Plasma levels of Hsp70 and anti-Hsp70 antibody predict risk of acute coronary syndrome

Although immune reactions against heat shock proteins have been implicated in the pathogenesis of atherosclerosis, conflicting associations between Hsp70, anti-Hsp70 antibody and coronary heart disease (CHD) have been reported. This study assessed whether there is a significant association between extracellular human Hsp70, anti-Hsp70 antibody and acute coronary syndrome (ACS) and stable angina (SA), and examined dynamic changes in Hsp70 and anti-Hsp70 antibody levels induced by acute myocardial infarction (AMI). Plasma Hsp70 and anti-Hsp70 antibody levels in 291 patients with ACS (179 AMI, 112 unstable angina), 126 patients with SA and 417 age and sex-matched healthy subjects, and in 40 patients after admission for AMI, and on day 2, 3, and 7 after the onset of AMI were determined using enzyme-linked immunosorbent assays. Hsp70 levels were significantly higher in ACS and SA and anti-Hsp70 antibody levels were only markedly lower in ACS than controls. After adjustment for traditional CHD risk factors, increasing levels of Hsp70 were significantly associated with an increased risk and severity of ACS (P for trend < 0.001), whereas increasing levels of anti-Hsp70 antibody were associated with a decreased risk of ACS (P for trend = 0.0003). High levels of Hsp70 combined with low levels of anti-Hsp70 antibody had a joint effect on the risk of ACS (OR, 5.14, 95% CI, 3.00-8.79; P < 0.0001). In patients with AMI, Hsp70 levels decreased rapidly from days 1-7 after onset, whereas anti-Hsp70 antibody levels increased in patients with AMI. These findings suggest that higher Hsp70 levels or lower anti-Hsp70 antibody levels are independently associated with a higher risk of ACS. Higher Hsp70 levels and lower anti-Hsp70 antibody levels combine to further increase this risk.     

Cellular localization of a Hsp90 homologue in Porphyromonas gingivalis

We previously reported an association between elevated serum antibody titers to the 90-kDa human heat shock protein (Hsp90), periodontal health and colonization by Porphyromonas gingivalis. In this study, we examined the cellular localization of the Hsp90 homologue of P. gingivalis. Cultures of P. gingivalis were heat-stressed (45 degrees C) and examined for localization of the Hsp90 homologue. Heat stress induced a 4-5-fold increase in anti-Hsp90 antibody reactivity over that of the unstressed controls. Western blot analysis revealed two bands (44 and 68 kDa) that reacted with anti-Hsp90 antibodies. The 68-kDa band was heat-inducible, while the 44-kDa band was not. Immunogold staining revealed that the Hsp90 homologue localized principally to the membrane and extracellular vesicles. Subcellular fractionation confirmed that the Hsp90 homologue was primarily membrane-associated.     

Circulating anti-heat-shock-protein antibodies in normal pregnancy and preeclampsia

It has been previously reported that circulating anti-heat-shock-protein (Hsp) antibody levels are elevated in cardiovascular disorders. The aim of the present study was to determine circulating antihuman Hsp60, antimycobacterial Hsp65, and antihuman Hsp70 antibody levels in healthy pregnant women and preeclamptic patients and to investigate their relationship to the clinical characteristics of the study subjects, as well as to the markers of inflammation (C-reactive protein (CRP)), endothelial activation (von Willebrand factor antigen), or endothelial injury (fibronectin), oxidative stress (malondialdehyde) and to serum Hsp70 levels. Ninety-three preeclamptic patients and 127 normotensive healthy pregnant women were involved in this case control study. Serum anti-Hsp60, anti-Hsp65, anti-Hsp70, and Hsp70 levels were measured with enzyme-linked immunosorbent assay (ELISA). Serum CRP levels were determined by an autoanalyzer using the manufacturer’s kit. Plasma von Willebrand factor antigen levels were quantified by ELISA, while plasma fibronectin concentration by nephelometry. Plasma malondialdehyde levels were measured by the thiobarbituric-acid-based colorimetric assay. For statistical analyses, nonparametric methods were applied. Anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibodies were detected in all of our serum samples. There were no significant differences in serum anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibody levels between the control and preeclamptic groups. Serum levels of Hsp70 and CRP, as well as plasma levels of VWF antigen, fibronectin, and malondialdehyde, were significantly higher in preeclamptic patients than in normotensive healthy pregnant women. Serum anti-Hsp60 antibody levels showed significant correlations with serum anti-Hsp65 antibody levels both in the control and the preeclamptic groups (Spearman R = 0.55 and 0.59; p < 0.001, respectively). However, no other relationship was found between clinical features (maternal age, smoking status, parity, body mass index, gestational age at blood draw, systolic and diastolic blood pressure, gestational age at delivery, and fetal birth weight) and measured laboratory parameters of the study subjects and serum anti-Hsp antibody levels in either study group. In conclusion, anti-Hsp60 and anti-Hsp70 antibodies as naturally occurring autoantibodies are present in the peripheral circulation of healthy pregnant women. Nevertheless, humoral immunity against heat shock proteins was not associated with preeclampsia. Further studies are warranted to explore the role of heat shock proteins and immune reactivity to them in the immunobiology of normal pregnancy and preeclampsia.     

