Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides

1985 ◽  
Vol 5 (6) ◽  
pp. 1229-1237
Author(s):  
W J Welch ◽  
J R Feramisco
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Stress Proteins ◽  
Stress Protein ◽  
Exchange Chromatography ◽  
Native Form ◽  
Step Procedure ◽  
Rapid Purification ◽  
Stress Dependent ◽  
Tetramethylrhodamine Isothiocyanate

A new and rapid purification procedure has been developed for the mammalian 70,000-dalton (70-kDa) heat-shock (or stress) proteins. Both the constitutive 73-kDa protein and the stress-induced 72-kDa protein have been purified by a two-step procedure employing DE52 ion-exchange chromatography followed by affinity chromatography on ATP-agarose. The two proteins, present in approximately equal amounts in either the 12,000 X g supernatant or pellet of hypotonically lysed heat-shock-treated HeLa cells, were found to copurify in relatively homogenous form. The purified proteins were covalently labeled with the fluorescent dye tetramethylrhodamine isothiocyanate, and the fluorescently labeled proteins were introduced back into living rat embryo fibroblasts via microinjection. The microinjected cells maintained at 37 degrees C showed only diffuse nuclear and cytoplasmic fluorescence. After heat-shock treatment of the cells, fluorescence was observed throughout the nucleus and more prominently within the nucleolus. This result is consistent with our earlier indirect immunofluorescence studies which showed a nuclear and nucleolar distribution of the endogenous 72-kDa stress protein in heat-shock-treated mammalian cells. The result also indicates that, for at least the 72-kDa protein, (i) the protein has been purified in apparently "native" form and (ii) its nucleolar distribution is stress dependent.

scholarly journals Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides.

1985 ◽  
Vol 5 (6) ◽  
pp. 1229-1237 ◽  
Author(s):  
W J Welch ◽  
J R Feramisco
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Stress Proteins ◽  
Stress Protein ◽  
Exchange Chromatography ◽  
Native Form ◽  
Step Procedure ◽  
Rapid Purification ◽  
Stress Dependent ◽  
Tetramethylrhodamine Isothiocyanate

A new and rapid purification procedure has been developed for the mammalian 70,000-dalton (70-kDa) heat-shock (or stress) proteins. Both the constitutive 73-kDa protein and the stress-induced 72-kDa protein have been purified by a two-step procedure employing DE52 ion-exchange chromatography followed by affinity chromatography on ATP-agarose. The two proteins, present in approximately equal amounts in either the 12,000 X g supernatant or pellet of hypotonically lysed heat-shock-treated HeLa cells, were found to copurify in relatively homogenous form. The purified proteins were covalently labeled with the fluorescent dye tetramethylrhodamine isothiocyanate, and the fluorescently labeled proteins were introduced back into living rat embryo fibroblasts via microinjection. The microinjected cells maintained at 37 degrees C showed only diffuse nuclear and cytoplasmic fluorescence. After heat-shock treatment of the cells, fluorescence was observed throughout the nucleus and more prominently within the nucleolus. This result is consistent with our earlier indirect immunofluorescence studies which showed a nuclear and nucleolar distribution of the endogenous 72-kDa stress protein in heat-shock-treated mammalian cells. The result also indicates that, for at least the 72-kDa protein, (i) the protein has been purified in apparently "native" form and (ii) its nucleolar distribution is stress dependent.

Stress protein systems of mammalian cells

1986 ◽  
Vol 250 (1) ◽  
pp. C1-C17 ◽  
Author(s):  
J. R. Subjeck ◽  
T. T. Shyy
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Mammalian Cells ◽  
Stress Proteins ◽  
Heat Exposure ◽  
Stress Protein ◽  
Protective Role ◽  
Living Organisms ◽  
Shock Proteins ◽  
Glucose Regulated Proteins

