Serum and lymphocyte levels of heat shock protein 70 in aging: a study in the normal Chinese population

Heat shock proteins (Hsps) have been reported to play an important role in both physiological and pathological processes. Hsps also may serve as biomarkers for evaluating disease states and exposure to environmental stresses. Whether Hsp levels in serum and lymphocytes are correlated with age and sex is largely unknown. In this study, we analyzed serum Hsp70 (the most abundant mammalian Hsp) levels by using Western dot blot in 327 healthy male donors aged between 15 and 50 years. We also investigated the association between Hsp70 levels and age in lymphocytes of 80 normal individuals aged between 40 and 77 years because various chronic diseases increase after the age of 40 years. Our data showed that serum Hsp70 levels were positively correlated with age in subjects aged between 15 and 30 years (P < 0.05) but negatively correlated with age in subjects aged between 30 and 50 years (P < 0.05). Serum Hsp70 levels were the highest in individuals aged between 25 and 30 years among all age groups. In the lymphocyte study there also was a significant age-related decrease in Hsp70 levels in lymphocytes of individuals older than 40 years. The Hsp70 levels were negatively correlated with age (r = -3.708, P < 0.0001) but not with sex (r = -10.536, P = 0.452). This suggests that both serum and lymphocyte Hsp70 levels are age-related and that these may be linked to age-related stress. Thus, age is an important factor in using serum and lymphocyte Hsp70 as biomarkers to evaluate the disease states or exposure to environmental stresses (or both).     

Structural Insights into the Chaperone Activity of the 40-kDa Heat Shock Protein DnaJ: BINDING AND REMODELING OF A NATIVE SUBSTRATE*

Hsp40 chaperones bind and transfer substrate proteins to Hsp70s and regulate their ATPase activity. The interaction of Hsp40s with native proteins modifies their structure and function. A good model for this function is DnaJ, the bacterial Hsp40 that interacts with RepE, the repressor/activator of plasmid F replication, and together with DnaK regulates its function. We characterize here the structure of the DnaJ-RepE complex by electron microscopy, the first described structure of a complex between an Hsp40 and a client protein. The comparison of the complexes of DnaJ with two RepE mutants reveals an intrinsic plasticity of the DnaJ dimer that allows the chaperone to adapt to different substrates. We also show that DnaJ induces conformational changes in dimeric RepE, which increase the intermonomeric distance and remodel both RepE domains enhancing its affinity for DNA.     

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 degrees C 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 degrees C, those exposed to heat stress (42 degrees C 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.     

CD95-mediated alteration in Hsp70 levels is dependent on protein stabilization

Engagement of death receptors induces caspase activation and apoptosis. A recent study reported altered protein expression, including increased Hsp70 levels during CD95-mediated apoptosis. Here, we examined the mechanism underlying increased Hsp70 levels in cells challenged with a monoclonal antibody directed against the CD95 receptor. Levels of Hsp70 were found to increase in a dose-dependent manner, occurring independently of either heat shock factor 1 activation or the accumulation of Hsp70 messenger ribonucleic acid (mRNA), suggesting the involvement of posttranslational modifications. Inhibition of translation and de novo protein synthesis by cycloheximide resulted in Hsp70 protein levels diminishing over time in control cells, whereas its level remained constant during CD95 signaling. In addition, death receptor activation through exposure of cells to tumor necrosis factor-related apoptosis-inducing ligand did not alter Hsp70 levels. These findings demonstrate that receptor-specific signaling through the CD95 increases the stability of Hsp70 protein, rather than mRNA, when compared with control cells. The results describe a novel mechanism of heat shock protein accumulation, where increased protein stability and reduced turnover, is the mechanism by which Hsp70 accumulates in cells during CD95-mediated apoptosis.     

Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

In the eukaryotic cytosol, Hsp70 and Hsp90 cooperate with various co-chaperone proteins in the folding of a growing set of substrates, including the glucocorticoid receptor (GR). Here, we analyse the function of the co-chaperone Tpr2, which contains two chaperone-binding TPR domains and a DnaJ homologous J domain. In vivo, an increase or decrease in Tpr2 expression reduces GR activation, suggesting that Tpr2 is required at a narrowly defined expression level. As shown in vitro, Tpr2 recognizes both Hsp70 and Hsp90 through its TPR domains, and its J domain stimulates ATP hydrolysis and polypeptide binding by Hsp70. Furthermore, unlike other co-chaperones, Tpr2 induces ATP-independent dissociation of Hsp90 but not of Hsp70 from chaperone-substrate complexes. Excess Tpr2 inhibits the Hsp90-dependent folding of GR in cell lysates. We propose a novel mechanism in which Tpr2 mediates the retrograde transfer of substrates from Hsp90 onto Hsp70. At normal levels substoichiometric to Hsp90 and Hsp70, this activity optimizes the function of the multichaperone machinery.     

Synergistic and differential modulation of immune responses by Hsp60 and lipopolysaccharide

Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation of an efficient immune response. In this study we show that Hsp60 mediates immune stimulation by different mechanisms, dependent and independent of lipopolysaccharide (LPS). We have demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific interferon (IFN) gamma release in T cells. Here we show that in contrast to LPS Hsp60 induces IFNalpha production in professional APC. Neutralization of IFNalpha as well as the absence of functional IFNalphabeta receptor on APC and T cells interfered with Hsp60-mediated IFNgamma secretion in antigen-dependent T cell activation, strongly suggesting that IFNalpha represents one factor contributing to Hsp60-specific immune stimulation. On the other hand, we show that Hsp60 bound to the cell surface of APC colocalizes with the LPS co-receptor CD14 and LPS binding sites. Hsp60 specifically binds bacterial LPS and both molecules synergistically enhanced IL-12p40 production in APC and IFNgamma release in antigen-dependent T cell activation. This effect was Hsp60-specific and dependent on LPS-binding by Hsp60. Furthermore, we show that Hsp60 exclusively binds to macrophages and DC but not to T or B lymphocytes and that both, T cell stimulation by Hsp60 as well as Hsp60/LPS complexes, strictly depends on the presence of professional APC and is not mediated by B cells. Taken together, our data support an extension of the concept of Hsp60 as an endogenous danger signal: besides its function as a classical danger signal indicating unplanned tissue destruction to the innate immune system, in the incident of bacterial infection extracellular Hsp60 may bind LPS and facilitate microbe recognition by lowering the threshold of pathogen-associated molecular pattern (PAMP) detection and enhancing Toll-like receptor (TLR) signaling.     

Hsp70B' regulation and function

Heat shock protein (Hsp) 70B' is a human Hsp70 chaperone that is strictly inducible, having little or no basal expression levels in most cells. Using siRNAs to knockdown Hsp70B' and Hsp72 in HT-29, SW-480, and CRL-1807 human colon cell lines, we have found that the two are regulated coordinately in response to stress. We also have found that proteasome inhibition is a potent activator of Hsp70B'. Flow cytometry was used to assay Hsp70B' promoter activity in HT-29eGFP cells in this study. Knockdown of both Hsp70B'- and Hsp72-sensitized cells to heat stress and increasing concentrations of proteasome inhibitor. These data support the conclusion that Hsp72 is the primary Hsp70 family responder to increasing levels of proteotoxic stress, and Hsp70B' is a secondary responder. Interestingly ZnSO4 induces Hsp70B' and not Hsp72 in CRL-1807 cells, suggesting a stressor-specific primary role for Hsp70B'. Both Hsp70B' and Hsp72 are important for maintaining viability under conditions that increase the accumulation of damaged proteins in HT-29 cells. These findings are likely to be important in pathological conditions in which Hsp70B' contributes to cell survival.     

