Heat shock proteins (Hsps) have provoked interest not only because of their involvement in human diseases but also for their potential as biomarkers of environmental pollution. Whereas the former interest is covered by numerous reports, the latter is an exciting new field of research. We report the isolation of the full-length cpn60 messenger ribonucleic acid (mRNA) and partial genomic deoxyribonucleic acid from the free-living, environmental sentinel nematode Plectus acuminatus, a species used in classical ecotoxicity tests. Although the primary sequence displays high identity scores to other nematodes and human Cpn60 (75% and 70%, respectively), the intron-exon structure differs markedly. Furthermore, although mRNA levels remained constant after exposure to ZnCl₂ (0–330 μM) under laboratory conditions, protein levels increased significantly in a dose-dependent manner. In conclusion, this first account of molecular genetic similarities and differences of Cpn60 in a neglected nematode taxon provides a valuable insight into its potential uses in gene-based ecotoxicological risk assessment exercises.

Heat shock proteins (Hsps) are overexpressed in a wide range of human cancers and are implicated in tumor cell proliferation, differentiation, invasion, metastasis, death, and recognition by the immune system. We review the current status of the role of Hsp expression in cancer with special emphasis on the clinical setting. Although Hsp levels are not informative at the diagnostic level, they are useful biomarkers for carcinogenesis in some tissues and signal the degree of differentiation and the aggressiveness of some cancers. In addition, the circulating levels of Hsp and anti-Hsp antibodies in cancer patients may be useful in tumor diagnosis. Furthermore, several Hsp are implicated with the prognosis of specific cancers, most notably Hsp27, whose expression is associated with poor prognosis in gastric, liver, and prostate carcinoma, and osteosarcomas, and Hsp70, which is correlated with poor prognosis in breast, endometrial, uterine cervical, and bladder carcinomas. Increased Hsp expression may also predict the response to some anticancer treatments. For example, Hsp27 and Hsp70 are implicated in resistance to chemotherapy in breast cancer, Hsp27 predicts a poor response to chemotherapy in leukemia patients, whereas Hsp70 expression predicts a better response to chemotherapy in osteosarcomas. Implication of Hsp in tumor progression and response to therapy has led to its successful targeting in therapy by 2 main strategies, including: (1) pharmacological modification of Hsp expression or molecular chaperone activity and (2) use of Hsps in anticancer vaccines, exploiting their ability to act as immunological adjuvants. In conclusion, the present times are of importance for the field of Hsps in cancer, with great contributions to both basic and clinical cancer research.

To test the temperature sensitivity of molecular chaperones in poikilothermic animals, we purified the molecular chaperone Hsc70 from 2 closely related notothenioid fishes—the Antarctic species Trematomus bernacchii and the temperate New Zealand species Notothenia angustata—and characterized the effect of temperature on Hsc70 adenosine triphosphatase (ATPase) activity. Hsc70 ATPase activity was measured using [α-³²P]-adenosine triphosphate (ATP)–based in vitro assays followed by separation of adenylates by thin-layer chromatography. For both species, a significant increase in Hsc70 ATPase activity was observed across a range of temperatures that was ecologically relevant for each respective species. Hsc70 from T bernacchii hydrolyzed 2-fold more ATP than did N angustata Hsc70 at 0°C, suggesting that the Antarctic molecular chaperone may be adapted to function more efficiently at extreme cold temperatures. In addition, Q₁₀ measurements indicate differential temperature sensitivity of the ATPase activity of Hsc70 from these differentially adapted fish that correlates with the temperature niche inhabited by each species. Hsc70 from T bernacchii was relatively temperature insensitive, as indicated by Q₁₀ values calculated near 1.0 across each temperature range measured. In the case of Hsc70 purified from N angustata, Q₁₀ values indicated thermal sensitivity across the temperature range of 0°C to 10°C, with a Q₁₀ of 2.714. However, Hsc70 from both T bernacchii and N angustata exhibited unusually high thermal stabilities with ATPase activity at temperatures that far exceeded temperatures encountered by these fish in nature. Overall, as evidenced by in vitro ATP hydrolysis, Hsc70 from T bernacchii and N angustata displayed biochemical characteristics that were supportive of molecular chaperone function at ecologically relevant temperatures.

