Stimulation of multiple mitogen-activated protein kinase sub-families by oxidative stress and phosphorylation of the small heat shock protein, HSP25/27, in neonatal ventricular myocytes.

We investigated the activation of three subfamilies of mitogen-activated protein kinases (MAPKs), namely the stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs), the extracellularly responsive kinases (ERKs) and p38-MAPK, by oxidative stress as exemplified by H2O2 in primary cultures of neonatal rat ventricular myocytes. The 46 and 54 kDa species of SAPKs/JNKs were activated 5- and 10-fold, respectively, by 0.1 mM H2O2 (the maximally effective concentration). Maximal activation occurred at 15-30 min, but was still detectable after 2 h. Both ERK1 and ERK2 were activated 16-fold by 0.1 mM H2O2 with a similar time course to the SAPKs/JNKs, and this was comparable with their activation by 1 microM PMA, the most powerful activator of ERKs that we have so far identified in these cells. The activation of ERKs by H2O2 was inhibited by PD98059, which inhibits the activation of MAPK (or ERK) kinases, and by the protein kinase C (PKC) inhibitor, GF109203X. ERK activation was also inhibited by down-regulation of PMA-sensitive PKC isoforms. p38-MAPK was activated by 0.1 mM H2O2 as shown by an increase in its phosphorylation. However, maximal phosphorylation (activation) was more rapid (<5 min) than for the SAPKs/JNKs or the ERKs. We studied the downstream consequences of p38-MAPK activation by examining activation of MAPK-activated protein kinase 2 (MAPKAPK2) and phosphorylation of the MAPKAPK2 substrate, the small heat shock protein HSP25/27. As with p38-MAPK, MAPKAPK2 was rapidly activated (maximal within 5 min) by 0.1 mM H2O2. This activation was abolished by 10 microM SB203580, a selective inhibitor of certain p38-MAPK isoforms. The phosphorylation of HSP25/27 rapidly followed activation of MAPKAPK2 and was also inhibited by SB203580. Phosphorylation of HSP25/27 was associated with a decrease in its aggregation state. These data indicate that oxidative stress is a powerful activator of all three MAPK subfamilies in neonatal rat ventricular myocytes. Activation of all three MAPKs has been associated with the development of the hypertrophic phenotype. However, stimulation of p38-MAPK and the consequent phosphorylation of HSP25/27 may also be important in cardioprotection.

Crystal Structures of Xanthomonas Small Heat Shock Protein Provide a Structural Basis for an Active Molecular Chaperone Oligomer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystallization and preliminary X-ray diffraction analysis of XAC1151, a small heat-shock protein from Xanthomonas axonopodis pv. citri belonging to the alpha-crystallin family

The hspA gene (XAC1151) from Xanthomonas axonopodis pv. citri encodes a protein of 158 amino acids that belongs to the small heat-shock protein ( sHSP) family of proteins. These proteins function as molecular chaperones by preventing protein aggregation. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 angstrom resolution using a synchrotron-radiation source. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 128.7, c = 55.3 angstrom. The crystal structure was solved by molecular-replacement methods. Structure refinement is in progress.

Use of the immunodominant 18-kiloDalton small heat shock protein as a serological marker for exposure to Mycobacterium ulcerans

While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is actually exposed to this disease. Immunological cross-reactivity among mycobacterial species complicates the development of a specific serological test. Among immunodominant proteins recognized by a panel of anti-M. ulcerans monoclonal antibodies, the M. ulcerans homologue of the M. leprae 18-kDa small heat shock protein (shsp) was identified. Since this shsp has no homologues in M. bovis and M. tuberculosis, we evaluated its use as a target antigen for a serological test. Anti-18-kDa shsp antibodies were frequently found in the sera of Buruli ulcer patients and of healthy household contacts but rarely found in controls from regions where the infection is not endemic. The results indicate that only a small proportion of M. ulcerans-infected individuals contract the clinical disease.

