Heat Shock Stress Response Research Articles

An overview of molecular stress response mechanisms in Escherichia coli contributing to survival of Shiga toxin-producing Escherichia coli during raw milk cheese production.

The ability of foodborne pathogens to survive in certain foods mainly depends on stress response mechanisms. Insight into molecular properties enabling pathogenic bacteria to survive in food is valuable for improvement of the control of pathogens during food processing. Raw milk cheeses are a potential source for human infections with Shiga toxin-producing Escherichia coli (STEC). In this review, we focused on the stress response mechanisms important for allowing STEC to survive raw milk cheese production processes. The major components and regulation pathways for general, acid, osmotic, and heat shock stress responses in E. coli and the implications of these responses for the survival of STEC in raw milk cheeses are discussed.

CGGBP1 regulates cell cycle in cancer cells

BackgroundCGGBP1 is a CGG-triplet repeat binding protein, which affects transcription from CGG-triplet-rich promoters such as the FMR1 gene and the ribosomal RNA gene clusters. Earlier, we reported some previously unknown functions of CGGBP1 in gene expression during heat shock stress response. Recently we had found CGGBP1 to be a cell cycle regulatory midbody protein required for normal cytokinetic abscission in normal human fibroblasts, which have all the cell cycle regulatory mechanisms intact.ResultsIn this study we explored the role of CGGBP1 in the cell cycle in various cancer cell lines. CGGBP1 depletion by RNA interference in tumor-derived cells caused an increase in the cell population at G0/G1 phase and reduced the number of cells in the S phase. CGGBP1 depletion also increased the expression of cell cycle regulatory genes CDKN1A and GAS1, associated with reductions in histone H3 lysine 9 trimethylation in their promoters. By combining RNA interference and genetic mutations, we found that the role of CGGBP1 in cell cycle involves multiple mechanisms, as single deficiencies of CDKN1A, GAS1 as well as TP53, INK4A or ARF failed to rescue the G0/G1 arrest caused by CGGBP1 depletion.ConclusionsOur results show that CGGBP1 expression is important for cell cycle progression through multiple parallel mechanisms including the regulation of CDKN1A and GAS1 levels.

DCPIP (2,6-dichlorophenolindophenol) as a genotype-directed redox chemotherapeutic targeting NQO1*2 breast carcinoma

Accumulative experimental evidence suggests feasibility of chemotherapeutic intervention targeting human cancer cells by pharmacological modulation of cellular oxidative stress. Current efforts aim at personalization of redox chemotherapy through identification of predictive tumour genotypes and redox biomarkers. Based on earlier research demonstrating that anti-melanoma activity of the pro-oxidant 2,6-dichlorophenolindophenol (DCPIP) is antagonized by cellular NAD(P)H:quinone oxidoreductase (NQO1) expression, this study tested DCPIP as a genotype-directed redox chemotherapeutic targeting homozygous NQO1*2 breast carcinoma, a common missense genotype [rs1800566 polymorphism; NP_000894.1:p.Pro187Ser] encoding a functionally impaired NQO1 protein. In a panel of cultured breast carcinoma cell lines and NQO1-transfectants with differential NQO1 expression levels, homozygous NQO1*2 MDA-MB231 cells were hypersensitive to DCPIP-induced caspase-independent cell death that occurred after early onset of oxidative stress with glutathione depletion and loss of genomic integrity. Array analysis revealed upregulated expression of oxidative (GSTM3, HMOX1, EGR1), heat shock (HSPA6, HSPA1A, CRYAB) and genotoxic stress response (GADD45A, CDKN1A) genes confirmed by immunoblot detection of HO-1, Hsp70, Hsp70B’, p21 and phospho-p53 (Ser15). In a murine xenograft model of human homozygous NQO1*2-breast carcinoma, systemic administration of DCPIP displayed significant anti-tumour activity, suggesting feasibility of redox chemotherapeutic intervention targeting the NQO1*2 genotype.

