the murine leucemic cell line, P388, trimerization of HSF1, transcriptional activation of the hsp70 gene, and expression of Hsp70 are achieved as a result of heat shock. In contrast, the small heat shock proteins Hsp25 and alpha B-crystallin are not expressed in these cells and cannot be induced upon heat shock. Furthermore, no hsp25 transcript can be detected, indicating that there is a defect in the hsp25 gene or a block in its expression. Comparison of the hsp25 gene structure between P388 and Hsp25-expressing Ehrlich ascites tumor (EAT) cells by Southern blot analysis revealed no differences in the structural organization of the gene and no changes in its localization in the genome. However, sequence analysis of the hsp25 promoter region in P388 cells demonstrates minor differences. Despite these differences, the hsp25 promoter from P388 cells mediates heat shock-induced activation of a reporter gene when transfected into human HeLa cells which is comparable to that of the hsp25 promoter from EAT cells. Furthermore, the hsp25 gene isolated from EAT or P388 cells can both be expressed in HeLa cells and lead to a similar heat shock-stimulated accumulation of Hsp25. Silencing of the hsp25 and alpha B-crystallin genes in P388 cells by DNA-methylation could also be excluded since 5-azacytidine treatment does not influence expression of these genes. Interestingly, when expressed in P388 cells the hsp25 promoter from EAT cells is not activated upon heat shock, whereas the human hsp70 promoter is activated. Taken together, the data suggest cell line-specific differences in a mechanism of regulation of hsp25 transcription, which interferes with the activation of the promoter by HSF1 and which may also affect the alpha B-crystallin gene.

Animals↗
PubMed1998

Small heat-shock protein family: function in health and disease.

In summary, several important issues were discussed at the workshop. These included the importance of oligomer size for different functions of the sHsps, the highly significant observation that the sHsps can block apoptosis; and that some mechanisms of sHsps protection involve glutathione while others may be related to microfilament stability. Further evidence was presented for the chaperone functions of sHsps, and structural studies provided additional information relating sHsps' large oligomeric structure to chaperone function. Regulation of oligomer size by phosphorylation was also a prominent topic of discussion, as was the importance of the relationship of oligomer size to differing functions of Hsp27. An interesting contrast was noted between Hsp27, whose oligomer size is regulated by phosphorylation, and alpha beta-crystallin, whose large oligomer structure is unaffected by phosphorylation. This may prove to be of physiological significance, particularly in cell types that express both of these proteins. Observations of the translocation of MAPKAP kinase 2 from the nucleus to the cytoplasm in live cells suggest that the dynamics of phosphorylation of the sHsps may be more complex than previously thought. One topic that seems to have been settled is that the binding site of at least dimers of sHsps resides in the carboxyl-terminal region of the proteins. Ever-increasing numbers of studies are reporting interesting patterns of sHsp expression, phosphorylation, and subcellular localizations of the sHsps in various cells or tissues in normal and diseased states. Unfortunately, very little is understood about what these observations may mean. It can be expected, however, that as more is learned about the function and regulation of sHsps, the relationships between sHsps and cellular response to disease will become better understood. Lastly, the discovery that specific Hsp27-binding proteins may exist should open completely new avenues of investigation into the functions of sHsps. If the observations of unique subcellular localizations of sHsps in various cells types (e.g., striated muscle and Sertoli cells) can be related to expression of specific Hsp-binding proteins, considerable advances in our understanding of sHsps should result. It was abundantly clear from results presented and from ensuing discussions in the workshop that the study of sHsps is an exceedingly dynamic area of research (FIG. 2) with an ever-expanding significance for a number of other areas of current biological research.

Animals↗
PubMed1998

Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation.

