In this study we characterized the chaperone functions of Xenopus recombinant Hsp30C and Hsp30D by using an in vitro rabbit reticulocyte lysate (RRL) refolding assay system as well as a novel in vivo Xenopus oocyte microinjection assay. Whereas heat- or chemically denaturated luciferase (LUC) did not regain significant enzyme activity when added to RRL or microinjected into Xenopus oocytes, compared with native LUC, denaturation of LUC in the presence of Hsp30C resulted in a reactivation of enzyme activity up to 80–100 %. Recombinant Hsp30D, which differs from Hsp30C by 19 amino acids, was not as effective as its isoform in preventing LUC aggregation or maintaining it in a folding-competent state. Removal of the first 17 amino acids from the N-terminal region of Hsp30C had little effect on its ability to maintain LUC in a folding-competent state. However, deletion of the last 25 residues from the C-terminal end dramatically reduced Hsp30C chaperone activity. Coimmunoprecipitation and immunoblot analyses revealed that Hsp30C remained associated with heat-denatured LUC during incubation in reticulocyte lysate and that the C-terminal mutant exhibited reduced affinity for unfolded LUC. Finally, we found that Hsc70 present in RRL interacted only with heat-denatured LUC bound to Hsp30C. These findings demonstrate that Xenopus Hsp30 can maintain denatured target protein in a folding-competent state and that the C-terminal end is involved in this function.

According to new hypotheses, extracellular heat shock proteins (Hsps) may represent an ancestral danger signal of cellular death or lysis-activating innate immunity. Recent studies demonstrating a dual role for Hsp70 as both a chaperone and cytokine, inducing potent proinflammatory response in human monocytes, provided support for the hypothesis that extracellular Hsp is a messenger of stress. Our previous work focused on the complement-activating ability of human Hsp60. We demonstrated that Hsp60 complexed with specific antibodies induces a strong classical pathway (CP) activation. Here, we show that another chaperone molecule also possesses complement-activating ability. Solid-phase enzyme-linked immunosorbent assay was applied for the experiments. Human Hsp70 activated the CP independently of antibodies. No complement activation was found in the case of human Hsp90. Our data further support the hypothesis that chaperones may messenger stress to other cells. Complement-like molecules and primitive immune cells appeared together early in evolution. A joint action of these arms of innate immunity in response to free chaperones, the most abundant cellular proteins displaying a stress signal, may further strengthen the effectiveness of immune reactions.

Human diploid fibroblasts (HDFs) exposed to subcytotoxic stresses under H₂O₂, tert-butylhydroperoxide (t-BHP), and ethanol (EtOH) undergo stress-induced premature senescence (SIPS) characterized by many biomarkers of HDFs replicative senescence. Among these biomarkers are a growth arrest, an increase in the senescence-associated β-galactosidase activity, a senescent morphology, an overexpression of p21^waf-1 and the subsequent inability to phosphorylate pRb, the presence of the common 4977-bp mitochondrial deletion, and an increase in the steady-state level of several senescence-associated genes such as apolipoprotein J (apo J). Apo J has been described as a survival gene against cytotoxic stress. In order to study whether apo J would be protective against cytotoxicity SIPS and replicative senescence in human fibroblasts, a full-length complementary deoxyribonucleic acid of apo J was transfected into WI-38 HDFs and SV40-transformed WI-38 HDFs. The overexpression of apo J resulted in an increased cell survival after t-BHP and EtOH stresses at cytotoxic concentrations. In addition, when WI-38 HDFs were exposed to 5 subcytotoxic stresses with EtOH or t-BHP, in conditions that were previously shown to induce SIPS, a lower induction of 2 biomarkers of SIPS was observed in HDFs overexpressing apo J. No effect of apo J overexpression was observed on the proliferative life span of HDFs, even if apo J overexpression triggered osteonectin (SPARC) overexpression, which was shown to decrease the mitogenic potential of platelet-derived growth factor but not of other common growth-inducing conditions. Apo J senescence–related overexpression is proposed to have antiapoptotic rather than antiproliferative effects.

Emotional stress affects cellular integrity in many tissues including the heart. Much less is known about the effects of social stress. We studied the effect of emotional (immobilization with or without cold exposure) or social (intermale confrontation) stress in mice. Tissue injury was measured by means of the release of enzyme activities to blood plasma: lactate dehydrogenase (LDH), creatine kinase (CK), aspartate transaminase (AST), and alanine transaminase (ALT). Tape-immobilization increased all these activities in the plasma. AST-ALT ratio was also increased in these animals. Electrophoretic analysis of CK isoenzymes showed the appearance of CK-MB. These results indicate that the heart was injured in immobilized mice. Analysis of LDH isoenzymes and measurement of α-hydroxybutyrate dehydrogenase (HBDH) activity suggests that other tissues, in addition to the heart, contribute to the increase in plasma LDH activity. Restraint in small cylinders increased plasma LDH, CK, AST, and ALT activities, but to lower levels than in tape immobilization. Because the decrease in liver glycogen and the increase in plasma epidermal growth factor (EGF) were also smaller in restraint than in the tape-immobilization model of emotional stress, we conclude that the former is a less intense stressor than the latter. Cold exposure during the restraint period altered the early responses to stress (it enhanced liver glycogen decrease, but abolished the increase in plasma EGF concentration). Cold exposure during restraint enhanced heart injury, as revealed by the greater increase in CK and AST activities. Intermale confrontation progressively decreased liver glycogen content. Plasma EGF concentration increased (to near 100 nM from a resting value of 0.1 nM) until 60 minutes, and decreased thereafter. Confrontation also affected cellular integrity in some tissues, as indicated by the rise in plasma LDH activity. However, in this type of stress, the heart appeared to be specifically protected because there was no increase in plasma CK activity, and both AST and ALT increased, but the AST-ALT ratio remained constant. Habituation to restraint (1 h/d, 4 days) made mice resistant to restraint-induced tissue injury as indicated by the lack of an increase in plasma LDH, CK, AST, or ALT activities. Similar general protection against homotypic stress-induced injury was observed in mice habituated to intermale confrontation.

The eukaryotic Hsp60 cytoplasmic chaperonin CCT (chaperonin containing the T-complex polypeptide–1) is essential for growth in budding yeast, and mutations in individual CCT subunits have been shown to affect assembly of tubulin and actin. The present research focused mainly on the expression of the CCT subunits, CCTα and CCTβ, in yeast (Saccharomyces cerevisiae). Previous studies showed that, unlike most other chaperones, CCT in yeast does not undergo induction following heat shock. In this study, messenger ribonucleic acid (mRNA) and protein levels of CCT subunits following exposure to low temperatures, were examined. The Northern blot analysis indicated a 3- to 4-fold increase in mRNA levels of CCTα and CCTβ genes after cold shock at 4°C. Interestingly, Western blot analysis showed that cold shock induces an increase in the CCTα protein, which is expressed at 10°C, but not at 4°C. Transfer of 4°C cold-shocked cells to 10°C induced a 5-fold increase in the CCTα protein level. By means of fluorescent immunostaining and confocal microscopy, we found CCTα to be localized in the cortex and the cell cytoplasm of S. cerevisiae. Localization of CCTα was not affected at low temperatures. Co-localization of CCT and filaments of actin and tubulin was not observed by microscopy. The induction pattern of the CCTα protein suggests that expression of the chaperonin may be primarily important during the recovery from low temperatures and the transition to growth at higher temperatures, as found for other Hsps during the recovery phase from heat shock.

In the ligand-binding inactive state, the steroid receptor heterocomplex contains Hsp90, Hsp70, high–molecular weight immunophilins, and other proteins. Hsp90 acts in association with co-chaperones to maintain the native state of the receptor within the cells. It was reported earlier that Hsp90 might not be as important for the androgen receptor (AR) activity as for the glucocorticoid receptor (GR) and the progesterone receptor (PR) activities. We used the Hsp90 inhibitor geldanamycin (GA) to explore the role of Hsp90 in the function of the AR heterocomplex. GA selectively binds to Hsp90 and inhibits its activity, leading to the loss of steroid receptor activity, and frequently, its degradation. In our study, LNCaP prostate cancer cells were treated with GA for 30 minutes or 24 hours, in the presence of mibolerone, a synthetic androgen. GA reduced the androgen-induced AR protein levels to 15 % after 24 hours of treatment. Several androgen up-regulated genes, including immunophilin FKBP51 and prostate specific antigen (PSA), were reduced by GA treatment. In cells treated with GA after transfection with a PSA promoter or an androgen response element–driven reporter gene, AR-mediated transactivation of reporter gene expression was reversibly inhibited by GA. Loss of androgen-binding ability and AR levels was attributed to reduced transcription of AR-regulated gene expression. Degradation rate of ³⁵S-labeled AR was significantly increased by GA in the presence or absence of mibolerone. GA induced the degradation of AR through the proteasomal pathway. AR in cells treated with proteasomal inhibitor lactacystin, was insoluble in Nonidet P-40 (NP40)-based buffer and could not restore the androgen-binding ability. We report here that GA treatment disrupted both hormone-binding activity and receptor protein stability, resulting in a dramatic loss of androgen-induced gene activation. These results show that Hsp90 activity is important for both the chaperone-mediated folding of the AR into a high-affinity ligand-binding conformation and the functional activity of the AR.

We have previously characterized the unique organization of the U14 small nucleolar ribonucleic acid (snoRNA) gene in Chinese hamster HA-1 cells. The single copy of the hsc70/U14 gene is the only source for the production of both U14 snoRNA species and hsc70 messenger ribonucleic acid (mRNA) in these cells. Here we report that the accumulations of U14 snoRNA and hsc70 mRNA are different in response to various stress conditions, although both of them are transcribed in a single primary transcript. Heat shock induced an increased accumulation of both U14 snoRNA and hsc70 mRNA. On the other hand, exposure to sodium arsenite or azetidine induced an increased accumulation of hsc70 mRNA, but did not lead to a concomitant increase in the level of U14 snoRNA. Under normal growth conditions, the variations in the levels of U14 snoRNA and hsc70 mRNA, in the different phases of the cell cycle, are correlated. The increased expression of U14 snoRNA and hsc70 mRNA, and the hsc70 protein induced specifically by heat shock suggest that they participate in the repair process of heat-induced damage to macromolecular complexes involved in the synthesis and processing of ribosomal RNA.

This paper is dedicated to the memory of Professor David A. Walsh (1945–2000), of the University of New South Wales, Australia, who pioneered research on the heat shock response during early mammalian development.

Stressful stimuli can elicit 2 distinct reactive cellular responses, the heat shock (stress) response and the activation of cell death pathways. Most studies on the effects of hyperthermia on the mammalian nervous system have focused on the heat shock response, characterized by the transient induction of Hsps, which play roles in repair and protective mechanisms. This study examines the effect of hyperthermia on the induction of cell death via apoptosis, assayed by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling and active caspase 3 cytochemistry, in the adult rat brain, testis, and thymus. Results show that a fever-like increase in temperature triggered apoptosis in dividing cell populations of testis and thymus, but not in mature, postmitotic cells of the adult cerebellum. These differential apoptotic responses did not correlate with whole-tissue levels of Hsp70 induction. We further investigated whether dividing neural cells were more sensitive to heat-induced apoptosis by examining the external granule cell layer of the cerebellum at postnatal day 7 and the neuroepithelial layers of the neocortex and tectum at embryonic day 17. These proliferative neural regions were highly susceptible to hyperthermia-induced apoptosis, suggesting that actively dividing cell populations are more prone to cell death induced by hyperthermia than fully differentiated postmitotic neural cells.

The benzoquinone ansamycin geldanamycin (GA) stimulates proteasome-mediated degradation of plasma membrane–associated ErbB2, a receptor tyrosine kinase. Drug sensitivity is mediated by ErbB2's kinase domain and occurs subsequent to the disruption of Hsp90 interaction with this domain. Full-length ErbB2 is efficiently processed via the endoplasmic reticulum (ER) and Golgi network, so that at steady state most of the detectable protein is plasma membrane associated. However, previous studies have also demonstrated the GA sensitivity of newly synthesized ErbB2, normally a minor component of the total cellular pool of the kinase. Drug sensitivity of nascent ErbB2 is distinguished by 2 characteristics—protein instability and inability to traverse the ER. As nascent ErbB2 can associate with both cytoplasmic Hsp90 and its ER luminal homolog Grp 94, also a GA-binding protein, the purpose of this study was to examine the relative contributions of the cytoplasmic and ER luminal domains of ErbB2 to the GA sensitivity of the nascent kinase. By studying the drug sensitivity of ErbB2/DK, a construct lacking ErbB2's cytoplasmic kinase domain, and by examining the activity of a GA derivative that preferentially binds Hsp90, we conclude that both the stability and the maturation of nascent ErbB2 are regulated by its cytoplasmic, Hsp90-interacting domain.

Using homologous molecular probes, we examined the influence of equivalent temperature shifts on the in vivo expression of genes coding for a constitutive heat shock protein (Hsc70), heat shock proteins (Hsps) (Hsp70 and Hsp90), and polyubiquitin, after acclimation in the American lobster, Homarus americanus. We acclimated sibling, intermolt, juvenile male lobsters to thermal regimes experienced during overwintering conditions (0.4 ± 0.3°C), and to ambient Pacific Ocean temperatures (13.6 ± 1.2°C), for 4–5 weeks. Both groups were subjected to an acute thermal stress of 13.0°C, a temperature shift previously found to elicit a robust heat shock response in ambient-acclimated lobsters. Animals were examined after several durations of acute heat shock (0.25–2 hours) and after several recovery periods (2–48 hours) at the previous acclimation temperature, following a 2-hour heat shock. Significant inductions in Hsp70, Hsp90, and polyubiquitin messenger RNA (mRNA) levels were found for the ambient-acclimated group. Alternatively, for the cold-acclimated group, an acute thermal stress over an equivalent interval resulted in no induction in mRNA levels for any of the genes examined. For the ambient-acclimated group, measurements of polyubiquitin mRNA levels showed that hepatopancreas, a digestive tissue, incurred greater irreversible protein damage relative to the abdominal muscle, a tissue possessing superior stability over the thermal intervals tested.

In the present study we used a murine melanoma model to investigate the effect of the 25-kDa heat shock protein (Hsp25) on natural killer (NK) cytotoxicity. The melanoma lines K1735-Cl23 (low metastatic potential) and K1735-M2 (high metastatic potential) were transfected with hsp25 and a control plasmid. Highly purified interleukin (IL)-2–stimulated DX-5 NK cells showed enhanced lysis of Hsp25-overexpressing K1735-Cl23 targets in comparison with controls. In contrast, there was no difference in susceptibility to lysis by purified IL-2–stimulated DX-5 NK cells between Hsp25-overexpressing and control-transfected K1735-M2 targets. Fluorescence-activated cell sorter analysis revealed that Hsp25 is displayed on the cell surface independently of Hsp25 overexpression and metastatic phenotype. Thus, surface localization of Hsp25 does not correlate with the target cell susceptibility to killing. To sum up, a cytoplasmic overexpression of Hsp25 is associated with an increased susceptibility to lysis by DX-5 NK cells in the low-metastatic murine melanoma model investigated.

We have isolated a complementary deoxyribonucleic acid clone that encodes the protein disulfide isomerase of Bombyx mori (bPDI). This protein has a putative open reading frame of 494 amino acids and a predicted size of 55.6 kDa. In addition, 2 thioredoxin active sites, each with a CGHC sequence, and an endoplasmic reticulum (ER) retention signal site with a KDEL motif were found at the C-terminal. Both sites are typically found in members of the PDI family of proteins. The expression of bPDI messenger ribonucleic acid (mRNA) was markedly increased during ER stress induced by stimulation with calcium ionophore A23187, tunicamycin, and dithiothreitol, all of which are known to cause an accumulation of unfolded proteins in the ER. We also examined the tissue distribution of bPDI mRNA and found pronounced expression in the fat body of insects. Hormonal regulation studies showed that juvenile hormone, insulin, and a combination of juvenile hormone and transferrin (although not transferrin alone) affected bPDI mRNA expression. A challenge with exogenous bacteria also affected expression, and the effect peaked 16 hours after infection. These results suggest that bPDI is a member of the ER-stress protein group, that it may play an important role in exogenous bacterial infection of the fat body, and that its expression is hormone regulated.