Expression of Heat Shock and Other Stress Response Proteins in Ticks and Cultured Tick Cells in Response toAnaplasmaspp. Infection and Heat Shock

Ticks are ectoparasites of animals and humans that serve as vectors of Anaplasma and other pathogens that affect humans and animals worldwide. Ticks and the pathogens that they transmit have coevolved molecular interactions involving genetic traits of both the tick and the pathogen that mediate their development and survival. In this paper, the expression of heat shock proteins (HSPs) and other stress response proteins (SRPs) was characterized in ticks and cultured tick cells by proteomics and transcriptomics analyses in response to Anaplasma spp. infection and heat shock. The results of these studies demonstrated that the stress response was activated in ticks and cultured tick cells after Anaplasma spp. infection and heat shock. However, in the natural vector-pathogen relationship, HSPs and other SRPs were not strongly activated, which likely resulted from tick-pathogen coevolution. These results also demonstrated pathogen- and tick-specific differences in the expression of HSPs and other SRPs in ticks and cultured tick cells infected with Anaplasma spp. and suggested the existence of post-transcriptional mechanisms induced by Anaplasma spp. to control tick response to infection. These results illustrated the complexity of the stress response in ticks and suggested a function for the HSPs and other SRPs during Anaplasma spp. infection.

Direct interaction between hnRNP‐M and CDC5L/PLRG1 proteins affects alternative splice site choice

Heterogeneous nuclear ribonucleoprotein-M (hnRNP-M) is an abundant nuclear protein that binds to pre-mRNA and is a component of the spliceosome complex. A direct interaction was detected in vivo between hnRNP-M and the human spliceosome proteins cell division cycle 5-like (CDC5L) and pleiotropic regulator 1 (PLRG1) that was inhibited during the heat-shock stress response. A central region in hnRNP-M is required for interaction with CDC5L/PLRG1. hnRNP-M affects both 5' and 3' alternative splice site choices, and an hnRNP-M mutant lacking the CDC5L/PLRG1 interaction domain is unable to modulate alternative splicing of an adeno-E1A mini-gene substrate.

Virus–host cell interactions in vaccine production cell lines infected with different human influenza A virus variants: A proteomic approach

A proteomic approach was used to investigate the dynamic cellular host cell response induced by influenza virus infection in two different vaccine production cell lines, MDCK and Vero. For identification of proteins possibly involved in global host cell response mechanisms and virus-host cell interactions in these producer cell lines, quantitative 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were performed. In particular, host cell proteome alterations caused by infection with influenza virus variants showing differences in replication characteristics in MDCK cells were compared. Moreover, the host cell response to virus infection in Vero cells with respect to their deficiency in interferon (IFN) production and the need for virus adaptation to optimize productivity of this cell line were analyzed. Several proteins with differential abundance profiles were identified and Western blot analysis was performed for further confirmation of selected proteins. IFN-induced signal transduction, cytoskeleton remodeling, vesicle transport and proteolysis were the principal pathways that changed in infected MDCK cells. In Vero cells alterations of cell interactions, heat shock and oxidative stress response were detected. The findings will improve understanding of host cell response mechanisms during influenza vaccine production and viral strategies to evade these responses and to replicate efficiently in different cell lines.

Abstract PL5-1: Targeting the nexus of heat shock and metabolic stress responses in cancer