UNLABELLED: To study the intracellular localization of MAPKAP kinase 2 (MK2), which carries a putative bipartite nuclear localization signal (NLS), we constructed a green fluorescent protein-MAPKAP kinase 2 fusion protein (GFP-MK2). In transfected cells, this protein is located predominantly in the nucleus; unexpectedly, upon stress, it rapidly translocates to the cytoplasm. This translocation can be blocked by the p38 MAP kinase inhibitor SB203580, indicating its regulation by phosphorylation. Molecular mimicry of MK2 phosphorylation at T317 in GFP-MK2 led to a mutant which is located almost exclusively in the cytoplasm of the cell, whereas the mutant T317A shows no stress-induced redistribution. Since leptomycin B, which inhibits the interaction of exportin 1 with the Rev-type leucine-rich nuclear export signal (NES), blocks stress-dependent translocation of GFP-MK2, it is supposed that phosphorylation-induced export of the protein causes the translocation. We have identified the region responsible for nuclear export in MK2 which is partially overlapping with and C-terminal to the autoinhibitory motif. This region contains a cluster of hydrophobic amino acids in the characteristic spacing of a leucine-rich Rev-type NES which is necessary to direct GFP-MK2 to the cytoplasm. However, unlike the Rev-type NES, this region alone is not sufficient for nuclear export. The data obtained indicate that MK2 contains a constitutively active NLS and a stress-regulated signal for nuclear export. KEYWORDS: nuclear export/nuclear import/protein phosphorylation/signal transduction/stress response

3T3 Cells↗
PubMed1998

Analysis of the role of Hsp25 phosphorylation reveals the importance of the oligomerization state of this small heat shock protein in its protective function against TNFalpha- and hydrogen peroxide-induced cell death.

The role of murine Hsp25 phosphorylation in the protection mediated by this protein against TNFalpha- or H2O2-mediated cytotoxicity was investigated in L929 cell lines expressing wild type (wt-) or nonphosphorylatable (mt-) Hsp25. We show that mt-Hsp25, in which the phosphorylation sites, serines 15 and 86, were replaced by alanines, is still efficient in decreasing intracellular reactive oxygen species levels and in raising glutathione cellular content, leading the protective activity of mt-Hsp25 against oxidative stress to be identical to that of wt-Hsp25. To independently investigate the role of Hsp25 phosphorylation, we blocked TNFalpha-induced phosphorylation of wt-Hsp25 using SB203580, a specific inhibitor of the P38 MAP kinase. This treatment did not abolish the protective activity of Hsp25 against TNFalpha. The pattern of Hsp25 oligomerization was also analyzed, showing mt-Hsp25 to constitutively display large native sizes, as does wt-Hsp25 after TNFalpha treatment in the presence of SB203580. Our results, therefore, are consistent with the possibility that the hyperaggregated form of Hsp25 is responsible for the protective activity against oxidative stress and that the phosphorylation of serines 15 and/or 86 by interfering with this structural reorganization, may lead to the inactivation of Hsp25 protective activity.

Animals↗
PubMed1998

Selective NMR Experiments on Macromolecules: Implementation and Analysis of QUIET-NOESY.

The QUIET-NOESY experiment (Zwahlen et al., J. Am. Chem Soc. 116, 362-368, 1994) is applied to measure the mobility of the flexible extensions in the large aggregate (800 kDa) of a small heat-shock protein. The proper choices of the experimental protocol and parameters are discussed in order to employ a simplified data analysis procedure. Further experimental verification of the proposed strategy is also presented using the cyclic peptide gramicidin S as a model compound. Under suitable conditions, the determinations based on the analysis of QUIET-NOESY data are affected to a negligible extent by the approximations that are introduced by the proposed approach. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Phosphorylation is not essential for protection of L929 cells by Hsp25 against H2O2-mediated disruption actin cytoskeleton, a protection which appears related to the redox change mediated by Hsp25.

Small stress proteins protect against the cytotoxicity mediated by oxidative stress. The relationship between Hsp25 expression and the integrity of the actin network was studied in H2O2-treated murine L929 fibrosarcoma cells overexpressing endogenous wild-type (wt-) or non-phosphorylatable mutant (mt-) Hsp25. We show here that both proteins prevented actin network disruption induced by a 1 h treatment with 400 microM H2O2. In contrast, SB203580, a p38 MAPkinase inhibitor which suppresses Hsp25 phosphorylation, abolished the protective activity conferred by both wt- and mt-Hsp25. Hence, phosphorylation does not appear essential for Hsp25 protective activity against H2O2-induced actin disruption, and SB203580-sensitive events other than Hsp25 phosphorylation may be important for actin network regulation. Since, in L929 cells, wt- or mt-Hsp25 expression modulates intracellular glutathione levels, analyses were performed which revealed a direct correlation between glutathione and the integrity of the actin network. Moreover, laser scanning confocal immunofluorescences revealed that only a small fraction of wt- or mt-Hsp25 colocalized with actin microfilaments. Taken together, our results suggest that, in L929 cells, the protection against actin network disruption is probably a consequence of the redox change mediated by Hsp25 rather than a direct effect of this stress protein towards actin.

