Stress-inducible Heat Shock Protein Research Articles

Hsp70 and cardiac surgery: molecular chaperone and inflammatory regulator with compartmentalized effects

Open heart surgery is a unique model to study the interplay between cellular injury, regulation of inflammatory responses and tissue repair. Stress-inducible heat shock protein 70-kDa (Hsp70) provides a molecular link between these events. In addition to molecular chaperoning, Hsp70 exerts modulatory effects on endothelial cells and leukocytes involved in inflammatory networks. Hsp70 residing in the intracellular compartment is part of an inhibitory feedback loop that acts on nuclear factor kappaB (NF-kappaB). In contrast, extracellular Hsp70 is recognized by multiple germline-encoded immune receptors, e.g., Toll-like receptor (TLR) 2, TLR4, LOX-1, CD91, CD94, CCR5 and CD40. Hsp70 is thereby able to enhance chemotaxis, phagocytosis and cytolytic activity of innate immune cells and stimulate antigen-specific responses. These apparent contradictory pro- and anti-inflammatory effects of endogenous Hsp70 in the context of cardiac surgery are still not fully understood. An all-embracing model of the compartmentalized effects of endogenous Hsp70 in the orchestration of inflammatory responses in cardiac surgery is proposed.

Structural and Functional Diversities between Members of the Human HSPB, HSPH, HSPA, and DNAJ Chaperone Families

Heat shock proteins (HSPs) were originally identified as stress-responsive proteins required to deal with proteotoxic stresses. Besides being stress-protective and possible targets for delaying progression of protein folding diseases, mutations in chaperones also have been shown to cause disease (chaperonopathies). The mechanism of action of the "classical", stress-inducible HSPs in serving as molecular chaperones preventing the irreversible aggregation of stress-unfolded or disease-related misfolded proteins is beginning to emerge. However, the human genome encodes several members for each of the various HSP families that are not stress-related but contain conserved domains. Here, we have reviewed the existing literature on the various members of the human HSPB (HSP27), HSPH (HSP110), HSPA (HSP70), and DNAJ (HSP40) families. Apart from structural and functional homologies, several diversities between members and families can be found that not only point to differences in client specificity but also seem to serve differential client handling and processing. How substrate specificity and client processing is determined is far from being understood.

Autoimmunity in atherosclerosis: a protective response losing control?

Atherosclerosis is a chronic inflammatory disease characterized by accumulation of oxidized lipoproteins, increased cell death and hypertrophic degeneration of the arterial intima. The disease process is associated with local formation of modified self antigens that are targeted by both innate and adaptive immune responses. Although it remains to be firmly established it is likely that these autoimmune responses initially have a beneficial effect facilitating the removal of potentially harmful rest products from oxidized LDL and dying cells. However, studies performed on hypercholesterolaemic mice deficient in different components of the immune system uniformly suggest that the net effect of immune activation is pro-atherogenic and that atherosclerosis, at least to some extent, should be regarded as an autoimmune disease. These observations point to the possibility of developing new treatments for atherosclerosis based on modulation of immune responses against plaque antigens, an approach presently tested clinically for several other chronic inflammatory diseases with autoimmune components. Pilot studies in animals have provided promising results for both parental and oral vaccines based on oxidized LDL antigens. The time when this concept is ready for clinical testing is rapidly approaching but it will be important not to underestimate the difficulties that will be encountered in transferring the promising results from experimental animals into humans.