The antioxidant/electrophile response element (ARE/EpRE) is a cis-acting element involved in redox regulation of c-Ha-ras gene. Protein binding to the ARE/EpRE may be credited to deoxyribonucleic acid sequence; therefore, studies were conducted to evaluate the influence of internal and flanking regions to the 10-bp human c-Ha-ras ARE/ EpRE core (hHaras10) on nuclear protein binding in oxidant-treated vascular smooth muscle cells. A protein doublet bound to an extended oligonucleotide comprising the ARE/EpRE core in genomic context (hHaras27), whereas a single complex bound to hHaras10. Protein binding involved specific interactions of 25- and 23-kDa proteins with hHaras10, and binding of 80-, 65-, and 55-kDa proteins to hHaras27. Competition assays with hNQO₁ and rGSTA2 confirmed the specificity of deoxyribonucleic acid–protein interactions and indicated preferred binding of p25 and p23 to the c-Ha-ras ARE/EpRE. “NNN” sequences within the core afforded unique protein-binding profiles to the c-Ha-ras ARE/ EpRE. In addition, Nrf2 and heat shock protein 90β (p80) were identified as components of the c-Ha-ras ARE/EpRE heterocomplex. We conclude that both internal bases and flanking sequences regulate nuclear protein recruitment and complex assembly on the c-Ha-ras ARE/EpRE.

In the living and working environment, stressful factors, such as noise, can cause health problems including cardiovascular diseases and noise-induced hearing loss. Some heat shock proteins (Hsps) play an important role in protecting cardiac cells against ischemic injury, and antibodies against these Hsps are associated with the development and prognosis of atherogenesis, coronary heart disease, and hypertension. Whether the presence of such antibodies is associated with abnormal electrocardiography (ECG) in stressed autoworkers exposed to chronic noise is presently unknown. Therefore, we investigated the association between the levels of plasma anti-Hsp60 and anti-Hsp70 with electrocardiograph abnormality in 396 autoworkers exposed to different noise levels by using Western blot, ECG, and multivariate logistic regression analysis. The results showed that the increase in levels of anti-Hsp70 was associated with a higher risk of ECG abnormalities characteristic of chronic myocardial ischemia (P < 0.05), conductive abnormality (P < 0.01), or heart displacement (P < 0.05); in contrast, elevated anti-Hsp60 was related to ECG abnormalities characteristic of sinus arrhythmia, chronic myocardial ischemia, and ectopic rhythm (P < 0.01 for all). Overall, high levels of both anti-Hsp70 and anti-Hsp60 were associated with significantly increased risk of ECG abnormalities (odds ratio [OR] = 1.73 and 95% confidence interval [CI] = 1.04–2.86 for anti-Hsp70 and OR = 1.36 and 95% CI = 1.07– 1.72 for anti-Hsp60) with and without adjustment for cumulative noise exposure (OR = 1.96 and 95% CI = 1.20–3.21 for anti-Hsp70 and OR = 3.93 and 95% CI = 1.72–8.92 for anti-Hsp60). These findings suggest that the production of both anti-Hsp70 and anti-Hsp60 may be independent risk factors for the development and progression of abnormal ECG and therefore possibly cardiovascular diseases in autoworkers exposed to occupational noise.