Actins↗
PubMed1997

CREB is activated by UVC through a p38/HOG-1-dependent protein kinase.

Changes in environmental conditions such as the addition of growth factors or irradiation of cells in culture first affect immediate response genes. We have shown previously that short wavelength UV irradiation (UVC) elicits massive activation of several growth factor receptor-dependent pathways. At the level of the immediate response gene c-fos, these pathways activate the transcription factor complex serum response factor (SRF)-p62TCF which mediates part of the UV-induced transcriptional response. These studies have, however, suggested that more that one pathway is required for full UV responsiveness of c-fos. Using appropriate promoter mutations and dominant-negative cAMP response element (CRE)-binding protein (CREB), we now find that UVC-induced transcriptional activation depends also on the CRE at position -60 of the c-fos promoter and on the functionality of a CREB. Upon UV irradiation, CREB and ATF-1 are phosphorylated at serines 133 and 63, respectively, preceded by and dependent on activation of p38/RK/HOG-1 and of a p38/RK/HOG-1-dependent p108 CREB kinase. Although p90RSK1 and MAPKAP kinase 2 are also activated by UV, p90RSK1 does not, at least not decisively, participate in this signalling pathway to CREB and ATF-1 as it is not p38/RK/HOG-1 dependent, and CREB is a poor substrate for MAPKAP kinase 2 in vitro. On the basis of resistance to the growth factor receptor inhibitor suramin and of several types of cross-refractoriness experiments, the UVC-induced CREB/ATF-1 phosphorylation represents an as yet unrecognized route of UVC-induced signal transduction, independent of suramin-inhibitable growth factor receptors and different from the Erk 1,2-p62TCF pathway.

Activating Transcription Factor 1↗
PubMed1997

Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation.

Small heat shock proteins (sHsps) are a conserved and ubiquitous protein family. Their ability to convey thermoresistance suggests their participation in protecting the native conformation of proteins. However, the underlying functional principles of their protective properties and their role in concert with other chaperone families remain enigmatic. Here, we analysed the influence of Hsp25 on the inactivation and subsequent aggregation of a model protein, citrate synthase (CS), under heat shock conditions in vitro. We show that stable binding of several non-native CS molecules to one Hsp25 oligomer leads to an accumulation of CS unfolding intermediates, which are protected from irreversible aggregation. Furthermore, a number of different proteins which bind to Hsp25 can be isolated from heat-shocked extracts of cells. Under permissive folding conditions, CS can be released from Hsp25 and, in cooperation with Hsp70, an ATP-dependent chaperone, the native state can be restored. Taken together, our findings allow us to integrate sHsps functionally in the cellular chaperone system operating under heat shock conditions. The task of sHsps in this context is to efficiently trap a large number of unfolding proteins in a folding-competent state and thus create a reservoir of non-native proteins for an extended period of time, allowing refolding after restoration of physiological conditions in cooperation with other chaperones.

Animals↗
PubMed1997

p38 mitogen activated protein kinase regulates endothelial VCAM-1 expression at the post-transcriptional level.

The cytokine tumor necrosis factor (TNF) alpha was found to stimulate the p38 mitogen activated protein (MAP) kinase signalling cascade in human umbilical vein endothelial cells. TNFalpha increased the activity of the p38 substrate MAP kinase-activated-protein (MAPKAP) kinase 2 and the subsequent phosphorylation of the small heat shock protein Hsp27 about two to three fold. This stimulation was blocked almost completely by the specific p38 MAP kinase inhibitor SB203580. This inhibitor also suppressed the TNFalpha-induced surface expression of the endothelial adhesion molecule vascular cell adhesion molecule (VCAM)-1. In contrast, inhibition of p38 MAP kinase had no effect on the stimulated surface expression of the intercellular cell adhesion molecule (ICAM)-1. VCAM-1 mRNA accumulation induced by TNFalpha was not affected by SB203580, suggesting that the p38 MAP kinase signalling cascade regulates the endothelial expression of VCAM-1 at the post-transcriptional level.

