Relationship between Hyperthermic Cell Killing and Protein Denaturation by Alcohols

Massicotte-Nolan, Patricia; Glofcheski, D.J.; Kruuv, J.; Lepock, James R.

doi:10.2307/3575584

Cited by 63 publications

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“…Although the cause of cell death is not definitely known, various pieces of indirect evidence suggest that the irreversible loss of vital protein structure and associated function is a major factor (1,2). When cells are pretreated by exposure to increased but nonlethal temperatures or by brief exposure to lethal temperatures followed by a period ofrecovery, the treated cells exhibit a dramatically enhanced rate of survival upon subsequent exposure to lethal temperatures.…”

mentioning

confidence: 99%

Nonspecific stabilization of stress-susceptible proteins by stress-resistant proteins: a model for the biological role of heat shock proteins.

Minton¹,

Karmin²,

Hahn³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

125

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It is demonstrated experimentally that addition of proteins that are themselves resistant to denaturation by heat or ethanol can nonspecifically stabilize other proteins that are ordinarily highly susceptible to inactivation. It is proposed that the diffusion-limited rate with which unfolded protein molecules encounter each other and become irreversibly crosslinked is reduced in the presence ofsubstantial concentrations ofan unreactive globular protein. We suggest that one of the functions of heat shock proteins, which are synthesized in large amounts after exposure of cells to increased temperature and other forms of stress, may be to stabilize other proteins kinetically in a similarly nonspecific fashion.When exposed to sufficiently high temperatures, cells die (as measured by reproductive assay) at a rate which, after an initial lag period, may be described by a first-order decay law. Although the cause of cell death is not definitely known, various pieces of indirect evidence suggest that the irreversible loss of vital protein structure and associated function is a major factor (1, 2). When cells are pretreated by exposure to increased but nonlethal temperatures or by brief exposure to lethal temperatures followed by a period ofrecovery, the treated cells exhibit a dramatically enhanced rate of survival upon subsequent exposure to lethal temperatures. This phenomenon is referred to as "acquired thermotolerance" (3).The mechanism by which cells acquire increased tolerance to thermal stress is unknown, but attention recently has focused on the possible role of heat shock proteins (hsp), a small number ('16) of specific proteins that are transcriptionally induced and synthesized in great quantity after exposure to elevated temperature ("heat shock") (4-6). The synthesis of hsp has been studied extensively as a model of gene regulation, particularly in Drosophila (6) but also in various organisms throughout the phylogenetic tree (4,(7)(8)(9)(10)(11)(12). Recently it has been shown that the time scale for the appearance and disappearance of hsp parallels the expression ofacquired thermotolerance in various cell types (11-13) and that thermotolerance is not acquired after heat shock if synthesis of hsp is inhibited (8,12,14).The synthesis of hsp is also induced by noxious stimuli other than heat shock, including arsenite (6, 14), oxygen deprivation (4, 6), and ethanol (14). It has been demonstrated that preliminary exposure to ethanol results in subsequently acquired resistance not only to ethanol but also to thermal stress (15). Moreover, thermal pretreatment appears to result in increased tolerance to ethanol (16). These findings suggest that hsp may play a role in a generalized cellular adaptation to stress.hsp are quite widespread within the cell. Low molecular weight hsp bind predominantly to nuclear chromatin; higher molecular weight hsp are found predominantly in the cytoplasm (4,7,(17)(18)(19)(20)(21)(22). No enzymatic activity of hsp has yet been identified (6, 7).Here we propose that hsp can con...

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confidence: 99%

Nonspecific stabilization of stress-susceptible proteins by stress-resistant proteins: a model for the biological role of heat shock proteins.