Human tumors frequently present heat shock protein 70 (Hsp70) on their cell membranes, whereas corresponding normal tissues fail to do so. Therefore, an Hsp70 membrane-positive phenotype provided a tumor-specific marker. Moreover, membrane-bound Hsp70 provides a target structure for the cytolytic attack mediated by natural killer (NK) cells. Vitamin A derivatives 13-cis retinoic acid (13-RA) and all-trans retinoic acid (ATRA) and sodium-butyrate (SBU) are known for their redifferentiating capacity. Therefore, we asked the question whether loss in tumorigenicity might be associated with a reduced Hsp70 membrane expression. For our studies we used epithelial colon (CX /CX−) and thyroid (ML-1) cancer cells, with initially different Hsp70 cell surface expression pattern. After treatment up to 7 weeks with freshly prepared 13-RA, ATRA, and SBU at nonlethal concentrations of 10 μM, 1 μM, and 0.5 mM, respectively, growth morphology, Hsp70 levels, and sensitivity toward Hsp70-specific NK cells were compared with that of untreated tumor cells. Significant growth delay was determined in CX tumor cells after 6 weeks treatment with 13-RA. Concomitantly, growth morphology changed from spheroid cell clusters to monolayers. Despite a weak increase in cytosolic Hsp70, the percentage of Hsp70 membrane-positive cells dropped significantly after repeated treatments with 13-RA and ATRA in CX and ML-1 but not in CX− tumor cells. Similar results were observed with SBU. Functionally, the decrease in Hsp70 membrane-positive CX and ML-1 cells correlated with a reduced sensitivity to lysis mediated by NK cells. In summary, redifferentiating agents predominantly affected Hsp70 membrane-positive tumors. The decrease in Hsp70 membrane positivity correlated with a lower sensitivity to NK lysis, growth delay, and altered growth morphology.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that is regulated under conditions of cellular stress. ASK1 phosphorylates c-Jun N-terminal kinase (JNK) and elicits an apoptotic response. ASK1 activity is regulated at multiple levels, 1 of which is through inhibition by cytosolic chaperones of the heat shock protein (Hsp) 70 family. Among the proteins that determine Hsp70 function, CHIP (C-terminus of Hsp70-interacting protein) is a cochaperone and ubiquitin ligase that interacts with Hsp70 through an amino-terminal tetratricopeptide repeat (TPR) domain. Prominent among the cellular functions mediated by CHIP is protection against physiologic stress. Because ASK1 is known to contain a TPR-acceptor site, we examined the role of CHIP in regulating ASK1 function. CHIP interacted with ASK1 in a TPR-dependent fashion and induced ubiquitylation and proteasome-dependent degradation of ASK1. Targeting of ASK1 by CHIP inhibited JNK activation in response to oxidative challenge and reduced ASK1-dependent apoptosis, whereas short interfering RNA (siRNA)-dependent depletion of CHIP enhanced JNK activation. Consistent with its ability to reduce cytoplasmic ASK1 levels, CHIP triggered the translocation of ASK1 partner protein death-associated protein (Daxx) into the nucleus, where it is known to activate an antiapoptotic response. These results indicate that CHIP regulates ASK1 activity by inducing its ubiquitylation and degradation, which, together with its effects on Daxx localization, provides a mechanism for the antiapoptotic effects of CHIP observed in the face of cellular and physiologic stress.

Mutations in the α-crystallin domain of 4 of the small heat shock proteins (sHsp) (Hsp27/HspB1, αA-crystallin/ HspB4, αB-crystallin/HspB5, and HspB8) are responsible for dominant inherited diseases in humans. One such mutation at a highly conserved arginine residue was shown to cause major conformational defects and intracellular aggregation of αA- and αB-crystallins and HspB8. Here, we studied the effect of this Arg mutation on the structure and function of Hsp27. Chinese hamster Hsp27 with Arg148 replaced by Gly (Hsp27R148G) formed dimers in vitro and in vivo, which contrasted with the 12- or 24-subunit oligomers formed by the wild-type protein (Hsp27WT). Despite these alterations, Hsp27R148G had a chaperone activity almost as high as Hsp27WT. The dimers of Hsp27R148G did not further deoligomerize on phosphorylation and like the dimers formed by phosphorylated Hsp27WT were not affected by the deletion of the N-terminal WD/EPF (single letter amino acid code) motif, suggesting that mutation of Arg148, deletion of the N-terminal WD/EPF motif, and phosphorylation of Ser90 may produce similar structural perturbations. Nevertheless, the structure of Hsp27R148G appeared unstable, and the mutated protein accumulated as aggregates in many cells. Both a lower basal level of phosphorylation of Hsp27R148G and the coexpression of Hsp27WT could reduce the frequency of formation of these aggregates, suggesting possible mechanisms regulating the onset of the sHsp-mediated inherited diseases.