Heat stress-induced heat shock protein 70 expression is dependent on ERK activation in zebrafish ( Danio rerio) cells

Heat shock response is a common event that occurs in many species. Despite its evolutionary conservation, comparative studies of heat shock response have been largely unexplored. In mammals, heat shock response decreases with age through unclear mechanisms. Understanding how the age-related decline in heat shock response occurs may provide information to understanding the biology of aging. We have previously shown that heat shock response similarly declines with age in zebrafish. However, signaling pathways that regulate the heat shock response in zebrafish are unknown. In mammals there is evidence that mitogen-activated protein kinases (MAPKs) of the ERK family alter Hsp70 transcription, serving as a potential regulator of the heat shock response. We explored if heat stress-induced Hsp70 expression is altered by activation of ERK in the zebrafish Pac2 fibroblast cell line as occurs in mammalian cells. Heat stress induced both Hsp70 mRNA expression and phosphorylation of both ERK1 and ERK2 (ERK1/2) in Pac2 cells. ERK inhibitors PD98059 and U0126 blocked both heat stress-induced and plated-derived growth factor (PDGF)-induced ERK1/2 phosphorylation, and also diminished heat-induced Hsp70 expression. Pac2 cell viability was not affected by either the ERK inhibitors or heat stress. These results demonstrate that induction of Hsp70 in response to heat stress is dependent on ERK activation in Pac2 cells. This suggests that the heat shock response in zebrafish utilizes a similar signaling pathway to that of mammals and that zebrafish are a good model for comparative studies of heat shock response.

Deletion of MK2 signalling in vivo inhibits small Hsp phosphorylation but not diabetic nephropathy

It is supposed that some stress-induced heat shock proteins (Hsps) are regulated through e.g. stimulation of the p38MAPK/MK(MAPKAP)-2 signalling pathway. It has been postulated from in vitro experiments that phosphorylation of Hsp25(rodents)/Hsp27(human), the major phosphorylation substrate of MK2, is responsible for mesangial contractility and glomerular hyperfiltration in the diabetic kidney. To verify this hypothesis in vivo we studied the renal function of nondiabetic and streptozotocin (STZ)-induced, diabetic MK2(-/-) mice in comparison to wild-type (WT) control mice. Following 8 weeks of hyperglycaemia, light microscopy showed increased glomerulosclerosis and tubulointerstitial renal fibrosis in both diabetic study groups. Protein analysis demonstrated that Hsp25 phosphorylation is stimulated upon high-glucose condition but inhibited in the diabetic MK2(-/-) mice. However, we found the kidney-body weight ratio significantly increased in diabetic WT and MK2(-/-) mice. No difference regarding the increased expression of the extracellular matrix proteins and TGF-beta1 between both diabetic study groups was observed. Importantly, diabetic MK2(-/-) mice showed no protection against renal hyperfiltration in the diabetic state and the development of diabetic albuminuria. Although activation of p38MAPK has been previously shown in diabetes mellitus, our results indicate that blockade of the downstream MK2/Hsp25 signalling pathway does not interfere with the development of early diabetic nephropathy.

Forced, not voluntary, exercise effectively induces neuroprotection in stroke

Previous treadmill exercise studies showing neuroprotective effects have raised questions as to whether exercise or the stress related to it may be key etiologic factors. In this study, we examined different exercise regimens (forced and voluntary exercise) and compared them with the effect of stress-only on stroke protection. Adult male Sprague-Dawley rats (n = 65) were randomly assigned to treatment groups for 3 weeks. These groups included control, treadmill exercise, voluntary running wheel exercise, restraint, and electric shock. Levels of the stress hormone, corticosterone, were measured in the different groups using ELISA. Animals from each group were then subjected to stroke induced by a 2-h middle cerebral artery (MCA) occlusion followed by 48-h reperfusion. Infarct volume was determined in each group, while changes in gene expression of stress-induced heat shock proteins (Hsp) 27 and 70 were compared using real-time PCR between voluntary and treadmill exercise groups. The level of corticosterone was significantly higher in both stress (P < 0.05) and treadmill exercise (P < 0.05) groups, but not in the voluntary exercise group. Infarct volume was significantly reduced (P < 0.01) following stroke in rats exercised on a treadmill. However, the amelioration of damage was not duplicated in voluntary exercise, even though running distance in the voluntary exercise group was significantly (P < 0.01) longer than that of the forced exercise group (4,828 vs. 900 m). Furthermore, rats in the electric shock group displayed a significantly increased (P < 0.01) infarct volume. Expression of both Hsp 27 and Hsp 70 mRNA was significantly increased (P < 0.01) in the treadmill exercise group as compared with that in the voluntary exercise group. These results suggest that exercise with a stressful component, rather than either voluntary exercise or stress alone, is better able to reduce infarct volume. This exercise-induced neuroprotection may be attributable to up-regulation of stress-induced heat shock proteins 27 and 70.