Suppression of the mTOR-raptor signaling pathway by the inhibitor of heat shock protein 90 geldanamycin

Heat shock protein 90 (Hsp90) was co-immunoprecipitated with raptor, the binding partner of the mammalian target of rapamycin (mTOR) from HEK293 cells. Hsp90 was detected in the anti-raptor antibody immunoprecipitates prepared from the cell extract by immunoblot analysis using the anti-Hsp90 antibody, and the association of these two proteins was confirmed by immunoprecipitation from the cells co-expressing Hsp90 and raptor as epitope-tagged molecules. Geldanamycin, a potent inhibitor of Hsp90, disrupted the in vivo binding of Hsp90 to raptor without affecting the association of raptor and mTOR, and suppressed the phosphorylation by mTOR of the downstream translational regulators p70 S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The protein kinase activity of S6K as well as the phosphorylation of the substrate, 40S ribosomal protein S6, were lowered in the geldanamycin-treated cells. These results indicate that Hsp90 is involved in the regulation of protein translation by facilitating the phosphorylation reaction of 4E-BP1 and S6K catalyzed by the mTOR/raptor complex through the association with raptor, and that the mTOR signaling pathway is a novel target of geldanamycin.     

Major stress protein Hsp70 interacts with NF-kB regulatory complex in human T-lymphoma cells

Polypeptides belonging to the Hsp70 major stress protein family and to the NF-kB/Rel multi-functional regulatory complex are known to be involved in cellular defense mechanisms. It was suggested that both systems may interact in cells that respond to injuring stimuli. To check this, Molt4 human lymphoma cells were heated at 43°C for 15 min and, after a 6 h post-shock recovery period, the cells were activated with phorbol ester or bacterial lipopolysaccharide. It as found that mild heat shock caused a substantial increase of the intracellular Hsp70 content with the concomitant suppression of NF-kB complexes, though the latter was properly activated in non-stressed cells. After a 24 h period of being inactive the complex fully recovered its activity and p65 and c-Rel subunits migrated to the nucleus. This new active period lasted even longer than that in non-heated control cells. As this suggested the existence of a Hsp70-related mechanism of NF-kB/Rel complex retention in cytoplasm, we carried out immunoprecipitation with the use of anti-Hsp70 and anti-Rel antibodies. All three Rel family members p65, c-Rel, p50, but not their precursors and IkBα inhibitory protein were shown to co-precipitate with the stress protein and anti-Hsp70 antibodies from both heated and non-heated cells. We conclude that the Hsp70 stress protein may confer a new mechanism of NF-kB regulation in cells affected by elevated temperature or other factors related to the cellular response to stress.     

TAT-Hsp40 inhibits oxidative stress-mediated cytotoxicity via the inhibition of Hsp70 ubiquitination

Heat shock protein 40 (Hsp40) functions as a co-chaperone of mammalian Heat shock protein 70 (Hsp70) and facilitates the ATPase activity of Hsp70, and also promotes the cellular protein folding and renaturation of misfolded proteins. In an effort to assess the effects of Hsp40, we generated TAT-fused Hsp40 (TAT-Hsp40). The cells were transduced with TAT-Hsp40 and exposed to H(2)O(2). We demonstrated that the TAT-Hsp40-transduced cells were more resistant to cellular cytotoxicity and cell death. In particular, the degradation of Hsp70 was significantly reduced in TAT-Hsp40-containing cells as a consequence of reduced ubiquitin-proteasome activity after oxidative injury. These data support the notion that Hsp40 may confer resistance to oxidative stress via the prevention of proteasome activity.     

Identification of alpha-tubulin as an hsp105alpha-binding protein by the yeast two-hybrid system

Hsp105alpha is a mammalian stress protein that belongs to the HSP105/110 family. Hsp105alpha prevents stress-induced apoptosis in neuronal cells and binds to Hsp70/Hsc70 and suppresses the Hsp70 chaperone activity in vitro. In this study, to further elucidate the function of Hsp105alpha, we searched for Hsp105alpha-binding proteins by screening a mouse FM3A cell cDNA library with full-length Hsp105alpha using the yeast two-hybrid system and obtained alpha-tubulin as an Hsp105alpha-binding protein. Hsp105alpha bound directly to alpha-tubulin both in vitro and in vivo. Indirect immunofluorescence analysis with anti-Hsp105 and anti-alpha-tubulin antibodies indicated that Hsp105alpha was colocalized with microtubules. Furthermore, the disorganization of microtubules induced by heat shock was prevented in Hsp105alpha-overexpressing COS-7 cells. These findings suggested that Hsp105alpha associates with alpha-tubulin and microtubules in cells and plays a role in protection of microtubules under conditions of stress.     

