The heat shock chaperones mortalin/mitochondrial heat shock protein 70 (mtHsp70) and Hsp60 are found in multiple subcellular sites and function in the folding and intracellular trafficking of many proteins. The chaperoning activity of these 2 proteins involves different structural and functional mechanisms. In spite of providing an excellent model for an evolutionarily conserved molecular “brotherhood,” their individual functions, although overlapping, are nonredundant. As they travel to various locations, both chaperones acquire different binding partners and exert a more divergent involvement in tumorigenesis, cellular senescence, and immunology. An understanding of their functional biology may lead to novel designing and development of therapeutic strategies for cancer and aging.

Heat shock protein (Hsp) 72 is a cytosolic protein that also is present in the circulation. Extracellular Hsp72 (eHsp72) is inducible by exercise and is suggested to act as a danger signal to the immune system. The adaptive response of eHsp72 to repeated exercise-heat exposures in humans remains to be determined. An intracellular animal study found a reduced Hsp72 response, with no change in resting levels, during heat stress after a single day of passive heat acclimation. The current study therefore tested whether adaptations in human eHsp72 levels would similarly occur 24 hours after a single exercise-heat exposure. Seven males completed cycle exercise (42.5% V̇_O2peak for 2 hours) in a hot, humid environment (38°C, 60% relative humidity) on each of 2 consecutive days. Blood samples were obtained from an antecubital vein before exercise and 0 hours and 22 hours postexercise for the analysis of eHsp72. Exercise-heat stress resulted in enhanced eHsp72, with a similar absolute increase found on both days (day 1: 1.26 ng/mL [0.80 ng/mL]; day 2: 1.29 ng/mL [1.60 ng/mL]). Resting eHsp72 decreased from rest on day 1 to day 2's 22-hour postexercise sample (P < 0.05). It is suggested that the reduction in resting eHsp72 after 2 consecutive exercise-heat exposures is possibly due to an enhanced removal from the circulation, for either immunoregulatory functions, or for improved cellular stress tolerance in this initial, most stressful period of acclimation.

We have examined the A431 (human epidermoid carcinoma) and HT29 (human colorectal carcinoma) cellular responses evoked by lectins of dietary origin, Jacalin of Artocarpus integrifolia (native jacalin; nJacalin), peanut agglutinin (PNA) of Arachis hypogea, and recombinant single-chain jacalin (rJacalin), which has the same protein backbone but ∼100-fold less affinity for carbohydrates than nJacalin. All three lectins (nJacalin, rJacalin, and PNA) are cycotoxic inhibitors of proliferation of A431 cells. However, cells recover once jacalin but not PNA have been removed from the growth medium. Treatment of nJacalin results in morphologically visible cell rounding while retaining the membrane integrity when treated at 40 μg ml⁻¹, but treatment with PNA did not induce such changes. The observed cell rounding was found to be due to stress as the phosphorylation of caveolin-1 (at tyr¹⁴), p38 but not c-Jun N-terminal kinase were up-regulated, while PNA did not up-regulate the phosphorylation of the same. Jacalin also down-regulated the phosphorylation of the epidermal growth factor receptor and extracellular signal regulated kinase in contrast to PNA, which failed to down-regulate the same. Confocal microscopic studies reveal that jacalin is not internalized, unlike the lectin of Agaricus bisporous. Analysis of the proteins that bind to an nJacalin-sepharose column revealed the binding of six to eight proteins, and significant among them is a protein at ∼110 kDa, which appears to be oxygen-regulated protein 150 (ORP150) (endoplasmic reticulum chaperone) as identified by its isoelectric point, two-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis and mass spectrometric analysis. This 110-kDa band is detectable with anti-Hsp70 antibody because ORP150 has homology with Hsp70. Confocal microscopic studies reveal the presence of Hsp70-like proteins on the surface of A431 cells as revealed by immunostaining with anti-Hsp70 antibody. Moreover, overexpression of ORP150 in A431 cells has resulted in a dramatic protection of A431 cells against jacalin-induced toxicity, confirming that the jacalin-induced cytotoxicity is mediated through ORP150, and impairment of ORP150 functions with the help of jacalin makes the cells more susceptible to death due to stress. Our studies suggest that the cellular responses, as a consequence of lectin binding, may not be exclusively mediated by carbohydrate binding property alone, but other factors such as protein-protein interactions may also contribute to the observed cellular responses.

Autophosphorylation of Hsp70 is detected in the process of substrate refolding in the presence of adenosine triphosphate (ATP) in the reaction mixture. But to date, the role and mechanism of Hsp70 autophosphorylation have not been elucidated. In this study we determined the site of histidine phosphorylation of Hsp70 as an intermediate in the process of phosphate transfer reaction by site-directed mutagenesis. We selected two possible sites (ie, His89 and His227) of intermediate histidine phosphorylation based on our hypothesis of the transfer of γ-phosphoryl groups and replacement by glycine and serine. Although an acid labile autophosphorylation intermediate of Hsp70 and its cytidine diphosphate–dependent dephosphorylation were detected in wild-type Hsp70, they were markedly suppressed in the H89S mutation of Hsp70, but not on the H227S mutation. The ATPase activity and ATP synthesis activity of Hsp70 were almost completely suppressed in the H89S and H89G mutations. The role of His89 in the phosphate transfer reaction of Hsp70 is discussed.

Because heat shock proteins (Hsps) are involved in protecting cells and in the pathophysiology of diseases such as inflammation, cancer, and neurodegenerative disorders, the use of regulators of the expression of Hsps in mammalian cells seems to be useful as a potential therapeutic modality. To identify compounds that modulate the response to heat shock, we analyzed several natural products using a mammalian cell line containing an hsp promoter-regulated reporter gene. In this study, we found that an extract from Fructus Arctii markedly suppressed the expression of Hsp induced by heat shock. A component of the extract arctigenin, but not the component arctiin, suppressed the response at the level of the activation of heat shock transcription factor, the induction of mRNA, and the synthesis and accumulation of Hsp. Furthermore, arctigenin inhibited the acquisition of thermotolerance in mammalian cells, including cancer cells. Thus, arctigenin seemed to be a new suppressive regulator of heat shock response in mammalian cells, and may be useful for hyperthermia cancer therapy.

Heat shock protein 70 (Hsp70) comprises proteins that have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli; however, little is known about whether Hsp70 protects against DNA damage. In this study, we investigated the relationship between Hsp70 expression and the levels of ultraviolet C (UVC)–induced DNA damage in A549 cells with normal, inhibited, and overexpressed Hsp70 levels. Hsp70 expression was inhibited by treatment with quercetin or overexpressed by transfection of plasmids harboring the hsp70 gene. The level of DNA damage was assessed by the comet assay. The results showed that the levels of DNA damage (shown as the percentage of comet cells) in A549 cells increased in all cells after exposure to an incident dose of 0, 10, 20, 40, and 80 J/m² whether Hsp70 was inhibited or overexpressed. This response was dose dependent: a protection against UVC-induced DNA damage in cells with overexpressed Hsp70 was observed at UVC dose 20 J/ m² with a maximum at 40 J/m² when compared with cells with normal Hsp70 levels and in quercetin-treated cells. This differential protection disappeared at 80 J/m². These results suggest that overexpressed Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from at UVC irradiation-induced DNA damage by Hsp70. The detailed mechanism how Hsp70 is involved in DNA damage and possible DNA repair warrants further investigation.

Simple independent enzyme-catalyzed reactions distributed homogeneously throughout an aqueous environment cannot adequately explain the regulation of metabolic and other cellular processes in vivo. Such an unstructured system results in unacceptably slow substrate turnover rates and consumes inordinate amounts of cellular energy. Current approaches to resolving compartmentalization in living cells requires the partitioning of the molecular species in question such that its localization can be resolved with fluorescence microscopy. Standard imaging approaches will not resolve localization of protein activity for proteins that are ubiquitously distributed, but whose function requires a change in state of the protein. The small heat shock protein sHSP27 exists as both dimers and large multimers and is distributed homogeneously throughout the cytoplasm. A fusion of the green fluorescent protein variant S65T and sHSP27 is used to assess the ability of diffusion rate histograms to resolve compartmentalization of the 2 dominant oligomeric species of sHSP27. Diffusion rates were measured by multiphoton fluorescence photobleaching recovery. Under physiologic conditions, diffusion rate histograms resolved at least 2 diffusive transport rates within a living cell potentially corresponding to the large and small oligomers of sHSP27. Given that oligomerization is often a means of regulation, compartmentalization of different oligomer species could provide a means for efficient regulation and localization of sHsp27 activity.

Wild-type inducible Hsp70 (WT) and 2 folding deficient mutants protect the brain against focal cerebral ischemia in vivo and brain cells from oxygen–glucose deprivation (OGD) in vitro, but the protective mechanisms remain unclear. Mitochondria are central to both normal physiological function and the regulation of cell death. We tested the effect of overexpressing Hsp70 and 2 mutants, Hsp70-K71E, an adenosine triphosphatase (ATPase)-deficient point mutant, and Hsp70-381–640, a deletion mutant lacking the ATPase domain on mitochondrial physiology under glucose deprivation (GD) stress in primary cultured astrocytes. Mitochondrial membrane potential was assessed using a potentiometric fluorescent dye tetramethylrhodamine ethyl ester (TMRE). By 5 hours of GD, the mitochondria in the LXSN control transfected astrocytes had markedly reduced membrane potential. However, in the Hsp70-WT, -K71E, and -381–640 groups, there was no apparent change in TMRE signal during 5 hours of GD. Oxygen consumption was measured to assess oxidative respiration. Overexpression of Hsp70-K71E and -381–640 prevented the decrease in state III respiration observed at 5 hours, and all 3 prevented the increase in state IV respiration found in LXSN controls after 5 hours of GD. Reactive oxygen species (ROS) production was assessed with hydroethidine. Hsp70 and its mutants all significantly reduced the increases in ROS accumulation during 5 hours of GD. The results demonstrate that the protective effect of the carboxyl-terminal half of Hsp70 and of the full-length molecule is associated with better maintained mitochondrial membrane potential, better maintained state IV respiration, and reduced ROS generation during GD.

Knowledge of the interactive domains on the surface of small heat shock proteins (sHSPs) is necessary for understanding the assembly of complexes and the activity as molecular chaperones. The primary sequences of 26 sHSP molecular chaperones were aligned and compared. In the interactive β3 sequence, ₇₃DRFSVNLDVKHFS₈₅ of human αB crystallin, Ser-76, Asn-78, Lys-82, and His-83 were identified as nonconserved residues on the exposed surface of the α crystallin core domain. Site-directed mutagenesis produced the mutant αB crystallins: S76E, N78G, K82Q, and H83F. Domain swapping with homologous β3 sequences, ₃₂EKFEVGLDVQFFT₄₄ from Caenorhabditis elegans sHSP12.2 or ₆₉DKFVIFLDVKHFS₈₁ from αA crystallin, resulted in the mutant αB crystallins, CE1 and αA1, respectively. Decreased chaperone activity was observed with the point mutants N78G, K82Q, and H83F and with the mutant, CE1, in aggregation assays using β_L crystallin, alcohol dehydrogenase (ADH), or citrate synthase (CS). The S76E mutant had minimal effect on chaperone activity, and domain swapping with αA crystallin had no effect on chaperone activity. The mutations that resulted in altered chaperone activity, produced minimal modification to the secondary, tertiary, and quaternary structure of human αB crystallin as determined by ultraviolet circular dichroism spectroscopy, chymotrypsin proteolysis, and size exclusion chromatography. Chaperone activity was influenced by the amount of unfolding of the target proteins and independent of complex size. The results characterized the importance of the exposed side chains of Glu-78, Lys-82, and His-83 in the interactive β3 sequence of the α crystallin core domain in αB crystallin for chaperone function.