Stress proteins in CNS inflammation

Stress proteins or heat shock proteins (HSPs) are ubiquitous cellular components that have long been known to act as molecular chaperones. By assisting proper folding and transport of proteins, and by assisting in the degradation of aberrant proteins, they play key roles in cellular metabolism. The frequent accumulation of insoluble protein aggregates during chronic neurodegenerative disorders suggests failure of HSP functions to be a common denominator among such diseases. Recent developments have clarified that functions of HSPs extend well beyond their role in protein folding and degradation alone. Stress-inducible HSPs also regulate apoptosis, antigen presentation, inflammatory signalling pathways and, intriguingly, also serve as extracellular mediators of inflammation. Several receptors have been identified for extracellular HSPs, which control inflammatory pathways similar to those activated by cytokines and chemokines. In this review, both the traditional and the exciting novel functions of HSPs are discussed, with a focus on their relevance for neurodegeneration and neuroinflammation. Recent advances in this field suggest that HSPs represent attractive novel targets as well as therapeutic entities for CNS disorders.

The Heat Shock Protein HSP70 Promotes Mouse NK Cell Activity against Tumors That Express Inducible NKG2D Ligands

The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells, in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells, which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus, HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor growth and suppression of metastatic disease.

143: Therapeutic induction of heme-oxygenase-1 in the lung using a hemoglobin-based oxygen carrier

Introduction: Success in lung transplantation is limited by obligatory ischemia and reperfusion (I/R) injury. Experimentally, endogenous and induced (hemin, hypoxia) overexpression of heme-oxygenase-1 (HO-1) provides protection against I/R injury, which is abolished with selective inhibition of this enzyme. Unfortunately, no clinically viable therapy exists to safely induce HO-1. We have previously reported that administration of a hemoglobin-based oxygen carrier (HBOC) upregulated HO-1 in isolated pulmonary microvascular endothelial cells. We therefore hypothesized that administration of a HBOC in vivo would induce expression of HO-1 in the lung. Methods: HBOC (PolyHeme®, human polymerized hemoglobin, pyridoxylated; Northfield Laboratories, Inc.) at 0% (control), 5% or 10% of calculated blood volume was administered intravenously (femoral vein) to anesthetized rats (N=5 per group). Recovering animals were subsequently maintained in a normothermic environment and sacrificed at 12 hours. Lungs were isolated, washed and then either flash frozen for tissue homogenization or frozen in embedding medium for histology. Immunoblot of tissue samples were probed for HO-1 and heat shock protein 72 (HSP72, stress-inducible). Densitometry was performed using ImageJ. Microscopic examination of tissue blocks were probed for HO-1 and CD 68 (anti-rat macrophage marker). Statistical analysis used ANOVA (*p<0.05). Results: Administration of HBOC produced nearly a 4-fold induction of lung HO-1 expression (* p<0.05) (see figure). However, there was no change in the stress-inducible HSP72. Immunohistochemistry confirmed upregulation of HO-1 and demonstrated the maximal upregulation of HO-1 to occur in macrophages. Conclusions: In this study, HBOC selectively induced expression of the protective enzyme HO-1 in the lung, without significant alteration of other stress-inducible heat-shock proteins. Significantly, we demonstrated that maximal production of HO-1 in response to treatment with HBOC occurs in macrophages. These data suggest a potential novel therapeutic strategy in lung transplantation. View Large Image Figure Viewer

Down-regulation of the Mixed-lineage Dual Leucine Zipper-bearing Kinase by Heat Shock Protein 70 and Its Co-chaperone CHIP

