Molecular chaperones, including the heat-shock proteins (Hsps), are a ubiquitous feature of cells in which these proteins cope with stress-induced denaturation of other proteins. Hsps have received the most attention in model organisms undergoing experimental stress in the laboratory, and the function of Hsps at the molecular and cellular level is becoming well understood in this context. A complementary focus is now emerging on the Hsps of both model and nonmodel organisms undergoing stress in nature, on the roles of Hsps in the stress physiology of whole multicellular eukaryotes and the tissues and organs they comprise, and on the ecological and evolutionary correlates of variation in Hsps and the genes that encode them. This focus discloses that (a) expression of Hsps can occur in nature, (b) all species have hsp genes but they vary in the patterns of their expression, (c) Hsp expression can be correlated with resistance to stress, and (d) species' thresholds for Hsp expression are correlated with levels of stress that they naturally undergo. These conclusions are now well established and may require little additional confirmation; many significant questions remain unanswered concerning both the mechanisms of Hsp-mediated stress tolerance at the organismal level and the evolutionary mechanisms that have diversified the hsp genes.

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Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology

The biochemistry and medical significance of the flavonoids.

T URNOVER OF AB ERR ANT PROT EINS IN E. COLI . . . . . . . . . . . . . . . . . 445 The Lon Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446 DnaK. DnaJ. GrpE. GroEL and GroES . . . . . . . . . . . . . . . . . . . . . . . . . . 447 The Clp Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448 The DegP Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

The Function of Heat-Shock Proteins in Stress Tolerance: Degradation and Reactivation of Damaged Proteins

Our cells and tissues are challenged constantly by exposure to extreme conditions that cause acute and chronic stress. Consequently, survival has necessitated the evolution of stress response networks to detect, monitor, and respond to environmental changes (Morimoto et al. 1990, 1994a; Baeuerle 1995; Baeuerle and Baltimore 1996; Feige et al. 1996; Morimoto and Santoro 1998). Prolonged exposure to stress interferes with efficient operations of the cell, with negative consequences on the biochemical properties of proteins that, under ideal conditions, exist in thermodynamically stable states. In stressed environments, proteins can unfold, misfold, or aggregate. Therefore, the changing demands on the quality control of protein biogenesis, challenges protein homeostasis, for which the heat shock response, through the elevated synthesis of molecular chaperones and proteases, repairs protein damage and assists in the recovery of the cell. The inducible transcription of heat shock genes is the response to a plethora of stress signals (Lis and Wu 1993; Morimoto 1993; Wu 1995) (Fig. 1), including (1) environmental stresses, (2) nonstress conditions, and (3) pathophysiology and disease states. Although changes in heat shock protein (HSP) expression are associated with certain diseases (Morimoto et al. 1990), these observations leave open the question of whether this is an adaptation to the particular pathophysiological state, a reflection of the suboptimal cellular environment associated with the disease, or serves to warn other cells and tissues of imminent danger. The protective role of HSPs is a measure of their capacity to assist in the repair of protein damage. Whether in prokaryotes, plants, or animals, overexpression of one or more HSPs is often sufficient to protect cells and tissues against otherwise lethal exposures to diverse environmental stresses including hydrogen peroxide and other oxidants, toxic chemicals, extreme temperatures, and ethanol-induced toxicity (Parsell and Lindquist 1994). In vertebrate tissue culture cells and animal models, elevating HSPs level, either by modulation of the heat shock response or by constitutive overexpression of specific heat shock proteins, restricts or substantially reduces the level of pathology and cell death (Mizzen and Welch 1988; Huot et al. 1991; Jaattela et al. 1992; Parsell and Lindquist 1994; Mestril et al. 1994; Plumier et al. 1995; Marber et al. 1995; Mehlen et al. 1995; Mosser et al. 1997). This has led to the recognition that HSPs, via their chaperoning effects on proteins, protect cells from many forms of stress-induced cell damage and could influence the course of disease.

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Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators

A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins.