Heterologous Expression of a Plant Small Heat-Shock Protein Enhances Escherichia Coli Viability under Heat And Cold Stress Ref.: 1

A small heat-shock protein (sHSP) that shows molecular chaperone activity in vitro was recently purified from mature chestnut (Castanea sativa) cotyledons. This protein, renamed here as CsHSP17.5, belongs to cytosolic class I, as revealed by cDNA sequencing and immunoelectron microscopy. Recombinant CsHSP17.5 was overexpressed in Escherichia coli to study its possible function under stress conditions. Upon transfer from 37°C to 50°C, a temperature known to cause cell autolysis, those cells that accumulated CsHSP17.5 showed improved viability compared with control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that such a protective effect in vivo is due to the ability of recombinant sHSP to maintain soluble cytosolic proteins in their native conformation, with little substrate specificity. To test the recent hypothesis that sHSPs may be involved in protection against cold stress, we also studied the viability of recombinant cells at 4°C. Unlike the major heat-induced chaperone, GroEL/ES, the chestnut sHSP significantly enhanced cell survivability at this temperature. CsHSP17.5 thus represents an example of a HSP capable of protecting cells against both thermal extremes. Consistent with these findings, high-level induction of homologous transcripts was observed in vegetative tissues of chestnut plantlets exposed to either type of thermal stress but not salt stress

The Chloroplast Small Heat-Shock Protein Oligomer Is Not Phosphorylated and Does Not Dissociate during Heat Stress in Vivo1

Plants synthesize several classes of small (15- to 30-kD monomer) heat-shock proteins (sHSPs) in response to heat stress, including a nuclear-encoded, chloroplast-localized sHSP (HSP21). Cytosolic sHSPs exist as large oligomers (approximately 200–800 kD) composed solely or primarily of sHSPs. Phosphorylation of mammalian sHSPs causes oligomer dissociation, which appears to be important for regulation of sHSP function. We examined the native structure and phosphorylation of chloroplast HSP21 to understand this protein's basic properties and to compare it with cytosolic sHSPs. The apparent size of native HSP21 complexes was > 200 kD and they did not dissociate during heat stress. We found no evidence that HSP21 or the plant cytosolic sHSPs are phosphorylated in vivo. A partial HSP21 complex purified from heat-stressed pea (Pisum sativum L.) leaves contained no proteins other than HSP21. Mature recombinant pea and Arabidopsis thaliana HSP21 were expressed in Escherichia coli, and purified recombinant Arabidopsis HSP21 assembled into homo-oligomeric complexes with the same apparent molecular mass as HSP21 complexes observed in heat-stressed leaf tissue. We propose that the native, functional form of chloroplast HSP21 is a large, oligomeric complex containing nine or more HSP21 subunits, and that plant sHSPs are not regulated by phosphorylation-induced dissociation.

Characterisation of amyloid fibril formation by small heat-shock chaperone proteins human alpha A-, alpha beta- and R120G alpha B-Crystallins

alpha B-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alpha B-crystallin in detail, and also that of alpha A-crystallin and the disease-related mutant R120G (alpha B-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alpha B-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. H-1 NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alpha B-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils. (C) 2007 Elsevier Ltd. All rights reserved.

Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.

Anti-60kDa heat shock protein antibodies in fetal serum : a biomarker for unexplained small for gestational age fetuses