Abstract Cancer results from genetic and epigenetic alterations that deregulate cell growth, checkpoints and threshold for cell death. The growing cancer mass exhibits metabolic and protein-misfolding/denaturing stress due to hypoxia, deprivation of nutrients, acidosis and increased reactive oxygen species (ROS). Ubiquitin/proteasome system clears the mass of misfolded proteins, which are continuously generated through mutations and various denaturing stresses in cancer cells. Heat shock proteins (hsp) 90 and hsp70 induced by heat shock factor-1 (HSF-1) are at the center of an ATP-dependent chaperone network that promotes protein refolding, prevents protein aggregation, as well as targets misfolded proteins for destruction through the proteasome. Indeed, inhibition of hsp90 and hsp70 not only promotes degradation of hsp90 client oncoproteins, e.g., HER-2, c-RAF, AKT, FLT-3, BCR-ABL, androgen and estrogen receptors and HIF-1, but also induces the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. Importantly, following exposure to chemical carcinogens and the stress of oncogene mutations, mice lacking HSF-1 exhibit a much lower incidence of tumors and the tumor burden. In cancer cells, the metabolic stress and higher levels of misfolded and ubiquitylated proteins also promote autophagy. The predominantly cytosolic, class II histone deacetylase 6 (HDAC6) has been recently recognized as the cellular stress surveillance factor. This activity of HDAC6 resides in its ability to bind and deacetylate hsp90 and hsp70, as well as on its high affinity for binding misfolded and polyubiquitylated proteins through its C-terminal BUZ domain. Following its association with the misfolded and polyubiquitylated proteins through its BUZ domain, HDAC6 dissociates from the complex that also includes hsp90 and HSF-1, releasing HSF-1 from hsp90 to translocate to nucleus and induce heat shock proteins. HDAC6 also shuttles the misfolded and polyubiquitylated proteins into the protective cellular organelle, the perinuclear aggresome, where these proteins are degraded and recycled. To carry out all of these functions, the catalytic activity of HDAC6 is necessary. Treatment of transformed cells with the pan-HDAC inhibitors, which also inhibit HDAC6, induces hyeracetylation and abrogation of chaperone function of hsp90 and hsp70, inhibits the aggresome formation, as well as induces ROS and apoptosis. Hyperacetylation of hsp70 has also been recently found to promote the initiation of the phagaphore formation through stabilization of the complex between Vps34, Beclin-1 and hsp70. These novel insights into the roles of HDAC6 and hsp90/hsp/70 chaperone network in the non-oncogenic stress phenotype have also highlighted potential combinations as novel therapies. These include combined therapy with pan-HDAC or hsp90 inhibitors and/or proteasome or autophagy inhibitors. Overall, how these novel therapies should be integrated with other targeted agents in the treatment of advanced cancer. Citation Information: Clin Cancer Res 2010;16(7 Suppl):PL5-1

Comprehensive analysis of rice DREB2-type genes that encode transcription factors involved in the expression of abiotic stress-responsive genes

DREB2s (dehydration-responsive element-binding protein 2s) are transcription factors that interact with a cis-acting DRE (dehydration-responsive element)/CRT (C-repeat) sequence and activate the expression of downstream genes involved in water- and heat-shock stress responses and tolerance in Arabidopsis thaliana. In this study, we performed a comprehensive analysis of all five DREB2-type genes in rice (OsDREB2 s: OsDREB2A, OsDREB2B, OsDREB2C, OsDREB2E and OsABI4) to determine which of them contribute to plant stress responses. We analysed the expression patterns of these genes under abiotic stress conditions, and we examined the subcellular localisation and transcriptional activation activity of their translational products in protoplasts. Only OsDREB2A and OsDREB2B showed abiotic stress-inducible gene expression. In addition, OsDREB2B showed nuclear specific localisation and the highest transactivation activity. OsDREB2B has functional and non-functional forms of its transcript similar to its orthologues in the grass family, and the functional form of its transcript was markedly increased during stress conditions. We analysed the splicing mechanism of OsDREB2B with transgenic rice that express the non-functional transcript and we found that the non-functional form is not a precursor of the functional form; thus, stress-inducible alternative splicing of pre-mRNA is an important mechanism for the regulation of OsDREB2B. Transgenic Arabidopsis plants overexpressing OsDREB2B showed enhanced expression of DREB2A target genes and improved drought and heat-shock stress tolerance. These results suggest that OsDREB2B is a key gene that encodes a stress-inducible DREB2-type transcription factor that functions in stress-responsive gene expression in rice.

Inhibitory Effects of Low-Energy Pulsed Ultrasonic Stimulation on Cell Surface Protein Antigen C through Heat Shock Proteins GroEL and DnaK inStreptococcus mutans

This study concerns the use of low-energy pulsed ultrasound as nondestructive photodynamic antimicrobial therapy for controlling dental plaque. We examined the antibacterial and bactericidal effects of low-energy pulsed ultrasound on mutans streptococci and its inhibitory effects on bacterial cell adhesion of Streptococcus mutans. The results indicated weak antibacterial and bactericidal effects. However, ultrasonic stimulation for less than 20 min markedly decreased bacterial cell adhesion. To analyze the mechanism underlying the inhibitory effect, we examined cell surface protein antigen C (PAc) and glucosyltransferase I (GTF-I) expression in S. mutans. The levels of PAc gene and protein expression were markedly decreased by ultrasonic stimulation for 20 min. However, no change in GTF-I expression was observed. The expression of stress response heat shock proteins GroEL and DnaK was also examined. GroEL and DnaK levels were significantly decreased by ultrasonic stimulation, and the expression of the PAc protein was also diminished upon the addition of GroEL or DnaK inhibitors without ultrasonic stimulation. These observations suggest that the expression of the PAc protein in S. mutans may be dependent on heat shock proteins. Thus, low-energy pulsed ultrasound decreases bacterial adhesion by the inhibitory effect on the PAc protein and heat shock protein expression and may be useful as photodynamic antimicrobial chemotherapy in controlling dental plaque.