Dual leucine zipper-bearing kinase (DLK) is a mixed-lineage kinase family member that acts as an upstream activator of the c-Jun N-terminal kinases. As opposed to other components of this pathway, very little is currently known regarding the mechanisms by which DLK is regulated in mammalian cells. Here we identify the stress-inducible heat shock protein 70 (Hsp70) as a negative regulator of DLK expression and activity. Support for this notion derives from data showing that Hsp70 induces the proteasomal degradation of DLK when both proteins are co-expressed in COS-7 cells. Hsp70-mediated degradation occurs with expression of wild-type DLK, which functions as a constitutively activated protein in these cells but not kinase-defective DLK. Interestingly, the Hsp70 co-chaperone CHIP, an E3 ubiquitin ligase, seems to be indispensable for this process since Hsp70 failed to induce DLK degradation in COS-7 cells expressing a CHIP mutant unable to catalyze ubiquitination or in immortalized fibroblasts derived from CHIP knock-out mice. Consistent with these data, we have found that endogenous DLK becomes sensitive to CHIP-dependent proteasomal degradation when it is activated by okadaic acid and that down-regulation of Hsp70 levels with an Hsp70 antisense attenuates this sensitivity. Therefore, our studies suggest that Hsp70 contributes to the regulation of activated DLK by promoting its CHIP-dependent proteasomal degradation.

Hsp70 protects against UVB induced apoptosis by preventing release of cathepsins and cytochrome c in human melanocytes

Stress-induced heat shock protein 70 (Hsp70) effectively protects cells against apoptosis, although the anti-apoptotic mechanism is still undefined. Exposure of human melanocytes to heat and subsequent UVB irradiation increased the level of Hsp70 and pre-heating reduced UVB induced apoptosis. Immunofluorescence staining of Hsp70 in combination with staining of lysosomes (Lamp2) or mitochondria (Mitotracker) in pre-heated UVB exposed cells showed co-localization of Hsp70 with both lysosomes and mitochondria in the surviving cell population. Furthermore, UVB induced apoptosis was accompanied by lysosomal and mitochondrial membrane permeabilization, detected as release of cathepsin D and cytochrome c, respectively, which were prevented by heat pre-treatment. In purified fractions of lysosomes and mitochondria, recombinant Hsp70 attached to both lysosomal and mitochondrial membranes. Moreover, in apoptotic cells Bax was translocated from a diffuse cytosolic location into punctate mitochondrial-like structures, which was inhibited by Hsp70 induction. Such inhibition of Bax translocation was abolished by transfection with Hsp70 siRNA. Furthermore, Hsp70 siRNA eliminated the apoptosis preventive effect observed after pre-heating. These findings show Hsp70 to rescue melanocytes from UVB induced apoptosis by preventing release of cathepsins from lysosomes, Bax translocation and cytochrome c release from mitochondria.

Deletion of the Inducible 70-kDa Heat Shock Protein Genes in Mice Impairs Cardiac Contractile Function and Calcium Handling Associated With Hypertrophy

