Role of HSF activation for resistance to heat, cold and high-temperature knock-down

Regulation of heat shock proteins (Hsps) by the heat shock factor (HSF) and the importance of these proteins for resistance to heat stress is well documented. Less characterized is the importance of Hsps for cold stress resistance although Hsp70 is known to be induced following long-term cold exposure in Drosophila melanogaster. In this study, a temperature-sensitive HSF mutant line was used to investigate the role of HSF activation following heat hardening, rapid cold hardening (RCH) and long-term cold acclimation (LTCA) on heat and cold resistance, and this was correlated with Hsp70 expression. In addition, the effect of HSF activation on high-temperature knock-down resistance was evaluated. We found a significantly decreased HSF activation in the mutant line as compared to a corresponding control line following heat hardening, and this was correlated with decreased heat resistance of the mutant line. However, we did not find this difference in HSF activity to be important for resistance to cold stress or high-temperature knock-down. The findings indicate that induction of stress genes regulated by HSF, such as Hsps, although occurring following LTCA, are not of major importance for cold stress resistance and neither for RCH nor high-temperature knock-down resistance in D. melanogaster.     

The evolution of heat shock genes and expression patterns of heat shock proteins in the species from temperature contrasting habitats

Heat shock genes are the most evolutionarily ancient among the systems responsible for adaptation of organisms to a harsh environment. The encoded proteins (heat shock proteins, Hsps) represent the most important factors of adaptation to adverse environmental conditions. They serve as molecular chaperones, providing protein folding and preventing aggregation of damaged cellular proteins. Structural analysis of the heat shock genes in individuals from both phylogenetically close and very distant taxa made it possible to reveal the basic trends of the heat shock gene organization in the context of adaptation to extreme conditions. Using different model objects and nonmodel species from natural populations, it was demonstrated that modulation of the Hsps expression during adaptation to different environmental conditions could be achieved by changing the number and structural organization of heat shock genes in the genome, as well as the structure of their promoters. It was demonstrated that thermotolerant species were usually characterized by elevated levels of Hsps under normal temperature or by the increase in the synthesis of these proteins in response to heat shock. Analysis of the heat shock genes in phylogenetically distant organisms is of great interest because, on one hand, it contributes to the understanding of the molecular mechanisms of evolution of adaptogenes and, on the other hand, sheds the light on the role of different Hsps families in the development of thermotolerance and the resistance to other stress factors.     

Role of Escherichia coli molecular chaperones in the protection of bacterial cells against irreversible aggregation induced by heat shock

All living organisms respond to environmental stresses, such as heat or ethanol by increasing the synthesis of a specific group of proteins termed heat shock proteins (Hsps) or stress proteins. Major Hsps are molecular chaperones and proteases. Molecular chaperones facilitate the proper folding of polypeptides, protect other proteins from inactivation, and reactivate aggregated proteins. Heat shock proteases eliminate proteins irreversibly damaged by stress. This review describes the role of heat shock proteins of the model bacterial cell, E. coli in the protection of other proteins against aggregation and in the mechanism of removal of protein aggregates from the cell. This mechanism remains unclear and it is believed to involve substrate renaturation and proteolysis by molecular chaperones and heat shock proteases. Recently, many studies have been focused on the disaggregation and reactivation of proteins by a bi-chaperone system consisting of DnaK/DnaJ/GrpE and ClpB, an ATPase from the AAA superfamily of proteins.     

Biochemical and biophysical characterization of small heat shock proteins from sugarcane. Involvement of a specific region located at the N-terminus with substrate specificity

When cells are submitted to an increase in temperature, heat shock proteins (Hsp) are synthesized to help heat stress resistance. Small Hsps, which are diverse and abundant in plants, have the major function of preventing irreversible protein aggregation. The diversity of small Hsps in plants is intriguing and characterization of their chaperone activity is important to understand plant tolerance to heat stress. A previous study showed that small Hsps, mainly represented by class I (cytosolic), correspond to about 5% of all sugarcane Expressed Sequencing Tags belonging to the molecular chaperone category. Here, we present biochemical and biophysical characterization of two sugarcane small Hsps from class I, which were named SsHsp17.2 and SsHsp17.9 according to their monomer molecular mass of 17.2 and 17.9 kDa, respectively. The recombinant proteins have identity of about 75% to each other and similar structural characteristics. However, their stability and their chaperone activity were not equivalent: SsHsp17.9 was more efficient in protecting citrate synthase and malate dehydrogenase from aggregation whereas SsHsp17.2 was more efficient in protecting luciferase from aggregation. There is only one region, which is located at the N-terminus, of low homology between these two proteins. Based on that and on previous works pointing to multiple sites, mainly at the N-terminus, involved with substrate specificity in small Hsps, we suggest that this specific region is one of these sites. In addition, this is the first report on the chaperone activity of sugarcane small Hsps.     

Apoptosis-resistant phenotype in HL-60-derived cells HCW-2 is related to changes in expression of stress-induced proteins that impact on redox status and mitochondrial metabolism

The onset of resistance to drug-induced apoptosis of tumour cells is a major problem in cancer therapy. We studied a drug-selected clone of promyelocytic HL-60 cells, called HCW-2, which display a complex resistance to a wide variety of apoptosis-inducing agents and we found that these cells show a dramatic increase in the expression of heat shock proteins (Hsps) 70 and 27, while the parental cell line does not. It is known that stress proteins such as Hsps can confer resistance to a variety of damaging agents other than heat shock, such as TNF-alpha, monocyte-induced cytotoxicity, and also play a role in resistance to chemotherapy. This elevated expression of Hsps is paralleled by an increased activity of mitochondrial metabolism and pentose phosphate pathway, this latter leading to high levels of glucose-6-phosphate dehydrogenase and, consequently, of glutathione. Thus, the apoptotic-deficient phenotype is likely because of the presence of high levels of stress response proteins and GSH, which may confer resistance to apoptotic agents, including chemotherapy drugs. Moreover, the fact that in HCW-2 cells Hsp70 are mainly localised in mitochondria may account for the increased performances of mitochondrial metabolism. These observations could have some implications for the therapy of cancer, and for the design of combined strategies that act on antioxidant defences of the neoplastic cell.     

Phototransduction genes are up-regulated in a global gene expression study of Drosophila melanogaster selected for heat resistance

The genetic architecture underlying heat resistance remains partly unclear despite the well-documented involvement of heat shock proteins (Hsps). It was previously shown that factors besides Hsps are likely to play an important role for heat resistance. In this study, gene expression arrays were used to make replicate measurements of gene expression before and up to 64 hours after a mild heat stress treatment, in flies selected for heat resistance and unselected control flies, to identify genes differentially expressed in heat resistance-selected flies. We found 108 genes up-regulated and 10 down-regulated using the Affymetrix gene expression platform. Among the up-regulated genes, a substantial number are involved in the phototransduction process. Another group of genes up-regulated in selected flies is characterized by also responding to heat shock treatment several hours after peak induction of known Hsps revert to nonstress levels. These findings suggest phototransduction genes to be critically involved in heat resistance, and support a role for components of the phototransduction process in stress-sensing mechanisms. In addition, the results suggest yet-uncharacterized genes responding to heat stress several hours after treatment to be involved in heat stress resistance. These findings mark an important increase in the understanding of heat resistance.     

Heat shock responses for understanding diseases of protein denaturation

Extracellular stresses induce heat shock response and render cells resistant to lethal stresses. Heat shock response involves induction of heat shock proteins (Hsps). Recently the roles of Hsps in neurodegenerative diseases and cancer are attracting increasing attention and have accelerated the study of heat shock response mechanism. This review focuses on the stress sensing steps, molecules involved in Hsps production, diseases related to Hsp malfunctions, and the potential of proteomics as a tool for understanding the complex signaling pathways relevant to these events.     

Changes in Ubiquitin Conjugates and Hsp72 Levels During Arm Regeneration in Echinoderms

All organisms show a common defensive mechanism that results in the expression of conserved heat shock proteins (Hsps). These proteins function in a wide range of stressful conditions. We have monitored their levels in species of regenerating echinoderms with different mechanisms of regeneration and from different geographical locations. The effect of an artificial higher temperature on expression of Hsps was also studied. Two stress proteins (Hsp72 and ubiquitin) that are important in processes such as development and protein degradation were investigated. Using Western blot analysis and immunocytochemistry, we found significant changes in the level (Hsp72) and pattern of conjugation (ubiquitin) that corresponded with the repair phase (early regenerative stages) and with the later growth and regeneration of new tissues. Animals from the intertidal environment showed a distinctly sustained expression pattern of Hsp72 compared with benthic animals which suggests a functionally adaptative and dynamic stress response program. Accepted March 1, 2000.     

Agonistic encounters and cellular angst: social interactions induce heat shock proteins in juvenile salmonid fish

Juvenile salmonid fish readily form dominance hierarchies when faced with limited resources. While these social interactions may result in profound behavioural and physiological stress, it is unknown if this social stress is evident at the level of the cellular stress response—specifically, the induction of stress or heat shock proteins (Hsps). Thus, the goal of our study was to determine if Hsps are induced during hierarchy formation in juvenile rainbow trout (Oncorhynchus mykiss). To this end, we measured levels of three Hsps, Hsp70, Hsc (heat shock cognate)70 and Hsp90 in the white muscle, liver and brain of trout that had been interacting for 36 h, 72 h or 6 days. Our data indicate that Hsps are induced in both dominant and subordinate fish in a time- and tissue-specific manner. In further mechanistic experiments on fasted and cortisol-treated fish, we demonstrated that high plasma cortisol does not affect Hsp induction in trout white muscle or liver, but both conditions may be part of the mechanism for Hsp induction with social stress in the brain. We conclude that the behavioural and physiological stress experienced by juvenile rainbow trout in dominance hierarchies can be extended to the induction of Hsps.     

The evolutionary and ecological role of heat shock proteins

Most heat shock proteins (Hsp) function as molecular chaperones that help organisms to cope with stress of both an internal and external nature. Here, we review the recent evidence of the relationship between stress resistance and inducible Hsp expression, including a characterization of factors that induce the heat shock response and a discussion of the associated costs. We report on studies of stress resistance including mild stress, effects of high larval densities, inbreeding and age on Hsp expression, as well as on natural variation in the expression of Hsps. The relationship between Hsps and life history traits is discussed with special emphasis on the ecological and evolutionary relevance of Hsps. It is known that up‐regulation of the Hsps is a common cellular response to increased levels of non‐native proteins that facilitates correct protein folding/refolding or degradation of non‐functional proteins. However, we also suggest that the expression level of Hsp in each species and population is a balance between benefits and costs, i.e. a negative impact on growth, development rate and fertility as a result of overexpression of Hsps. To date, investigations have focused primarily on the Hsp70 family. There is evidence that representatives of this Hsp family and other molecular chaperones play significant roles in relation to stress resistance. Future studies including genomic and proteonomic analyses will increase our understanding of molecular chaperones in stress research.     

Mobile phones, heat shock proteins and cancer

There are several reports which indicate that electromagnetic radiation (such as from mobile phones) at non-thermal levels may elicit a biological effect in target cells or tissues. Whether or not these biological effects lead to adverse health effects, including cancer, is unclear. To date there is limited scientific evidence of health issues, and no mechanism by which mobile phone radiation could influence cancer development. In this paper, we develop a theoretical mechanism by which radiofrequency radiation from mobile phones could induce cancer, via the chronic activation of the heat shock response. Upregulation of heat shock proteins (Hsps) is a normal defence response to a cellular stress. However, chronic expression of Hsps is known to induce or promote oncogenesis, metastasis and/or resistance to anticancer drugs. We propose that repeated exposure to mobile phone radiation acts as a repetitive stress leading to continuous expression of Hsps in exposed cells and tissues, which in turn affects their normal regulation, and cancer results. This hypothesis provides the possibility of a direct association between mobile phone use and cancer, and thus provides an important focus for future experimentation.     

Induction of heat shock proteins in differentiated human and rodent neurons by celastrol

Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis have been termed protein misfolding disorders that are characterized by the neuronal accumulation of protein aggregates. Manipulation of the cellular stress-response involving induction of heat shock proteins (Hsps) in differentiated neurons offers a therapeutic strategy to counter conformational changes in neuronal proteins that trigger pathogenic cascades resulting in neurodegenerative diseases. Hsps are protein repair agents that provide a line of defense against misfolded, aggregation-prone proteins. These proteins are not induced in differentiated neurons by conventional heat shock. We have found that celastrol, a quinine methide triterpene, induced expression of a wider set of Hsps, including Hsp70B', in differentiated human neurons grown in tissue culture compared to cultured rodent neuronal cells. Hence the beneficial effect of celastrol against human neurodegenerative diseases may exceed its potential in rodent models of these diseases.     

PKC epsilon is a unique regulator for hsp90 beta gene in heat shock response

An early event in cellular heat shock response is the transmittance of stress signals from the cell surface into the nuclei, resulting in the induction of heat shock proteins (Hsps). Protein kinase C (PKC) has been implicated as a key player in transducing stress signals. However, mechanism(s) by which PKC regulates heat shock-induced events remains largely unknown. Here we present data that pan-PKC inhibitor GF109203X, but not classic PKC inhibitor G?6976, specifically repressed heat shock-induced accumulation of mRNA as well as promoter activity of hsp90 beta, but not hsp90 alpha, in Jurkat cells. Subcellular fractionation studies revealed that heat shock exclusively induced PKC-epsilon membrane translocation. Consistently, expression of a constitutively active PKC-epsilon(A159E) resulted in an enhanced promoter activity of hsp90 beta upon heat shock, whereas a dominant-negative PKC-epsilon(K437R) abolished this effect. In contrast, constitutively active-PKC-alpha or dominant-negative-PKC-alpha had no effects on heat shock induction of the gene. The effect of PKC-epsilon on hsp90 beta expression seems to be stimuli-specific, as phorbol myristate acetate-mediated hsp90 beta expression was PKC-epsilon-independent. We conclude that PKC-epsilon is specifically required in the signaling pathway leading to the induction of hsp90 beta gene in response to heat shock.     

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.     

Heat-shock proteins are induced in unstressed leaves of Nicotiana attenuata (Solanaceae) when distant leaves are stressed

In Nicotiana attenuata, systemic induction of heat-shock proteins (Hsps) was detected in response to the treatment of single leaves by either heat shock, mechanical damage, or exogenous application of methyl jasmonate (MJ). All treatments increased the abundance of members of the 70-kD Hsp (Hsp70) family and induced synthesis of one or more of the small Hsps (sHsp) (16-23 kD) in both treated and untreated leaves. These results provide the first evidence that Hsps can be systemically induced in plants and suggest that systemic induction of Hsps may be important in pre-adapting leaves to stress.