Transcriptional induction of GRP78/BiP by histone deacetylase inhibitors and resistance to histone deacetylase inhibitor–induced apoptosis

Histone deacetylase (HDAC) inhibitors are emerging as effective therapies in the treatment of cancer, and the role of HDACs in the regulation of promoters is rapidly expanding. GRP78/BiP is a stress inducible endoplasmic reticulum (ER) chaperone with antiapoptotic properties. We present here the mechanism for repression of the Grp78 promoter by HDAC1. Our studies reveal that HDAC inhibitors specifically induce GRP78, and the induction level is amplified by ER stress. Through mutational analysis, we have identified the minimal Grp78 promoter and specific elements responsible for HDAC-mediated repression. We show the involvement of HDAC1 in the negative regulation of the Grp78 promoter not only by its induction in the presence of the HDAC inhibitors trichostatin A and MS-275 but also by exogenous overexpression and small interfering RNA knockdown of specific HDACs. We present the results of chromatin immunoprecipitation analysis that reveals the binding of HDAC1 to the Grp78 promoter before, but not after, ER stress. Furthermore, overexpression of GRP78 confers resistance to HDAC inhibitor-induced apoptosis in cancer cells, and conversely, suppression of GRP78 sensitizes them to HDAC inhibitors. These results define HDAC inhibitors as new agents that up-regulate GRP78 without concomitantly inducing the ER or heat shock stress response, and suppression of GRP78 in tumors may provide a novel, adjunctive option to enhance anticancer therapies that use these compounds.

CoREST Represses the Heat Shock Response Mediated by HSF1

The stress response in cells involves a rapid and transient transcriptional activation of stress genes. It has been shown that Hsp70 limits its own transcriptional activation functioning as a corepressor of heat shock factor 1 (HSF1) during the attenuation of the stress response. Here we show that the transcriptional corepressor CoREST interacts with Hsp70. Through this interaction, CoREST represses both HSF1-dependent and heat shock-dependent transcriptional activation of the hsp70 promoter. In cells expressing short hairpin RNAs directed against CoREST, Hsp70 cannot repress HSF1-dependent transcription. A reduction of CoREST levels also provoked a significant increase of Hsp70 protein levels and an increase of HSF1-dependent transactivation of hsp70 promoter. Via chromatin immunoprecipitation assays we show that CoREST is bound to the hsp70 gene promoter under basal conditions and that its binding increases during heat shock response. In conclusion, we demonstrated that CoREST is a key regulator of the heat shock stress response.

Stress-Dependent Dynamics of Global Chromatin Remodeling in Yeast: Dual Role for SWI/SNF in the Heat Shock Stress Response

Although chromatin structure is known to affect transcriptional activity, it is not clear how broadly patterns of changes in histone modifications and nucleosome occupancy affect the dynamic regulation of transcription in response to perturbations. The identity and role of chromatin remodelers that mediate some of these changes are also unclear. Here, we performed temporal genome-wide analyses of gene expression, nucleosome occupancy, and histone H4 acetylation during the response of yeast (Saccharomyces cerevisiae) to different stresses and report several findings. First, a large class of predominantly ribosomal protein genes, whose transcription was repressed during both heat shock and stationary phase, showed strikingly contrasting histone acetylation patterns. Second, the SWI/SNF complex was required for normal activation as well as repression of genes during heat shock, and loss of SWI/SNF delayed chromatin remodeling at the promoters of activated genes. Third, Snf2 was recruited to ribosomal protein genes and Hsf1 target genes, and its occupancy of this large set of genes was altered during heat shock. Our results suggest a broad and direct dual role for SWI/SNF in chromatin remodeling, during heat shock activation as well as repression, at promoters and coding regions.