Hspa1a and Hspa1b genes encode stress-inducible 70-kDa heat shock proteins (Hsp70) that protect cells from insults such as ischemia. Mice with null mutations of both genes (KO) were generated, and their cardiac phenotype was explored. Heart rate and blood pressures were normal in the KO mice. Hearts from KO mice were more susceptible to both functional and cellular damage by ischemia/reperfusion. Cardiac hypertrophy developed in Hsp70-KO mice. Ca2+ transients in cardiomyocytes of KO mice showed a delayed (120%) calcium decline and decreased sarcoplasmic reticulum calcium content. Cell shortening was decreased by 35%, and rates of contraction and relaxation were slower by 40%. These alterations can be attributed to the absence of Hsp70 because viral expression of Hsp70 in KO cultured cardiomyocytes restored these parameters. One mechanism underlying myocyte dysfunction could be decreased SERCA2a expression. This hypothesis was supported by a prolonged calcium decline and decreased SERCA2a protein. Viral SERCA2a expression restored contractility and Ca2+ transients. We examined the involvement of Jun N-terminal kinase (JNK), p38-mitogen-activated protein kinase (p38-MAPK), Raf-1, and extracellular signal-regulated kinase (ERK) in SERCA2a downregulation and the cardiac phenotype of KO mice. Levels of phosphorylated JNK, p38-MAPK, Raf-1, and ERK were elevated in KO hearts. Activation of the Raf-1-ERK pathway in normal cardiomyocytes resulted in decreased SERCA2a. Absence of Hsp70 leads to dysfunctional cardiomyocytes and impaired stress response of Hsp70-KO hearts against ischemia/reperfusion. In addition, deletion of Hsp70 genes might induce cardiac dysfunction and development of cardiac hypertrophy through the activation of JNK, p38-MAPK, Raf-1, and ERK.

Viewing a Stressful Episode of ER

See related article, pages 1186–1193 How cardiac cells sense, respond, and adapt to acute and chronic changes of their metabolic and environmental milieu remain among the most enigmatic and fundamental questions in contemporary cardiovascular biology and medicine. With more than 700 000 deaths and 6 million hospital discharges annually in the U.S., ischemic heart disease remains a major public health problem. If prompt therapy is delayed, myocardial injury from depletion of high-energy phosphates from inhibition of oxidative phosphorylation is inevitable. Ischemia/reperfusion (I/R) also unleashes a cascade of cellular and molecular events whose sequalae may sustain organ function at diminished capacity but, beyond a critical balance, threatens the organism’s survival. Reactive oxygen and nitrogen species (ROS; RNS), released from the mitochondria and other sources, alter the tertiary and quaternary structures of proteins, exposing their hydrophobic residues to allow conformational tendencies toward protein misfolding and aggregation.1 Nevertheless, highly sophisticated schemes equipped with molecular sensors, rapid response pathways, and adaptor mechanisms have evolved to mitigate the accumulation of unfolded proteins in a compartment specific manner, principally the cytoplasm and endoplasmic reticulum (ER). Although the cardiovascular field has paid considerably more attention to the cytoprotective mechanisms of heat shock proteins in response to I/R, termed the “classical” heat shock response, a parallel series of events has been unfolding at a breathtaking pace in the ER, with arguably considerable excitement to warrant a sneak preview of the article published in this issue of Circulation Research . To place into perspective the current buzz being garnered by the ER requires a brief discussion of more familiar themes in the field, which were developed thanks to the pioneering efforts of cardiovascular scientists, and others, working on the complementary stress response network, best exemplified by heat shock “stress” proteins and their transcriptional regulators. First discovered by …

Control of Metastasized Pancreatic Carcinomas in SCID/Beige Mice with Human IL-2/TKD-Activated NK Cells

Pancreatic carcinoma, the fifth leading cause of cancer-related mortality, frequently presents the stress-inducible heat shock protein 70 (Hsp70) on the cell membrane. Therefore, we explored an immunological approach exploiting the efficacy of NK cells activated either with low dose IL-2 plus Hsp70-peptide TKDNNLLGRFELSG (TKD; IL-2/TKD) or with IL-2 alone in a xenograft pancreatic carcinoma model. An orthotopic injection of either 2.5 x 10(6) or 1 x 10(6) Colo357 cells in SCID/beige mice resulted in rapidly growing primary tumors and the development of hepatic metastases on days 5 and 10, respectively. In line with results of in vitro migration assays, these NK cells also had the capacity to infiltrate pancreatic tumors and liver metastases in tumor-bearing mice. In vitro, a combined treatment of NK cells with IL-2/TKD but neither of the two treatments alone causes a profound increase in the lytic capacity against Hsp70 membrane-positive Colo357 cells. In vivo, a single i.v. injection of these NK cells on day 15 post-tumor inoculation resulted in a significant reduction in tumor weights, a delayed onset of hepatic metastases, and a prolonged life expectancy. In contrast, identically treated T cells and NK cells treated with IL-2 alone were significantly less efficient in controlling pancreatic tumors and metastases. Most importantly, four repeated i.v. infusions of IL-2/TKD-activated NK cells eradicated primary tumors and prevented hepatic metastases. In summary, our mouse data have implicated that NK cells preactivated with IL-2/TKD might provide a novel therapeutic tool for the treatment of aggressive, Hsp70-positive pancreatic carcinoma.