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.     

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.     

Birth defects and anti-heat shock protein 70 antibodies in early pregnancy

Abstract It has been suggested that induction of the heat shock response in the mammalian embryo during the critical period of organogenesis can result in anatomical malformation. We measured serum heat shock protein 70 (Hsp70), anti-Hsp70, and anti-Hsp60 in samples taken from expectant mothers at 16 weeks gestation. Samples from women whose babies were born with a birth defect (n = 30) were compared with controls who gave birth to healthy babies (n = 46). Anti-Hsp70 levels were significantly elevated in patients who later gave birth to babies with cleft lip or palate or neurological abnormalities (n = 10): 260 (223-406) microg/mL compared to 150 (88-207) microg/mL in controls (P < 0.001). No significant differences were found in serum Hsp70 and anti-Hsp60 levels between cases and controls. This finding of increased maternal anti-Hsp70 in patients who later gave birth to babies with these abnormalities suggests a previous stressful event may have contributed to the pathogenesis. Further work is required to determine whether Hsp70 has a direct or indirect role in this pathogenesis or whether anti-Hsp70 is simply a marker of a prior increase in Hsp70 due to a physiological stress that itself resulted in the damage. This work is consistent with previous studies showing a buffering role for Hsps in evolution.     

In vivo chaperone activity of heat shock protein 70 and thermotolerance

Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.     

Human cerebral cysticercosis: immunolocalization of a sodium-dependent glucose cotransporter (SGLT) in larval and adult tapeworms.

Light microscopic immunocytochemistry was used to examine human brain cysticerci resected from the fourth ventricles of patients who had not been treated with anthelminthic drugs. Tissues were examined from 3 different patients undergoing surgery for treatment of hydrocephalus. A rabbit polyclonal antiserum to the peptide corresponding to amino acids 564-575 unique to the rabbit sodium-dependent, SGLT1 glucose cotransporter labeled with immunoperoxidase, localized immunoreactive SGLT epitopes. This antibody localizes SGLT1 in the apical brush borders of human enterocytes, but is negative in cytoplasm, as well as lateral and basal enterocyte membranes. Taenia solium neurocysticerci were SGLT positive; transporter protein was highly expressed on the surface microvilli of the external cyst wall. The well-developed network of small and larger osmoregulatory ducts within racemose larval cystcerci displayed high expression of SGLT cotransporter, consistent with a resorptive function for this system of tubules. Because water is cotransported with glucose molecules by the SGLT protein, its high expression in neurocysticerci may contribute to the expansive growth of these larvae in subarachnoid and intraventricular sites. The SGLT epitopes were also immunolocalized in gravid proglottids of Taenia saginata, indicating that cotransporter expression persisted in intestinal-dwelling, adult tapeworms. Cotransporter antibody was abundantly localized at the proglottid tegumentary surface and in the lateral osmoregulatory ducts, analogous to the SGLT localization in cysticerci. Furthermore, high expression of this cotransporter was seen in the branches of the uterus, suggesting that SGLT-mediated absorption of glucose and water has an important functional role within the reproductive system of adult tapeworms.     

Hsp70 promotes epithelial sodium channel functional expression by increasing its association with coat complex II and its exit from endoplasmic reticulum

The epithelial sodium channel (ENaC) plays an important role in the homeostasis of blood pressure and of the airway surface liquid, and inappropriate regulation of ENaC results in refractory hypertension (in Liddle syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsp70, is not completely understood. Building on the previous suggestion by our group that Hsp70 promotes ENaC functional and surface expression in Xenopus oocytes, we investigated the mechanism by which Hsp70 acts upon ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αβγ-ENaC and with tetracycline-inducible overexpression of Hsp70, treatment with 1 or 2 μg/ml doxycycline increased total Hsp70 expression ~2-fold and ENaC functional expression ~1.4-fold. This increase in ENaC functional expression corresponded to an increase in ENaC expression at the apical surface of the cells and was not present when an ATPase-deficient Hsp70 was similarly overexpressed. The increase in functional expression was not due to a change in the rate at which ENaC was retrieved from the apical membrane. Instead, Hsp70 overexpression increased the association of ENaC with the Sec24D cargo recognition component of coat complex II, which carries protein cargo from the endoplasmic reticulum to the Golgi. These data support the hypothesis that Hsp70 promotes ENaC biogenesis and trafficking to the apical surface of epithelial cells.