Host responses influence on the induction of lambda prophage

Inactivation of bacteriophage lambda CI repressor leads almost exclusively to lytic development. Prophage induction can be initiated either by DNA damage or by heat treatment of a temperature-sensitive repressor. These two treatments also cause a concurrent activation of either the host SOS or heat-shock stress responses respectively. We studied the effects of these two methods of induction on the lytic pathway by monitoring the activation of different lambda promoters, and found that the lambda genetic network co-ordinates information from the host stress response networks. Our results show that the function of the CII transcriptional activator, which facilitates the lysogenic developmental pathway, is not observed following either method of induction. Mutations in the cro gene restore the CII function irrespective of the induction method. Deletion of the heat-shock protease gene ftsH can also restore CII function following heat induction but not following SOS induction. Our findings highlight the importance of the elimination of CII function during induction as a way to ensure an efficient lytic outcome. We also show that, despite the common inhibitory effect on CII function, there are significant differences in the heat- and SOS-induced pathways leading to the lytic cascade.

Distinct heat-shock element arrangements that mediate the heat shock, but not the late-embryogenesis induction of small heat-shock proteins, correlate with promoter activation in root-knot nematode feeding cells

Genes coding small heat-shock proteins (sHSPs) show distinct behaviours with respect to environmental and developmental signals. Their transcriptional regulation depends on particular combinations of heat stress cis-elements (heat-shock elements; HSEs) but many aspects regarding their regulation remain unclear. Cyst and root-knot nematodes induce, in the roots of infected plants, the differentiation of special feeding cells with high metabolic activity (syncytia and giant cells, respectively), a process accompanied by extensive gene expression changes. The Hahsp17.7G4 (G4) promoter was active in giant cells and its HSE arrangements were crucial for this activation. In the present work, we provide further basis to associate giant cell expression with the heat-shock response of this gene class, by analysing additional promoters. The Hahsp17.6G1 (G1) promoter, not induced by heat shock, was silent in giant cells, while Hahsp18.6G2 (G2), which responds to heat shock, was specifically induced in giant cells. In addition, a mutated Hahsp17.7G4 promoter version (G4MutP) with a strong heat-shock induction was also induced in giant cells. The responses of the different promoters correlated with distinct HSE configurations, which might have implications on differential trans-activation. Furthermore, the shortest giant cell and heat-shock-inducible sHSP promoter version analysed in tobacco (-83pb Hahsp17.7G4) fully maintained its expression profile in Arabidopsis. Cyst nematodes did not induce the Hahsp17.7G4 promoter, revealing additional specificity in the nematode response. These findings, together with the fact that the class I sHSP products of endogenous genes accumulated specifically in tobacco giant cells, support the idea that these nematode-induced giant cells represent a transcriptional state very similar to that produced by heat shock regarding this class of genes. The high metabolic rate of giant cells may result in unfolded proteins requiring class I sHSPs as chaperones, which might, somehow, mimic heat-shock and/or other stress responses.

Preliminary Results of CNTO 328, an Anti-Interleukin (IL)-6 Monoclonal Antibody (mAb), in Combination with Bortezomib in the Treatment of Relapsed or Refractory Multiple Myeloma.