The release of Hsp70 from A431 carcinoma cells is mediated by secretory-like granules

In our earlier work we have demonstrated that the treatment of squamous carcinoma cell line A431 with a pharmacological inhibitor of phospholipase C activity, U73122, resulted in fast release of stress-inducible heat shock protein 70 (Hsp70) into the extracellular medium (Evdonin et al., Cancer Cell Int., 4, 2, 2004). The purpose of the present study was to identify cellular organelles involved in the release of Hsp70 from A431 cells. We determined that Hsp70 is present in granules located at the periphery of cells, which had been treated with U73122 or subjected to heat shock. An inhibitor of the classical protein export pathway, brefeldin A was found to prevent the U73122-induced appearance of Hsp70 in the extracellular medium and in the peripheral granules. These findings suggest that vesicular transport is involved in Hsp70 release. The Hsp70-containing granules did not carry markers specific for lipid bodies, endosomes, or lysosomes. However, they were positive for a marker of secretory granules, i.e. chromogranin A. The levels of extracellular Hsp70 and chromogranin A were found to increase simultaneously. The secretory-like granule-dependent transport of Hsp70 was also studied in minimally transformed human HaCaT keratinocytes. We found that after U73122 and heat stress treatment, HaCaT cells secreted Hsp70 in a manner similar to A431 cells. Collectively our results suggest that human keratinocyte-derived cells release Hsp70 in the extracellular medium through a pathway involving secretory-like granules.

Heat shock protein 70 binds its own messenger ribonucleic acid as part of a gene expression self-limiting mechanism

Expression of heat shock proteins is a cellular response to a variety of stressors. HSP70, the major stress-induced heat shock protein, is involved in repair and protection after the insult. However, the prolonged presence of this protein is detrimental. Consequently, Hsp70 expression must be tightly regulated. We have previously shown an increase in the degradation of Hsp70 messenger ribonucleic acid (mRNA) paralleling the accumulation of HSP70. Incubation of cells with transcriptional and translational inhibitors after heat shock resulted in a significant reduction in Hsp70 mRNA degradation. These observations suggest that newly synthesized, stress-induced factors might be involved in the decay of Hsp70 mRNA. We found that HSP70 binds directly to Hsp70 mRNA, as demonstrated by immunoprecipitation. This observation was confirmed by RNA gel-shift assays. These results are evidence for a novel and likely direct interaction between HSP70 and Hsp70 mRNA in cells after stress. This interaction may be part of a self-limiting mechanism to reduce HSP70 production, thus avoiding potential toxic effects of this protein in the absence of stress.

Stress-induced heat shock protein 27 expression and its role in dorsal root ganglion neuronal survival

Heat shock protein 27 (Hsp27), a molecular chaperone ubiquitously expressed in many cell types, has been shown to play a role in protecting neurons from cellular stresses. Unlike adult DRG neurons in vitro, neonatal DRG neurons require NGF for survival; withdrawal of NGF results in apoptosis of a majority of neonatal neurons. We hypothesized that Hsp27 contributes to the neurotrophin-independent survival of adult DRG neurons. Constitutive Hsp27 expression is higher in adult DRG neurons compared to neonates, although both upregulate Hsp27 expression after heat shock (HS). We found that increasing endogenous Hsp27 by HS in neonatal neurons was able to inhibit NGF withdrawal-induced apoptosis. Heat shock of adult and neonatal neurons also resulted in Akt activation, which could be a mechanism for the increased survival. Hsp27 siRNA treatment of adult neurons effected a decreased expression of Hsp27, which correlated with increased apoptosis in these neurons. Downregulation of Hsp27 via siRNA also blocked the HS-induced rescue of neonatal neurons after NGF withdrawal. These results indicate that physiologically induced upregulation of Hsp27 is sufficient to provide some degree of neuronal protection. Further, this induction appears to be regulated by the transcriptional activation of HSF1 as shown by HSF1 nuclear translocation and by EMSA analyses of HSF1 binding to nuclear protein.