Sea urchin deciliation induces thermoresistance and activates the p38 mitogen-activated protein kinase pathway

In this study, we demonstrate by a variety of approaches (ie, morphological analysis, Western blots, immunolocalization, and the use of specific antibodies) that hyperosmotic deciliation stress of sea urchin embryos induces a thermotolerant response. Deciliation is also able to activate a phosphorylation signaling cascade the effector of which might be the p38 stress-activated protein kinase because we found that the administration of the p38 inhibitor SB203580 to sea urchin deciliated gastrula embryos makes the hyperosmotic deciliation stress lethal.     

Radicicol, an inhibitor of Hsp90, enhances TRAIL-induced apoptosis in human epithelial ovarian carcinoma cells by promoting activation of apoptosis-related proteins

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various cancer cells. Hsp90 is known to be involved in cell survival and growth in tumor cells. Nevertheless, Hsp90 inhibitors exhibit a variable effect on the cytotoxicity of anticancer drugs. Furthermore, the combined effect of Hsp90 inhibitors on TRAIL-induced apoptosis in epithelial ovarian cancer cells has not been determined. To assess the ability of an inhibitor of Hsp90 inhibitor radicicol to promote apoptosis, we investigated the effect of radicicol on TRAIL-induced apoptosis in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. TRAIL induced a decrease in Bid, Bcl-2, Bcl-xL, and survivin protein levels, increase in Bax levels, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases (-8, -9, and -3), cleavage of PARP-1 and an increase in the tumor suppressor p53 levels. Radicicol enhanced TRAIL-induced apoptosis-related protein activation, nuclear damage and cell death. These results suggest that radicicol may potentiate the apoptotic effect of TRAIL on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-dependent pathway and the mitochondria-mediated apoptotic pathway, leading to caspase activation. Radicicol may confer a benefit in the TRAIL treatment of epithelial ovarian adenocarcinoma.     

Hsp70B' regulation and function

Heat shock protein (Hsp) 70B' is a human Hsp70 chaperone that is strictly inducible, having little or no basal expression levels in most cells. Using siRNAs to knockdown Hsp70B' and Hsp72 in HT-29, SW-480, and CRL-1807 human colon cell lines, we have found that the two are regulated coordinately in response to stress. We also have found that proteasome inhibition is a potent activator of hsp70B'. Flow cytometry was used to assay hsp70B' promoter activity in HT-29eGFP cells in this study. Knockdown of both Hsp70B' and Hsp72 sensitized cells to heat stress and increasing concentrations of proteasome inhibitor. These data support the conclusion that Hsp72 is the primary Hsp70 family responder to increasing levels of proteotoxic stress, and Hsp70B' is a secondary responder. Interestingly ZnSO4 induces Hsp70B' and not Hsp72 in CRL-1807 cells, suggesting a stressor-specific primary role for Hsp70B'. Both Hsp70B' and Hsp72 are important for maintaining viability under conditions that increase the accumulation of damaged proteins in HT-29 cells. These findings are likely to be important in pathological conditions in which Hsp70B' contributes to cell survival.     

CD40, an extracellular receptor for binding and uptake of Hsp70-peptide complexes

Tumor and viral antigens elicit a potent immune response by heat shock protein-dependent uptake of antigenic peptide with subsequent presentation by MHC I. Receptors on antigen-presenting cells that specifically bind and internalize a heat shock protein-peptide complex have not yet been identified. Here, we show that cells expressing CD40, a cell surface protein crucial for B cell function and autoimmunity, specifically bind and internalize human Hsp70 with bound peptide. Binding of Hsp70-peptide complex to the exoplasmic domain of CD40 is mediated by the NH(2)-terminal nucleotide-binding domain of Hsp70 in its ADP state. The Hsp70 cochaperone Hip, but not the bacterial Hsp70 homologue DnaK, competes formation of the Hsp70-CD40 complex. Binding of Hsp70-ADP to CD40 is strongly increased in the presence of Hsp70 peptide substrate, and induces signaling via p38. We suggest that CD40 is a cochaperone-like receptor mediating the uptake of exogenous Hsp70-peptide complexes by macrophages and dendritic cells.     