Background: Bortezomib represents a significant advance in the treatment of relapsed/refractory myeloma, but its efficacy is limited by a number of resistance mechanisms including activation of the anti-apoptotic heat shock protein (HSP) and stress response pathways. CNTO 328 is an anti-IL-6 chimeric monoclonal antibody shown to have anti - myeloma activities in vitro. Because IL-6 signaling augments the HSP response, downregulation of IL-6 signaling may enhance bortezomib's clinical anti-myeloma activity. Pre-clinical studies demonstrated that this combination had an additive to synergistic effect in inducing apoptosis in IL-6-dependent and independent multiple myeloma cell lines (Voorhees et al., 2007, in press).Methods: In this safety lead-in cohort of a pivotal trial (clinicaltrials.gov), CNTO 328, 6 mg/kg, was administered by IV every 2 weeks in combination with bortezomib, 1.3 mg/m2, given by IV on days 1, 4, 8, and 11 every 3 weeks. Pateints received a maximum of four, 6-week treatment cycles, after which the bortezomib schedule was reduced to four, once weekly doses in a 5-week maintenance cycle. When disease progression was documented, oral dexamethasone was added at 20 mg and given on the day of, and day after each bortezomib infusion, for a maximum of two cycles. All later cycles included 20 mg dexamethasone given only on the day of bortezomib administration.Results: Preliminary results from the first 6 patients are available. Pateint characteristics included median age of 68 years; B2M > 3.0 mg/L, (n = 4 pateints); CRP > 3.0 mg/L (3); a median of 2 prior regimens (range 1 – 3), and prior bone marrow or peripheral blood stem cell transplantation in three patients. Using the EBMT response criteria, three patients achieved confirmed partial responses (PRs). Additionally, two patients had unconfirmed PRs. One patient discontinued prior to confirmation, while a follow up assessment for the other patient is pending. The median number of cycles administered was 3. Two of the patients ended treatment prematurely, 1 due to disease progression and 1 due to adverse events. The remaining 4 patients continue on study. Grade 3/4 adverse events (AEs) considered possibly related to CNTO 328 and bortezomib include neutropenia (2 patients), leukopenia (1), lymphopenia (1), and bloody diarrhea (1).Conclusion: Treatment with anti-IL-6 CNTO 328 in combination with bortezomib has been evaluated for safety and efficacy in the first 6 patients of an ongoing pivotal trial for the treatment of relapsed or refractory multiple myeloma. Initial activity observed with this combination is encouraging, and enrollment is continuing to explore its full potential.

Selenoprotein H Is a Redox-sensing High Mobility Group Family DNA-binding Protein That Up-regulates Genes Involved in Glutathione Synthesis and Phase II Detoxification

Selenoprotein H is a recently identified member of the selenoprotein family whose function is not fully known. Previous studies from our laboratory and others showed that Drosophila melanogaster selenoprotein H is essential for viability and antioxidant defense. In this study we investigated the function of human selenoprotein H in murine hippocampal HT22 cells engineered to stably overexpress the protein. After treatment of cells with L-buthionine-(S,R)-sulfoximine to deplete glutathione, selenoprotein H-overexpressing cells exhibited higher levels of total glutathione, total antioxidant capacities, and glutathione peroxidase enzymatic activity than did vector control cells. Overexpression of selenoprotein H also up-regulated the mRNA levels of endogenous selenoprotein H, glutamylcysteine synthetase heavy and light chains, and glutathione S-transferases Alpha 2, Alpha 4, and Omega 1. The amino acid sequence of selenoprotein H contains four putative nuclear localization sequences and an AT-hook motif, a small DNA-binding domain first identified in high mobility group proteins. Chromatin immunoprecipitation using a green fluorescent protein-selenoprotein H fusion revealed binding to sequences containing heat shock and/or stress response elements. Thus, selenoprotein H is a redox-responsive DNA-binding protein of the AT-hook family and functions in regulating expression levels of genes involved in de novo glutathione synthesis and phase II detoxification in response to redox status.

High-level overexpression of the Arabidopsis HsfA2 gene confers not only increased themotolerance but also salt/osmotic stress tolerance and enhanced callus growth

Heat shock transcription factors (Hsfs) are the central regulators of the heat shock (HS) stress response in all eukaryotic organisms. HsfA2 is one of the Arabidopsis class A Hsfs, and the induction of HsfA2 expression in response to HS stress is highest among all 21 Arabidopsis Hsfs. In this study, it is reported that basal and acquired thermotolerance was significantly enhanced in high-level HsfA2-overexpressed transgenic lines (El2Omega::HsfA2) in comparison with wild-type plants. By contrast, the dominant negative mutants of HsfA2 (El2Omega::HsfA2DeltaC264) plants displayed reduced thermotolerance. These results indicate that the HsfA2 gene plays a role in the HS stress response. Microarray analysis of the El2Omega::HsfA2 plants identified putative target genes, which included HS stress-inducible genes and other stress-responsive genes. Salt and osmotic stress induced HsfA2 gene expression. In fact, the El2Omega::HsfA2 plants showed enhanced tolerance to these stresses, suggesting that HsfA2 was involved in multiple stress tolerance. El2Omega::HsfA2 plants showed accelerated callus growth from root explants compared with the wild type, unlike the El2Omega::HsfA2DeltaC264 plants whose growth was delayed. These observations suggest that HsfA2 plays, in addition to its role in stress tolerance, an important role in cell proliferation.