Rapport de synthèseObjectifsLe retard de croissance intrautérin (RCIU) est un problème affectant 10% des grossesses et est associé à une morbidité périnatale importante. Dans environ 80% des cas, une étiologie ou un facteur de risque majeur peuvent être identifiés. Mais près de 20% des cas sont considérés comme inexpliqués. La heat shock protéine 60kDa (HSP60) est une protéine fortement immunogène dont la synthèse est considérablement augmentée lors de conditions non- physiologiques. Les HSP60 humaines et bactériennes partagent un haut degré d'homologie de séquence ce qui peut engendrer une maladie auto-immune à la suite d'une infection bactérienne. Nous avons supposé que les RCIU inexpliqués pourraient être la conséquence d'une sensibilisation à l'HSP60 humaine.MéthodesLes RCIU inexpliqués ont été identifiés par mesure échographique avec un doppler normal, sans anomalies décelables chez la mère ou le foetus. Les sera foetaux ont été obtenus par cordocentèse, effectuée lors d'analyse du caryotype en cas de RCIU inexpliqué (groupe d'étude) ou pour le dépistage d'une incompatibilité Rhésus (groupe témoin). Ils ont été testés pour l'antigène HSP60 et les IgG et IgM anti-HSP60 par ELISA ainsi que pour d'autres paramètres immunitaires et hématologiques.RésultatsLes paramètres maternels sont similaires entre les 12 cas du groupe d'étude et les 23 cas du groupe contrôle. L'âge gestationnel moyen lors de la cordocentèse est de 29 semaines. Les IgM anti-HSP60 sont détectés dans 12 cas d'étude (100%) mais dans aucun cas contrôle (p <0,00017), les IgG anti-HSP60 dans 7 cas d'étude (58%) et un seul dans le groupe contrôle (p <0,001). Trois des quatre cas avec les taux d'IgM les plus élevés sont décédés. Il n'y a pas de différences entre les deux groupes quant aux taux d'antigène HSP60 ou d'autres marqueurs immunologiques ou hématologiques.ConclusionLes foetus avec un RCIU inexpliqué expriment un taux élevé d'anticorps IgM et IgG contre l'HSP60 humaine et le taux d'IgM est un facteur prédictif de la mortalité foetale. La détection de ces anticorps indique qu'une perturbation placentaire et une réaction auto-immune foetale liée à l'HSP60 sont associées à ce retard de développement chez le foetus.

Anti-60-kDa heat shock protein antibodies in fetal serum: a biomarker for unexplained small for gestational age fetuses.

Introduction: Small for gestational age (SGA) is an important problem affecting 10% of pregnancies and is associated with significant perinatal morbidity. In about 80% of cases, a probable etiology or a major risk factor can be identified. But almost 20% of SGA cases are considered unexplained. The 60-kDa heat shock protein (HSP60) is a highly immunogenic protein whose synthesis is greatly upregulated under nonphysiological conditions. Bacterial and human HSP60 share a high degree of sequence homology, and immunity to conserved epitopes may result in development of autoimmunity following a bacterial infection. We hypothesized that unexplained SGA could be the consequence of immune sensitization to human HSP60. Methods: Unexplained SGA fetuses were identified by ultrasound biometry with normal Doppler velocimetry and with no detectable maternal or fetal abnormalities. Fetal sera were obtained by cordocentesis performed for a karyotype analysis in cases of unexplained SGA (study group) or for screening of Rhesus incompatibility (control group). Fetal sera were tested for HSP60 antigen and for IgG and IgM anti-HSP60 by ELISA as well as for other immune and hematological parameters. Results: Maternal parameters were similar between the 12 study cases and the 23 control cases. The mean gestational age at cordocentesis was 29 weeks. IgM anti-HSP60 was detected in 12 cases (100%) and in no controls (p < 0.00017), while IgG anti-HSP60 was detected in 7 cases (58%) and only 1 control (p < 0.001). Three of the 4 cases with the highest IgM antibody levels died. There were no differences in fetal serum levels of HSP60 antigen or other immune and hematological markers between the two groups. Conclusion: Fetuses with unexplained SGA are positive for IgM and IgG antibody to human HSP60 and the specific IgM antibody level is predictive of fetal mortality. Detection of these antibodies indicates that a placental perturbation and a fetal autoimmune reaction to HSP60 are associated with this developmental delay.