Blotting, Northern↗
PubMed1997

PMA-induced activation of the p42/44ERK- and p38RK-MAP kinase cascades in HL-60 cells is PKC dependent but not essential for differentiation to the macrophage-like phenotype.

The signaling mechanisms leading to phorbol ester myristate (PMA)-induced differentiation of HL-60 cells to the macrophagelike phenotype were investigated by using different protein kinase inhibitors. The protein kinase C inhibitor Ro 31-8220 specifically blocks PMA-induced differentiation, activation of the p42/44ERK- and p38RK-MAP kinase cascades and Hsp27-phosphorylation in HL-60 cells. Because Ro 31-8220 does not inhibit activation of the MAP kinase cascades by protein kinase C (PKC)-independent signals such as epidermal growth factor (EGF), heat shock, or anisomycin in these cells, only PMA-induced activation of the MAP kinases can be downstream of PKC. The MEK1 inhibitor PD 098059 and the p38RK inhibitor SB 203580 also were used to analyze whether the PMA-induced PKC-dependent activation of MAP kinases is involved in the differentiation process. Under certain conditions, PD 098059 can completely block the PMA-induced activation of the p42ERK as monitored by immunoprecipitation kinase assay by using the substrate myelin basic protein. SB 203580 specifically inhibits activation of p38RK as judged by MAPKAP kinase 2 activity against the substrate Hsp27 and also blocks Hsp27 phosphorylation in the cells. In contrast, neither PD 098059 nor SB 203580 nor both inhibitors together prevent PMA-induced differentiation of the HL-60 cells to the macrophagelike phenotype. The results suggest the existence of a diversification of PMA-induced signaling in HL-60 cells downstream of PKC, leading to activation of MAP kinases that are not essential for differentiation and to phosphorylation of other, so far unidentified, targets responsible for differentiation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

The protein kinase inhibitor SB203580 uncouples PMA-induced differentiation of HL-60 cells from phosphorylation of Hsp27.

HL-60 cells are an attractive model for studies of human myeloid cell differentiation. Among the well-examined parameters correlated to differentiation of HL-60 cells are the expression and phosphorylation of the small heat shock protein Hsp27. Here we demonstrate that PMA treatment of HL-60 cells stimulates different MAP kinase cascades, leading to significant activation of ERK2 and p38 reactivating kinase (p38RK). Using the protein kinase inhibitor SB 203580, we specifically inhibited p38RK and, thereby, activation of its target MAP kinase-activated protein kinase 2 (MAPKAP kinase 2), which is the major enzyme responsible for small Hsp phosphorylation. As a result, PMA-induced Hsp27 phosphorylation is inhibited in SB 203580-treated HL-60 cells indicating that p38RK and MAPKAP kinase 2 are components of the PMA-induced signal transduction pathway leading to Hsp27 phosphorylation. We further demonstrate that, although PMA-induced phosphorylation is inhibited, SB 203580-treated HL-60 cells are still able to differentiate to the macrophage-like phenotype as judged by decrease in cell proliferation, induction of expression of the cell surface antigen CD11b and changes in cell morphology. These results indicate that, although correlated, Hsp27 phosphorylation is not required for HL-60 cell differentiation. However, the results do not exclude that increased Hsp27 expression is involved in HL-60 cell differentiation.

Antigens, Surface↗
PubMed1996

Hypo-osmotic cell swelling activates the p38 MAP kinase signalling cascade.

Hypo-osmotic swelling of human Intestine 407 cells leads to a significant increase of intracellular MAPKAP-kinase 2 activity and Hsp27 phosphorylation. Pre-treatment of the cells with the p38 MAP kinase inhibitor SB-203580 blocks this activation, indicating that the hypotonicity-induced activation of MAPKAP kinase 2 is, similarly to that described for hyperosmotic treatment, the result of an activated p38 MAP kinase cascade. The activation of MAPKAP kinase 2 proceeds with kinetics similar to that of one of the first physiological responses of hypo-osmotic treatment, the opening of compensatory Cl- channels. However, inhibition of the p38 MAP kinase cascade does not block the osmo-sensitive anion efflux and, vice versa, activation of p38 MAP kinase by cytokines and anisomycin does not increase the efflux. These results indicate that the p38 MAP kinase cascade is not directly involved in Cl- channel activation but instead may play a role in subsequent cellular repair processes.

Anisomycin↗