Minton¹,

Karmin²,

Hahn³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

125

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“…We have recently shown by differential scanning calorimetry (DSC) that mild heat shock denatures a small fraction of total cellular protein of Chinese hamster lung V79 cells (Lepock et al, 1988). The transition temperatures (T,) of protein denaturation in whole cells are lowered by short-chain alcohols, which are cellular sensitizers to heat shock, and raised by glycerol, which is a cellular protector (Massicotte-Nolan et al, 1981); this finding suggests that an increase in the T, of protein denaturation in thermotolerant cells might be detectable by DSC. In this paper we report that this hypothesis is correct; i.e., V79 cells made thermotolerant are more thermostable as determined by DSC.…”

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confidence: 99%

Increased thermostability of thermotolerant CHL V79 cells as determined by differential scanning calorimetry

Lepock

Frey

Heynen

et al. 1990

Journal Cellular Physiology

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Heat shock denatures cellular protein and induces both a state of acquired thermotolerance, defined as resistance to a subsequent heat shock, and the synthesis of a category of proteins referred to as heat-shock proteins (HSPs). Thermotolerance may be due to the stabilization of thermolabile proteins that would ordinarily denature during heat shock, either by HSPs or some other factors. We show by differential scanning calorimetry (DSC) that mild heat shock irreversibly denatures a small fraction of Chinese hamster lung V79-WNRE cell protein (i.e., the enthalpy change, which is proportional to denaturation, on scanning to 45 degrees C at 1 degree C/min is approximately 2.3% of the total calorimetric enthalpy). Thermostability, defined by the extent of denaturation during heat shock and determined from DSC scans of whole cells, increases as the V79 cells become thermotolerant. Cellular stabilization appears to be due to an increase in the denaturation temperature of the most thermolabile proteins; there is no increase in the denaturation temperatures of the most thermally resistant proteins, i.e., those denaturing above 65 degrees C. Cellular stabilization is also observed in the presence of glycerol, which is known to increase resistance to heat shock and to stabilize proteins in vitro. A model is presented, based on a direct relationship between the extent of hyperthermic killing and the denaturation or inactivation of a critical target that defines the rate-limiting step in killing, which predicts a transition temperature (Tm) of the critical target for control V79-WNRE cells of 46.0 degrees C and a Tm of 47.3 degrees C for thermotolerant cells. This shift of 1.3 degrees C is consistent with the degree of stabilization detected by DSC.

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“…During heat shock, proteins become denatured (Massicotte-Nolan, 1981;Nguyen et al, 1989;Lepock et al, 1990) and have exposed hydrophobic regions, forming aggregates (Scopes, 1987). Pelham (1986) suggested that binding of HSP70 to hydrophobic regions reduces the effective concentration of hydrophobic surfaces and consequently inhibits the aggregation process.…”

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Constitutive HSP70: Oligomerization and its dependence on ATP binding

Kim

Lee

Corry

1992

Journal Cellular Physiology

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The constitutive HSP70 purified from CHO cells, which indicated a single band in SDS-polyacrylamide gel electrophoresis, showed multiple bands in native-polyacrylamide gel electrophoresis. These results indicate that the protein may exist in oligomeric forms. After crosslinking the oligomers with glutaraldehyde, SDS-polyacrylamide gel electrophoresis showed three protein bands of molecular weight 70 kDa, 153 kDa, and 200 kDa corresponded to monomer, dimer, and trimer, respectively. The relative amount of oligomeric forms was dependent upon ATP concentrations: it increased upon hydrolysis of ATP or decreased upon incubation with high concentrations of ATP (1-10 mM). Autoradiographic analysis of the native polyacrylamide gel electrophoresis of HSP70 following incubation with [gamma-32P]ATP revealed that ATP bound to only monomer. These results suggest that the equilibrium between oligomeric forms is dependent on ATP concentrations. Nonetheless, during heat shock, both monomer and oligomer might be indistinguishably associated with some proteins, probably denatured proteins.

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Relationship between Hyperthermic Cell Killing and Protein Denaturation by Alcohols

Cited by 63 publications

References 0 publications

Nonspecific stabilization of stress-susceptible proteins by stress-resistant proteins: a model for the biological role of heat shock proteins.

Nonspecific stabilization of stress-susceptible proteins by stress-resistant proteins: a model for the biological role of heat shock proteins.

Increased thermostability of thermotolerant CHL V79 cells as determined by differential scanning calorimetry

Constitutive HSP70: Oligomerization and its dependence on ATP binding

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