Death versus survival: functional interaction between the apoptotic and stress-inducible heat shock protein pathways

Induction of heat shock proteins (Hsps) following cellular damage can prevent apoptosis induced by both the intrinsic and the extrinsic pathways. The intrinsic pathway is characterized by mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, apoptosome assembly, and caspase activation. Hsps promote cell survival by preventing MOMP or apoptosome formation as well as via regulation of Akt and JNK activities. Engagement of the TNF death receptors induces the extrinsic pathway that is characterized by Fas-associated death domain-dependent (FADD-dependent) caspase-8 activation or induction of NF-kappaB to promote cellular survival. Hsps can directly suppress proapoptotic signaling events or stabilizing elements of the NF-kappaB pathway to promote cellular survival.

Subproteomic analysis of metal-interacting proteins in human B cells

Metal-protein interactions are vitally important in all living organisms. Metalloproteins, including structural proteins and metabolic enzymes, participate in energy transfer and redox reactions or act as metallochaperones in metal trafficking. Among metal-associated diseases, T cell mediated allergy to nickel (Ni) represents the most common form of human contact hypersensitivity. With the aim to elucidate disease-underlying mechanisms such as Ni-specific T cell activation, we initiated a proteomic approach to identify Ni-interacting proteins in human B cells. As antigen presenting cells, B cells are capable of presenting MHC-associated Ni-epitopes to T cells, a prerequisite for hapten-specific T cell activation. Using metal-affinity enrichment, 2-DE and MS, 22 Ni-interacting proteins were identified. In addition to known Ni-binding molecules such as tubulin, actin or cullin-2, we unexpectedly discovered that at least nine of these 22 proteins belong to stress-inducible heat shock proteins or chaperonins. Enrichment was particularly effective for the hetero-oligomeric TRiC/CCT complex, which is involved in MHC class I processing. Blue Native/SDS electrophoresis analysis revealed that Ni-NTA-beads specifically retained the complete protein machinery, including the associated chaperonin substrate tubulin. The apparent Ni-affinity of heat shock proteins suggests a new function of these molecules in human Ni allergy, by linking innate and adaptive immune responses.

Preservation of heat stress induced myocardial hsp 72 in aged animals following caloric restriction

The major stress inducible Heat Shock Protein (HSP 72) confers myocardial protection from ischemia. A decreased ability to express HSP 72 during homeostatic disruptions has been suggested as a possible mechanism for the increased susceptibility of aged hearts to ischemic stress. Given that Caloric Restriction (CR) has been reported to reverse or delay age-associated cellular senescence, we examined the effect of CR on the ability of aged hearts to induce and accumulate HSP 72. Adult (6 months), aged (22 months) and CR aged (22 months) Fisher 344 rats were heat stressed by raising core temperature to 41 °C for 10 min. Immediately after heat stress, or 24 h later, the myocardium was examined for either activation of the heat shock transcription factor (HSF) or HSP 72 accumulation. Hearts from heat stressed CR animals demonstrated an increased HSF activation and an increased HSP 72 content when compared to hearts from heat stressed aged animals. The HSF response and HSP 72 content of the hearts from heat stressed aged CR animals was comparable to that observed in hearts from heat stressed adult animals. These results suggest CR may preserve the ability of the aged myocardium to activate and/or express HSP 72.