Hsp90 modulates CAG repeat instability in human cells

The Hsp90 molecular chaperone has been implicated as a contributor to evolution in several organisms by revealing cryptic variation that can yield dramatic phenotypes when the chaperone is diverted from its normal functions by environmental stress. In addition, as a cancer drug target, Hsp90 inhibition has been documented to sensitize cells to DNA-damaging agents, suggesting a function for Hsp90 in DNA repair. Here we explore the potential role of Hsp90 in modulating the stability of nucleotide repeats, which in a number of species, including humans, exert subtle and quantitative consequences for protein function, morphological and behavioral traits, and disease. We report that impairment of Hsp90 in human cells induces contractions of CAG repeat tracks by tenfold. Inhibition of the recombinase Rad51, a downstream target of Hsp90, induces a comparable increase in repeat instability, suggesting that Hsp90-enabled homologous recombination normally functions to stabilize CAG repeat tracts. By contrast, Hsp90 inhibition does not increase the rate of gene-inactivating point mutations. The capacity of Hsp90 to modulate repeat-tract lengths suggests that the chaperone, in addition to exposing cryptic variation, might facilitate the expression of new phenotypes through induction of novel genetic variation.     

Geldanamycin induces heat shock protein 70 and protects against MPTP-induced dopaminergic neurotoxicity in mice

As key molecular chaperone proteins, heat shock proteins (HSPs) represent an important cellular protective mechanism against neuronal cell death in various models of neurological disorders. In this study, we investigated the effect as well as the molecular mechanism of geldanamycin (GA), an inhibitor of Hsp90, on 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity, a mouse model of Parkinson disease. Neurochemical analysis showed that pretreatment with GA (via intracerebral ventricular injection 24 h prior to MPTP treatment) increased residual dopamine content and tyrosine hydroxylase immunoreactivity in the striatum 24 h after MPTP treatment. To dissect out the molecular mechanism underlying this neuroprotection, we showed that the GA-mediated protection against MPTP was associated with a reduction of cytosolic Hsp90 and an increase in Hsp70, with no significant changes in Hsp40 and Hsp25 levels. Furthermore, in parallel with the induction of Hsp70, striatal nuclear HSF1 levels and HSF1 binding to heat shock element sites in the Hsp70 promoter were significantly enhanced by the GA pretreatment. Together these results suggested that the molecular cascade leading to the induction of Hsp70 is critical to the neuroprotection afforded by GA against MPTP-induced neurotoxicity in the brain and that pharmacological inhibition of Hsp90 may represent a potential therapeutic strategy for Parkinson disease.     

Cilia regeneration in starved tetrahymena: an inducible system for studying gene expression and organelle biogenesis.

Deciliated starved Tetrahymena recover motility with kinetics similar to those of growing cells and, like growing cells, require RNA and protein synthesis for regeneration. Comparisons of polysome profiles and electrophoretic analyses of newly synthesized proteins indicate, however, that the basal level of protein synthesis in starved cells is markedly lower than that in growing cells. This difference allows demonstration of changes in protein synthesis following deciliation of starved cells which cannot be detected (if they occur at all) in growing cells. Deciliation of starved cells induces a specific and orderly program of protein synthesis. The synthesis of an 80,000 dalton protein (deciliation-induced protein, DIP) begins shortly after deciliation, comprises 15% of the protein synthesized from 20-60 min, and declines around 60 min after deciliation, shortly after most cells have begun to regenerate cilia. The synthesis of a 55,000 dalton protein is also induced during regeneration and has been identified as tubulin using a well characterized antibody made to ciliary tubulin. Tubulin synthesis is undetectable during the first hour after deciliation even though 60-80% of the cells regain mobility and regenerate short but clearly visible cilia. Tubulin synthesis begins 60 min after deciliation and continues for 2 hr. At its peak, tubulin comprises 7-8% of the protein synthesized. The results of actinomycin D addition at different times after deciliation suggest that RNA required for DIP synthesis is synthesized early (0-30 min), while RNA required for tubulin is synthesized later and over a longer period (30-90 min). Thus deciliation of starved cells, an event occurring at the cell periphery, initiates a well defined and reproducible series of events culminating in cilia formation. This system should be useful in elucidating the molecular mechanisms regulating gene expression and organelle biogenesis in Tetrahymena.     

Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity

Studies on the Hsp70 chaperone machine in eukaryotes have shown that Hsp70 and Hsp40/Hdj1 family proteins are sufficient to prevent protein misfolding and aggregation and to promote refolding of denatured polypeptides. Additional protein cofactors include Hip and Bag1, identified in protein interaction assays, which bind to and modulate Hsp70 chaperone activity in vitro. Bag1, originally identified as an antiapoptotic protein, forms a stoichiometric complex with Hsp70 and inhibits completely Hsp70-dependent in vitro protein refolding of an unfolded polypeptide. Given its proposed involvement in multiple cell signaling events as a regulator of Raf1, Bcl2, or androgen receptor, we wondered whether Bag1 functions in vivo as a negative regulator of Hsp70. In this study, we demonstrate that Bag1, expressed in mammalian tissue culture cells, has pronounced effects on one of the principal activities of Hsp70, as a molecular chaperone essential for stabilization and refolding of a thermally inactivated protein. The levels of Hsp70 and Bag1 were modulated either by transient transfection or conditional expression in stably transfected lines to achieve levels within the range detected in different mammalian tissue culture cell lines. For example, a twofold increase in the concentration of Bag1 reduced Hsp70-dependent refolding of denatured luciferase by a factor of 2. This effect was titratable, and higher levels of wild-type but not a mutant form of Bag1 further inhibited Hsp70 refolding by up to a factor of 5. The negative effects of Bag1 were also observed in a biochemical analysis of Bag1- or Hsp70-overexpressing cells. The ability of Hsp70 to maintain thermally denatured firefly luciferase in a soluble state was reversed by Bag1, thus providing an explanation for the in vivo chaperone-inhibitory effects of Bag1. Similar effects on Hsp70 were observed with other cytoplasmic isoforms of Bag1 which have in common the carboxyl-terminal Hsp70-binding domain and differ by variable-length amino-terminal extensions. These results provide the first formal evidence that Bag1 functions in vivo as a regulator of Hsp70 and suggest an intriguing complexity for Hsp70-regulatory events.     

Hsp27 inhibits 6-hydroxydopamine-induced cytochrome c release and apoptosis in PC12 cells

Cellular stress may stimulate cell survival pathways or cell death depending on its severity. 6-Hydroxydopamine (6-OHDA) is a neurotoxin that targets dopaminergic neurons that is often used to induce neuronal cell death in models of Parkinson's disease. Here we present evidence that 6-OHDA induces apoptosis in rat PC12 cells that involves release of cytochrome c and Smac/Diablo from mitochondria, caspase-3 activation, cleavage of PARP, and nuclear condensation. 6-OHDA also induced the heat shock response, leading to increased levels of Hsp25 and Hsp70. Increased Hsp25 expression was associated with cell survival. Prior heat shock or overexpression of Hsp27 (human homologue of Hsp25) delayed cytochrome c release, caspase activation, and reduced the level of apoptosis caused by 6-OHDA. We conclude that 6-OHDA induces a variety of responses in cultured PC12 cells ranging from cell survival to apoptosis, and that induction of stress proteins such as Hsp25 may protect cells from undergoing 6-OHDA-induced apoptosis.