MAPK-activated Protein Kinase-2 (MK2)-mediated Formation and Phosphorylation-regulated Dissociation of the Signal Complex Consisting of p38, MK2, Akt, and Hsp27

The p38 MAPK and heat shock protein 27 (hsp27) form a signaling complex with serine/threonine kinase Akt and MAPK-activated protein kinase-2 (MK2), which plays an important role in controlling stress-induced apoptosis and reorganizing actin cytoskeleton. However, regulation of the complex is poorly understood. In this study, the interaction between p38 and hsp27 was visualized in single living L929 cells using fluorescence resonance energy transfer technology, while their association with Akt was examined by immunoprecipitation analysis. Under normal growth conditions, p38 kinase constitutively interacts with hsp27. When cells were exposed to H(2)O(2) or stimulated by arachidonic acid, this interaction was disrupted. However, inhibition of the activation of p38 and Akt by selective inhibitors or overexpression of the kinase-dead mutant of p38 diminished such effects. Furthermore, mutation of phosphorylation sites of hsp27 renders the interaction resistant to H(2)O(2) and arachidonic acid. It was interesting to find that the interaction disappeared in the cells from MK2-knock-out mice or the cells treated with lemptomycin B that blocks export of MK2 from nucleus to cytosol. However, MK2 is not required for the association of hsp27 with Akt. This study suggests that MK2 mediates the incorporation of p38 into the pre-existing complex of hsp27 with Akt. Phosphorylation of hsp27 finally breaks the signaling complex.

Stressing the obvious? Cell stress and cell stress proteins in cardiovascular disease

It is only some forty years since the discovery of the heat shock or cell stress response and just over twenty years since the heat shock/cell stress response was linked to protein misfolding. The plethora of intracellular proteins which promote correct protein folding in the cell, variously termed molecular chaperones, heat shock proteins, or cell stress proteins, have only been identified in the last fifteen years. During this period it has also been discovered that: (i) molecular chaperones are potent immunogens with immunomodulatory activity and (ii) they can be secreted by cells and exhibit intercellular signaling actions. These various functions of molecular chaperones are increasingly being linked to the pathology of the cardiovascular system. Molecular chaperones within cells can exhibit cardioprotection if their levels are artificially elevated, suggesting that these proteins may have therapeutic activity. In contrast, there is evidence that atherogenesis may be linked to immunity to one specific molecular chaperone, Hsp60. This may offer the possibility of treating atherosclerosis by vaccination. However, there is also growing evidence that secreted molecular chaperones have pro- or anti-inflammatory actions that are relevant to cardiovascular pathology. This review brings these various strands of research together to provide an overview of the role of molecular chaperones in cardiovascular disease.

Protein Expression Profiles of Chlamydia pneumoniae in Models of Persistence versus Those of Heat Shock Stress Response

Chlamydia pneumoniae is an obligate intracellular pathogen that causes both acute and chronic human disease. Several in vitro models of chlamydial persistence have been established to mimic chlamydial persistence in vivo. We determined the expression patterns of 52 C. pneumoniae proteins, representing nine functional subgroups, from the gamma interferon (IFN-gamma) treatment (primarily tryptophan limitation) and iron limitation (IL) models of persistence compared to those following heat shock (HS) at 42 degrees C. Protein expression patterns of C. pneumoniae persistence indicates a strong stress component, as evidenced by the upregulation of proteins involved in protein folding, assembly, and modification. However, it is clearly more than just a stress response. In IFN persistence, but not IL or HS, amino acid and/or nucleotide biosynthesis proteins were found to be significantly upregulated. In contrast, proteins involved in the biosynthesis of cofactors, cellular processes, energy metabolism, transcription, and translation showed an increased in expression in only the IL model of persistence. These data represent the most extensive protein expression study of C. pneumoniae comparing the chlamydial heat shock stress response to two models of persistence and identifying the common and unique protein level responses during persistence.