Heat shock induces in cells the synthesis of specific proteins called heat shock proteins (HSPs) and a transient state of thermotolerance. The putative role of one of the HSPs, HSP27, as a protective molecule during thermal stress has been directly assessed by measuring the resistance to hyperthermia of Chinese hamster and mouse cells transfected with the human HSP27 gene contained in plasmid pHS2711. One- and two-dimensional gel electrophoresis of [3H]leucine- and [32P]orthophosphate-labeled proteins, coupled with immunological analysis using Ha27Ab and Hu27Ab, two rabbit antisera that specifically recognize the hamster and the human HSP27 protein respectively, were used to monitor expression and inducibility of the transfected and endogenous proteins. The human HSP27 gene cloned in pHS2711 is constitutively expressed in rodent cells, resulting in accumulation of the human HSP27 and all phosphorylated derivatives. No modification of the basal or heat-induced expression of endogenous HSPs is detected. The presence of additional HSP27 protein provides immediate protection against heat shock administered 48 h after transfection and confers a permanent thermoresistant phenotype to stable transfectant Chinese hamster and mouse cell lines. Mild heat treatment of the transfected cells results in an induction of the full complement of the endogenous heat shock proteins and a small increase in thermoresistance, but the level attained did not surpass that of heat-induced thermotolerant control cells. These results indicate that elevated levels of HSP27 is sufficient to give protection from thermal killing. It is concluded that HSP27 plays a major role in the increased thermal resistance acquired by cells after exposure to HSP inducers.

/pdf/heat-shock-resistance-conferred-by-expression-of-the-human-1r9l3poc3f.pdf

Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells.

Morris hepatoma 7777 cells, heat conditioned at 43 degrees for 0.5 hr, become gradually thermoresistant during an incubation at 37 degrees as judged by their ability to form colonies following a second heat challenge. Pulse incorporation of [35S]methionine into proteins at various times after the conditioning treatment and subsequent fractionation of the proteins by polyacrylamide gel electrophoresis indicate that the gradual putative modifications occurring at the cellular level and leading to the thermotolerance state are accompanied by an elevated synthesis above the normal level of a small set of polypeptides with apparent molecular weights of 27,000, 65,000, 68,000, 70,000, 89,000, and 107,000. Both thermotolerance development and protein induction are completed after a 6- to 8-hr period. At the end of this period, thermotolerance is at its maximum level and heat shock protein synthesis is returned to normal. This acquired thermal resistance eventually disappears between 60 and 80 hr following conditioning treatment. In a parallel manner, the heat shock-induced proteins synthesized during the first 4 hr following the conditioning treatment are maintained in the cells at a high level for several hr but become undetectable by 82 hr. The results provide strong circumstantial evidence that heat shock proteins are involved in the acquisition, maintenance, and decay of thermotolerance.

https://cancerres.aacrjournals.org/content/canres/42/6/2457.full.pdf

Synthesis and Degradation of Heat Shock Proteins during Development and Decay of Thermotolerance

A family of 10 thermoresistant cell lines cloned from Chinese hamster cells transfected with a plasmid containing the structural gene for the small human Mr 27,000 heat shock protein (HSP27) was used to assess the putative role of this heat shock protein in chemoresistance. These cells express varying amounts of human HSP27 in addition to the normal level of endogenous hamster HSP27. As previously observed in the case of thermoresistance, a significant positive linear correlation (P less than 0.05) was found between cell survival in response to doxorubicin and the total amount of HSP27 expressed. Some clones were also examined for resistance to other drugs and chemicals. A statistically significant increased survival relative to the parental cells was observed following treatment with daunorubicin (three clones studied), colchicine, vincristine, actinomycin D, hydrogen peroxide, and sodium arsenite (one clone studied). However, the clone which expressed the highest level of HSP27 was as sensitive as control cells to the cytotoxic action of bis-chloronitrosourea and 5-fluorouracil. The relationship between HSP27 overexpression and increased resistance to cytotoxic agents was also evaluated in three independent pooled cell populations stably transformed with both the human HSP27 and the xanthine-guanine phosphoribosyltransferase gene and selected on the basis of resistance to mycophenolic acid and aminopterin. The results indicated that these cells survived significantly better than the control cells transfected with the marker gene only when exposed to doxorubicin. HSP27-mediated cellular protection was not associated either with decreased drug accumulation or with overexpression of P-glycoprotein. It is suggested that HSP27 might be involved in some form of chemoresistance and could participate in the development of clinical resistance to antineoplastic drugs.

https://cancerres.aacrjournals.org/content/canres/51/19/5245.full.pdf

Increased survival after treatments with anticancer agents of Chinese hamster cells expressing the human Mr 27,000 heat shock protein.