Chloroplast small heat shock proteins: Evidence for atypical evolution of an organelle-localized protein

Knowledge of the origin and evolution of gene families is critical to our understanding of the evolution of protein function. To gain a detailed understanding of the evolution of the small heat shock proteins (sHSPs) in plants, we have examined the evolutionary history of the chloroplast (CP)-localized sHSPs. Previously, these nuclear-encoded CP proteins had been identified only from angiosperms. This study reveals the presence of the CP sHSPs in a moss, Funaria hygrometrica. Two clones for CP sHSPs were isolated from a F. hygrometrica heat shock cDNA library that represent two distinct CP sHSP genes. Our analysis of the CP sHSPs reveals unexpected evolutionary relationships and patterns of sequence conservation. Phylogenetic analysis of the CP sHSPs with other plant CP sHSPs and eukaryotic, archaeal, and bacterial sHSPs shows that the CP sHSPs are not closely related to the cyanobacterial sHSPs. Thus, they most likely evolved via gene duplication from a nuclear-encoded cytosolic sHSP and not via gene transfer from the CP endosymbiont. Previous sequence analysis had shown that all angiosperm CP sHSPs possess a methionine-rich region in the N-terminal domain. The primary sequence of this region is not highly conserved in the F. hygrometrica CP sHSPs. This lack of sequence conservation indicates that sometime in land plant evolution, after the divergence of mosses from the common ancestor of angiosperms but before the monocot–dicot divergence, there was a change in the selective constraints acting on the CP sHSPs.

The Small, Methionine-Rich Chloroplast Heat-Shock Protein Protects Photosystem II Electron Transport during Heat Stress1

Evidence suggests that the small chloroplast heat-shock protein (Hsp) is involved in plant thermotolerance but its site of action is unknown. Functional disruption of this Hsp using anti-Hsp antibodies or addition of purified Hsp to chloroplasts indicated that (a) this Hsp protects thermolabile photosystem II and, consequently, whole-chain electron transport during heat stress; and (b) this Hsp completely accounted for heat acclimation of electron transport in pre-heat-stressed plants. Therefore, this Hsp is a major adaptation to acute heat stress in plants.

Human cyclophilin 40 is a heat shock protein that exhibits altered intracellular localization following heat shock

The unactivated steroid receptors are chaperoned into a conformation that is optimal for binding hormone by a number of heat shock proteins, including Hsp90, Hsp70, Hsp40, and the immunophilin, FKBP52 (Hsp56). Together with its partner cochaperones, cyclophilin 40 (CyP40) and FKBP51, FKBP52 belongs to a distinct group of structurally related immunophilins that modulate steroid receptor function through their association with Hsp90. Due to the structural similarity between the component immunophilins, FKBP52 and cyclophilin 40, we decided to investigate whether CyP40 is also a heat shock protein. Exposure of MCF-7 breast cancer cells to elevated temperatures (42 degreesC for 3 hours) resulted in a 75-fold increase in CyP40 mRNA levels, but no corresponding increase in CyP40 protein expression, even after 7 hours of heat stress. The use of cycloheximide to inhibit protein synthesis revealed that in comparison to MCF-7 cells cultured at 37 degreesC, those exposed to heat stress (42 degreesC for 3 hours) displayed an elevated rate of degradation of both CyP40 and FKBP52 proteins. Concomitantly, the half-life of the CyP40 protein was reduced from more than 24 hours to just over 8 hours following heat shock. As no alteration in CyP40 protein levels occurred in cells exposed to heat shock, an elevated rate of degradation would imply that CyP40 protein was synthesized at an increased rate. hence the designation of human CyP40 as a heat shock protein. Application of heat stress elicited a marked redistribution of CyP40 protein in MCF-7 cells from a predominantly nucleolar localization, with some nuclear and cytoplasmic staining, to a pattern characterized by a pronounced nuclear accumulation of CyP40, with no distinguishable nucleolar staining. This increase in nuclear CyP40 possibly resulted from a redistribution of cytoplasmic and nucleolar CyP40, as no net increase in CyP40 expression levels occurred in response to stress. Exposure of MCF-7 cells to actinomycin D for 4 hours resulted in the translocation of the nucleolar marker protein, B23, from the nucleolus, with only a small reduction in nucleolar CyP40 levels. Under normal growth conditions, MCF-7 cells exhibited an apparent colocalization of CyP40 and FKBP52 within the nucleolus.

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Background: Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods: Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results: Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion: 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs. © 2009 Ishikawa et al; licensee BioMed Central Ltd.

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Abstract Background Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs.