The effect of 15 consecutive days of heat–exercise acclimation on heat shock protein 70

The purpose of this study was to investigate the alterations in serum heat shock protein (Hsp) 70 levels during a 15-consecutive-day intermittent heat–exercise protocol in a 29-year-old male ultra marathon runner. Heat acclimation, for the purpose of physical activities in elevated ambient temperatures, has numerous physiological benefits including mechanisms such as improved cardiac output, increased plasma volume and a decreased core temperature (T _c). In addition to the central adaptations, the role of Hsp during heat acclimation has received an increasing amount of attention. The acclimation protocol applied was designed to correspond with the athlete’s tapering period for the 2007 Marathon Des Sables. The subject (VO₂max = 50.7 ml·kg⁻¹·min⁻¹, peak power output [PPO] = 376 W) cycled daily for 90 min at a workload corresponding to 50% of VO₂max in a temperature-controlled room (average WBGT = 31.9 ± 0.9°C). Venous blood was sampled before and after each session for measurement of serum osmolality and serum Hsp70. In addition, T _c, heart rate (HR) and power output (PO) was measured throughout the 90 min to ensure that heat acclimation was achieved during the 15-day period. The results show that the subject was successfully heat acclimated as seen by the lowered HR at rest and during exercise, decreased resting and exercising T _c and an increased PO. The heat exercise resulted in an initial increase in Hsp70 concentrations, known as thermotolerance, and the increase in Hsp70 after exercise was inversely correlated to the resting values of Hsp70 (Spearman’s rank correlation = −0.81, p < 0.01). Furthermore, the 15-day heat–exercise protocol also increased the basal levels of Hsp70, a response different from that of thermotolerance. This is, as far as we are aware, the first report showing Hsp70 levels during consecutive days of intermittent heat exposure giving rise to heat acclimation. In conclusion, a relatively longer heat acclimation protocol is suggested to obtain maximum benefit of heat acclimation inclusive of both cellular and systemic adaptations.     

Application of magnetic field–induced heat shock protein 70 for presurgical cytoprotection

To develop an alternative to hyperthermia for the induction of hsp70 for presurgical cytoprotection, we investigated the optimal exposure conditions for magnetic field induction of hsp70. Normal human breast cells (HTB124) were exposed to 60-Hz magnetic fields and hsp70 levels were measured following three different exposure conditions: continuous exposure up to 3 h, a single 20-min exposure, and a single 20-min exposure followed by repeated 20-min exposures at different field strengths. In cells exposed continuously for 3 h, hsp70 levels peaked (46%) within 20 min and returned to control levels by 2 h. Following a single 20-min exposure, the return of hsp70 levels to control values extended to more than 3 h. When cells underwent a 20-min exposure followed by repeated 20-min exposures (restimulation) with different field strengths, additional increases in hsp70 levels were induced: 31% at 1 h, 41% at 2 h, and 30% at 3 h. J. Cell. Biochem. 71:577–583, 1998. © 1998 Wiley-Liss, Inc.     

Is heat shock protein re-induction during tolerance related to the stressor-specific induction of heat shock proteins?

The existence of stressor-specific induction programs of heat shock proteins (hsps) leads us to analyze the possible occurrence of a stressor-specific tolerance induced by either heat shock, arsenite, or cadmium. As a measure of this tolerance re-induction of hsps was studied. In this paper, we tested whether the refractory state is either valid for each specific hsp (implying independent regulation of every member of the heat shock protein family) or extends from small subsets of the hsp-family to even larger groups of proteins (indicating a more common denominator in their regulation). (Re-)induction of hsps does not seem to be regulated at the level of each individual hsp since differences in induced synthesis of hsps between two stressor conditions are not supplemented systematically upon the sequential application of the two stressors. The most notable example in this respect is hsp60. A pretreatment with cadmium, which hardly induces synthesis of this hsp, does induce a tolerance to (re)-induction by heat shock, which normally induces hsp60. This suggests the existence of a more common denominator regulating the coordinate expression of at least some hsps. From our data we conclude that the degree, but not the pattern, of hsp re-induction is influenced by the type of stressor used in the pretreatment. The pattern of hsps induced by a secondary applied stressor still shows most of its stressor-specificity and seems to be independent of any pretreatment. The possible implications of stressor-specificity are discussed. © 1996 Wiley-Liss, Inc.     

Heat shock factor binds to heat shock elements upstream of heat shock protein 70a and Samui genes to confer transcriptional activity in Bombyx mori diapause eggs exposed to 5°C

To understand the molecular mechanisms of how 5 °C-incubation activates mRNA expression of Hsp70a and Samui genes in Bombyx mori diapause eggs, we first searched the 5′-upstream regions of the Hsp70a and Samui genes for heat shock elements (HSEs) and found two regions [Hsp70aHSE-1 (−95 to −58) and -2 (−145 to −121), and SamuiHSE-1 (−84 to −55) and -2 (−304 to −290)] corresponding to HSEs (repeats of nGAAn and/or nTTCn). We cloned four cDNAs encoding heat shock factor (HSF)-a2 (627 amino acids), -b (685 aa), -c (682 aa) and -d (705 aa), which were produced by alternative splicing. When we exposed diapause eggs to 5 °C beginning at 2 day post-oviposition to break diapause, HSFd mRNA only increased after chilling for 6–8 days, a pattern very similar to those of Hsp70a and Samui mRNAs. To examine further whether HSFd binds to the respective HSEs, we carried out a gel shift assay using HSFd protein expressed in a cell-free system and the isolated HSEs; migration of the respective digoxigenin(DIG)-labeled HSE-1 and -2 of Hsp70a and Samui was retarded by addition of HSFd; the retarded bands disappeared after addition of the corresponding unlabeled HSE-1 and -2 as competitors, but were not affected by addition of the respective mutated unlabeled HSE-1 and -2. These results indicated that HSFd protein binds to the respective HSEs and may activate mRNA expression of Hsp70a and Samui genes upon exposure of diapause eggs to 5 °C.     

Inhibition of α-synuclein aggregation by small heat shock proteins

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies.     

Molecular characterization of heat shock proteins 90 (HSP83?) and 70 in tropical strains of Bombyx mori

Following the concept of whole organism, we have extracted total protein from the Bombyx mori for the identification and analysis of HSPs. Expression of 90 kDa HSP in first, second and third instars, 84 kDa in fourth instar and 90‐, 84‐, 62‐, 60‐, 52‐ and 33‐kDa HSPs in fifth instar larvae of tropical polyvoltine and bivoltine silkworm strains were obvious. Further, we have combined single and 2‐DE with MALDI‐TOF for analysis of BmHSPs. Ninety kilodalton band excised from 1‐DE gel was identified as HSP83 by MALDI‐TOF‐MS. The immunoblot analysis confirmed the expression of HSP90 in all the instars larvae of B. mori. Heat shock‐induced protein spots were excised from 2‐DE gels for MALDI‐TOF‐MS analysis. The Mascot search results are for HSP68, HSC70‐1 and HSP70Ba in Pure Mysore, and major HSP70Bbb, HSP68, HSC‐3 and HSP83 in NB₄D₂. Multiple sequence alignment explicit the variations in amino acid sequence between Pure Mysore and NB₄D₂. Notably, the PMF of spot 2 matched the coding sequence of B. mori and its gene annotation was determined on chromosome 9. With this novel approach, expression of BmHSP90 was confirmed in all the instars and uncovered isoforms of BmHSP70, which provided unequivocal insight to analyze and understand the biological significance in B. mori.     

Increased heat shock protein 70 expression following toxicant-mediated cytotoxicity: a ubiquitous marker of toxicant exposure?

The up-regulation of heat shock protein (HSP) expression has been proposed as a general biomarker of cellular protection against various environmental stresses and chemicals. The present study investigated the possibility of using HSP70 up-regulation as a biomarker of toxicant exposure in vitro. Cells of a rat hepatoma cell line (FGC4) were exposed to concentrations of 1,3-dichloroacetone, duroquinone, diquat dibromide, menadione, hydrogen peroxide, cadmium chloride (CdCl2) and sodium (meta)arsenite (NaAsO2) that elicited 20-50% cytotoxicity over a 24-hour period, and HSP70 levels were measured by ELISA. Up-regulation of HSP70 expression was demonstrated following treatment with menadione, CdCl2 and NaAsO2, but not with the other chemicals tested. A shorter exposure time (6 hours) and/or the use of non-toxic concentrations reduced the level of HSP70 up-regulation with menadione, CdCl2 and NaAsO2, but did not uncover any up-regulation with the other chemicals. Although the toxicity of the majority of the chemicals tested is believed to involve an oxidative stress component, the results of this study clearly demonstrate that up-regulation of HSP70 expression cannot be used as a general biomarker of toxicant exposure in vitro.     

Antimicrobial activity and mechanism of action of a novel cationic α-helical octadecapeptide derived from heat shock protein 70 of rice

Hsp70(241–258), an octadecapeptide derived from the heat shock protein 70 (Hsp70) of rice (Oryza sativa L. japonica), is a novel cationic α-helical antimicrobial peptide (AMP) that contains four lysine, two arginine, and two histidine residues. The antimicrobial activity of Hsp70(241–258) against Porphyromonas gingivalis, a periodontal pathogen, and Candida albicans, an opportunistic fungal pathogen, was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. The 50% growth-inhibitory concentrations of Hsp70(241–258) against P. gingivalis and C. albicans cells were 63 μM and 70 μM, respectively. Hsp70(241–258) had little or no hemolytic activity even at 1 mM, and showed negligible cytotoxicity up to 300 μM. The degrees of calcein leakage from large unilamellar vesicles, which mimic the membranes of Gram-negative bacteria, and 3,3′-dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of Hsp70(241–258) increased in a concentration-dependent manner. When Hsp70(241–258) was added to calcein-acetoxymethyl ester-loaded C. albicans cells, calcein release from the cells increased in a concentration-dependent manner. Flow cytometric analysis also showed that the percentages of C. albicans cells stained with propidium iodide, a DNA-intercalating dye, increased as the concentration of Hsp70(241–258) added was increased. Therefore, Hsp70(241–258) appears to exhibit antimicrobial activity against P. gingivalis and C. albicans through membrane disruption. These results suggest that Hsp70(241–258) could be useful as a safe and potent AMP against P. gingivalis and C. albicans in many fields of health care, especially in the control of oral infections.     

Heteromeric complexes of heat shock protein 70 (HSP70) family members,including Hsp70B′, in differentiated human neuronal cells

Human neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis have been termed “protein misfolding disorders.” Upregulation of heat shock proteins that target misfolded aggregation-prone proteins has been proposed as a potential therapeutic strategy to counter neurodegenerative disorders. The heat shock protein 70 (HSP70) family is well characterized for its cytoprotective effects against cell death and has been implicated in neuroprotection by overexpression studies. HSP70 family members exhibit sequence and structural conservation. The significance of the multiplicity of HSP70 proteins is unknown. In this study, coimmunoprecipitation was employed to determine if association of HSP70 family members occurs, including Hsp70B′ which is present in the human genome but not in mouse and rat. Heteromeric complexes of Hsp70B′, Hsp70, and Hsc70 were detected in differentiated human SH-SY5Y neuronal cells. Hsp70B′ also formed complexes with Hsp40 suggesting a common co-chaperone for HSP70 family members.     

Constitutive heat shock protein 70 interacts with α-enolase and protects cardiomyocytes against oxidative stress

Constitutive heat shock protein 70 (Hsc70) is a molecular chaperone that has been shown to protect cardiomyocytes against oxidative stress. However, the molecular mechanism responsible for this protection remains uncertain. To understand the mechanism associated with the myocardial protective role of Hsc70, we have embarked upon a systematic search for Hsc70-interacting proteins. Using adenosine diphosphate (ADP) affinity chromatography and mass spectrometry, we have identified α-enolase, a rate-limiting enzyme in glycolysis, as a novel Hsc70-interacting protein in the myocardium of both sham and myocardial ischemia-reperfused Sprague-Dawley rat hearts. This interaction was confirmed by co-immunoprecipitation (IP) assays in the myocardial tissues and H9c2 cardiomyocytes and protein overlay assay (POA). It was further shown that Hsc70-overexpression alleviated the H(2)O(2)-induced decrease of α-enolase activity and cell damage, and Hsc70 deficiency aggravated the decrease of α-enolase activity and cell damage in H(2)O(2) treated H9c2 cells. Our research suggests that the protective effect of Hsc70 on the cardiomyocytes against oxidative stress is partly associated with its interaction with α-enolase.     

Constitutive heat shock protein 70 interacts with α-enolase and protects cardiomyocytes against oxidative stress

Abstract

Constitutive heat shock protein 70 (Hsc70) is a molecular chaperone that has been shown to protect cardiomyocytes against oxidative stress. However, the molecular mechanism responsible for this protection remains uncertain. To understand the mechanism associated with the myocardial protective role of Hsc70, we have embarked upon a systematic search for Hsc70-interacting proteins. Using adenosine diphosphate (ADP) affinity chromatography and mass spectrometry, we have identified α-enolase, a rate-limiting enzyme in glycolysis, as a novel Hsc70-interacting protein in the myocardium of both sham and myocardial ischemia-reperfused Sprague–Dawley rat hearts. This interaction was confirmed by co-immunoprecipitation (IP) assays in the myocardial tissues and H9c2 cardiomyocytes and protein overlay assay (POA). It was further shown that Hsc70-overexpression alleviated the H₂O₂-induced decrease of α-enolase activity and cell damage, and Hsc70 deficiency aggravated the decrease of α-enolase activity and cell damage in H₂O₂ treated H9c2 cells. Our research suggests that the protective effect of Hsc70 on the cardiomyocytes against oxidative stress is partly associated with its interaction with α-enolase.     

Targeting HSP70: The second potentially druggable heat shock protein and molecular chaperone?

The HSF1-mediated stress response pathway is steadily gaining momentum as a critical source of targets for cancer therapy. Key mediators of this pathway include molecular chaperones such as heat shock protein (HSP) 90. There has been considerable progress in targeting HSP90 and the preclinical efficacy and signs of early clinical activity of HSP90 inhibitors have provided proof-of-concept for targeting this group of proteins. The HSP70 family of molecular chaperones are also key mediators of the HSF-1-stress response pathway and have multiple additional roles in protein folding, trafficking and degradation, as well as regulating apoptosis. Genetic and biochemical studies have supported the discovery of HSP70 inhibitors which have the potential for use as single agents or in combination to enhance the effects of classical chemotherapeutic or molecularly targeted agents including HSP90 inhibitors. Here we provide a perspective on the progress made so far in designing agents which target the HSP70 family.     

Why do cells require heat shock proteins to survive heat stress?

The cellular response to heat stress includes the induction of a group of proteins called the Heat Shock Proteins, whose functions include the synthesis of the thermoprotectant trehalose, refolding of denatured proteins, and ubiquitin- and proteasome-dependent degradation. Recent studies show that simply increasing the activity of ubiquitin- and proteasome-dependent degradation can replace the essential functions played by the induction of heat shock proteins during a heat stress. These results suggest that accumulation of denatured or aggregated proteins is the reason for the loss of cell viability due to heat stress.     

Metal chelatorNNN’ N’-tetrakis-(2-pyridylmethyl)ethylene diamine inhibits the induction of heat shock protein 70 synthesis by heat in cultured keratinocytes

Heat shock protein (HSP) synthesis results from various types of injury, including heat shock (HS) and some oxidants. The intracellular signals leading to HSP synthesis are not yet fully elucidated. We have studied the influence ofNNN’N’-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), a metal chelator known to induce cellular zinc and copper deprivation, on resistance to heat and on hsp70 synthesis in HaCaT keratinocytes. TPEN was shown to sensitize HaCaT cells to heat shock. The effect of TPEN was neutralized by equimolar Zn²⁺. By the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Western blotting characterization of hsp70, it was shown that cultured HaCaT cells constitutively express the inducible form of hsp70. The application of TPEN alone slightly increases the level of hsp70 but inhibits its induction by HS. This inhibitory effect is related to metal deprivation, because it is eliminated when Cu²⁺ or Zn²⁺ ions are supplied together with TPEN. These results suggest that these metals are involved in the expression by keratinocytes of a stress protein which has a protective action against environmental stress.     

Monophosphoryl lipid A induces pharmacologic ‘preconditioning’ in rabbit hearts without concomitant expression of 70-kDa heat shock protein

The purpose of this study was to evaluate the protective effect of a new endotoxin analogue, monophosphoryl lipid A (MLA) in a rabbit model of myocardial ischemia/reperfusion and to show if this protection was mediated via synthesis of 70 kDa heat shock protein (HSP 70). Three groups of New Zealand White rabbits underwent 30 min coronary occlusion, followed by 4 hours reperfusion. First group of rabbits (n = 6) were treated with 0.35 ml vehicle (40 % propylene glycol, 10 % ethanol in water). The second and third group of rabbits (n = 6–8) were treated with MLA (35 g/kg, i.v.) 12 and 24 hours prior to ischemia and reperfusion. MLA treatment either 12 or 24 h prior to ischemia/reperfusion demonstrated significantly reduced infarct size (12.5 ± 1.7 and 14.7 ± 2.1% for 12 and 24 h) when compared with vehicle control (40.4 ± 8.6%, mean ± S.E.M, p < 0.05). No significant differences in the infarct size was observed between the 12 and 24 h MLA treated groups. The area at risk was not significantly different between the three groups. Baseline values of heart rate, systolic and diastolic blood pressure were not significantly different between the control and MLA treated groups. However, the systolic as well as diastolic blood pressure during reperfusion were significantly lower in rabbits treated with MLA. Western blot analysis of the protein extracts of the hearts (n = 2/group) demonstrated no increase in the expression of the inducible form of HSP 70 following treatment with MLA. We conclude that MLA has significant anti-infarct effect in rabbit which is not mediated by the cardioprotective protein HSP 70. The anti-infarct effect of this drug is superior to the reported protective effects of delayed ischemic or heat stress preconditioning. We hypothesize that the pharmacologic preconditioning afforded by MLA is accomplished via a unique pathway that bypasses the usual intracellular signaling pathways which lead to the myocardial protection with the expression of heat shock proteins.A preliminary form of this paper was presented at the XV World Congress of the International Society for Heart Research held in Prague, Czech Republic [17].This work was supported in part by grants HL 46763 and 51045 from Natinal Institute of Health and a grant from RIBI ImmunoChem Research, Hamilton, Montana. JBS was supported from a training grant HL 07537 from National Institutes of Health.This article was published in Molecular and Cellular Biochemistry 156: 1–8, 1996. Kluwer Academic Publishers regret the publication of the incorrect version.     

The protective role of heat shock protein against cardiomyocyte apoptosis by mitochondrial pathway

Apoptosis is an important contributor to heart diseases in which ischemia and hypoxia are key elements. Previous studies have shown that cardiomyocyte can be protected from ischemia-reperfusion (I/R) injury by heat shock proteins (HSP). However, to date the protective roles of HSP against cardiomyocyte apoptosis has not been confirmed.The present study was designed to explore the effects of mitochondrial pathway in the protective role of heat shock protein against cardiomyocyte apoptosis Cardiomyocyte apoptosis was induced by in vivo mouse heart ischemia-reperfusion (I/R) injury and by hydrogen peroxide (H2O2, 0.5mM) in cultured neonatal rat cardiomyocyte and C2C12 cell line. (2) Cardilmyocyte apoptosis was determined