The small molecular weight heat shock protein HSP27 was recently shown to confer a stable thermoresistant phenotype when expressed constitutively in mammalian cells after structural gene transfection. These results suggested that HSP27 may also play an important role in the development of thermotolerance, the transient ability to survive otherwise lethal heat exposure after a mild heat shock. In Chinese hamster O23 cells increased thermoresistance is first detected at 2 h after a triggering treatment of 20 min at 44 degrees C, attains a maximum at 5 hours, and decays thereafter with a half-life of 10 h. We found that the development and decay of transient thermotolerance cannot be solely explained on the basis of changes in the cellular concentration of HSP27. The cellular HSP27 concentration is not increased appreciably at 2 h after heat shock and attains a maximum at 14 h. Similar results were obtained in the case of another heat shock protein, HSP70. HSP70 follows slightly faster kinetics of accumulation (peaks at 10 h) and decays much more rapidly (ti/2 = 4h) than HSP27 (t1/2 = 13h). HSP27 has 3 isoelectric variants A, B, and C of which B and C are phosphorylated. In cells maintained at normal temperature, HSP27A represents more than 90% of all HSP27. Shifting the cell culture temperature from 37 to 44 degrees C induces the incorporation of 32P into the more acidic B and C forms, a process that occurs very rapidly since the reduction in the concentration of the A form and a corresponding increase in the level of B and C is detectable by immunoblot analysis within 2.5 min at 44 degrees C. Analyses performed at various times during development and decay of transient thermotolerance revealed a close relationship between the effect of heat shock on HSP27 phosphorylation and cell ability to survive. For example, fully thermotolerant cells (5 h post-induction) are refractory to induction of HSP27 phosphorylation by a 20-min heat shock. The induction of HSP27 phosphorylation was also studied in a family of clonal cell lines of O23 cells that are thermoresistant as a result of the constitutive expression of a transfected human HSP27 gene. In these thermoresistant cells, phosphorylation of the endogenous hamster HSP27 is induced to a level comparable to that found in the thermosensitive parental cells. However, phosphorylation of the exogenous human protein, which represents more than 80% of total HSP27 in these cells, was much less induced.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of HSP27 during development and decay of thermotolerance in Chinese hamster cells.

Four heat-resistant variants were isolated after treatment of Chinese hamster lung cells with the mutagen ethyl methane sulfonate, followed by a single-step selection procedure consisting in a severe hyperthermic treatment of 4 h at 44 degrees C. The isolated clones had a stable resistant phenotype for at least 150 generations during which they showed a 5,000-fold increased survival to a 4-h treatment at 44 degrees C when compared to wild-type cells. Comparative two-dimensional electrophoretic analyses of proteins revealed that, like induced thermotolerant wild-type cells (i.e., cells induced to a transient physiological state of thermotolerance by a sublethal heat conditioning treatment administered 18 h before), the heat-resistant variants had, at normal temperature, an increased content of a heat-shock protein with Mr of 27,000 (HSP27). In three of the four heat-resistant variants, the increased content of HSP27 was correlated with a two-fold increase in the constitutive level of the mRNA encoding HSP27. Chinese hamster HSP27 is composed of three species that differ in their relative isoelectric point, among which the two most acidic forms are phosphoproteins. In both the heat-resistant variant and wild-type cells, heat shock induces a rapid enhancement of the phosphorylation of HSP27: maximal phosphorylation occurs within 10 min upon changing the incubation temperature from 35 degrees to 44 degrees C. A concomitant shift in silver-staining intensity is rapidly detectable between the three isoforms, which seems to indicate that the two phosphorylated species represent post-translational modifications of the more basic species. It is concluded that most likely the enhanced expression of HSP27 is linked to the resistant phenotype of the variants. The study provides supporting evidence that both the content and phosphorylation status of HSP27 are determining factors in the ability of cells to survive hyperthermic treatments.

Pierre Chrétien

Papers

Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells.

Synthesis and Degradation of Heat Shock Proteins during Development and Decay of Thermotolerance

Increased survival after treatments with anticancer agents of Chinese hamster cells expressing the human Mr 27,000 heat shock protein.

Phosphorylation of HSP27 during development and decay of thermotolerance in Chinese hamster cells.

Enhanced constitutive expression of the 27-kDa heat shock proteins in heat-resistant variants from Chinese hamster cells.