Living organisms are known to react to a heat stress by the selective induction in the synthesis of several polypeptides. In this review we list the major stress proteins of mammalian cells that are induced by heat shock and other environments and categorize these proteins into specific subgroups: the major heat shock proteins, the glucose-regulated proteins, and the low-molecular-weight heat shock proteins. Characteristics of the localization and expression of proteins in each of these subgroups are presented. Specifically, the nuclear/nucleolar locale of certain of the major heat shock proteins is considered with respect to their association with RNA and the recovery of cells after a heat exposure. The induction of these major heat shock proteins and the repression of the glucose-regulated proteins as a result of reoxygenation of anoxic cells or by the addition of glucose to glucose-deprived cultures is described. Changes in the expression of these protein systems during embryogenesis and differentiation in mammalian and nonmammalian systems is summarized, and the protective role that some of these proteins appear to play in protecting the animal against the lethal effects of a severe heat treatment and against teratogenesis is critically examined.

scholarly journals Characterization of the thermotolerant cell. I. Effects on protein synthesis activity and the regulation of heat-shock protein 70 expression.

1988 ◽  
Vol 106 (4) ◽  
pp. 1105-1116 ◽  
Author(s):  
L A Mizzen ◽  
W J Welch
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Stress Proteins ◽  
Stress Protein ◽  
Shock Treatment ◽  
Heat Shock Treatment ◽  
Normal Medium ◽  
Growth Environment ◽  
Mammalian Cell Lines ◽  
C 30

Exposure of mammalian cells to a nonlethal heat-shock treatment, followed by a recovery period at 37 degrees C, results in increased cell survival after a subsequent and otherwise lethal heat-shock treatment. Here we characterize this phenomenon, termed acquired thermotolerance, at the level of translation. In a number of different mammalian cell lines given a severe 45 degrees C/30-min shock and then returned to 37 degrees C, protein synthesis was completely inhibited for as long as 5 h. Upon resumption of translational activity, there was a marked induction of heat-shock (or stress) protein synthesis, which continued for several hours. In contrast, cells first made thermotolerant (by a pretreatment consisting of a 43 degrees C/1.5-h shock and further recovery at 37 degrees C) and then presented with the 45 degrees C/30-min shock exhibited considerably less translational inhibition and an overall reduction in the amount of subsequent stress protein synthesis. The acquisition and duration of such "translational tolerance" was correlated with the expression, accumulation, and relative half-lives of the major stress proteins of 72 and 73 kD. Other agents that induce the synthesis of the stress proteins, such as sodium arsenite, similarly resulted in the acquisition of translational tolerance. The probable role of the stress proteins in the acquisition of translational tolerance was further indicated by the inability of the amino acid analogue, L-azetidine 2-carboxylic acid, an inducer of nonfunctional stress proteins, to render cells translationally tolerant. If, however, analogue-treated cells were allowed to recover in normal medium, and hence produce functional stress proteins, full translational tolerance was observed. Finally, we present data indicating that the 72- and 73-kD stress proteins, in contrast to the other major stress proteins (of 110, 90, and 28 kD), are subject to strict regulation in the stressed cell. Quantitation of 72- and 73-kD synthesis after heat-shock treatment under a number of conditions revealed that "titration" of 72/73-kD synthesis in response to stress may represent a mechanism by which the cell monitors its local growth environment.

Synthesis of stress protein 70 (Hsp70) in rainbow trout (Oncorhynchus mykiss) red blood cells

1997 ◽  
Vol 200 (3) ◽  
pp. 607-614 ◽  
Author(s):  
S Currie ◽  
B Tufts
Keyword(s):  
Protein Synthesis ◽  
Rainbow Trout ◽  
Heat Shock ◽  
Oncorhynchus Mykiss ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Stress Proteins ◽  
Stress Protein ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Hsp70 Synthesis

Unlike enucleated mammalian red blood cells (rbcs), the nucleated rbcs of lower vertebrates are capable of protein synthesis and may, therefore, serve as a valuable model to investigate the adaptive significance of stress protein synthesis in cells. This study examined the synthesis of stress protein 70 (Hsp70) in rbcs of the temperature-sensitive rainbow trout Oncorhynchus mykiss in response to heat shock and anoxia. Through western blot analysis, we have demonstrated that rainbow trout rbcs synthesize Hsp70 both constitutively and in response to an increase in temperature. Radioisotopic labelling experiments indicated that the temperature at which Hsp70 synthesis was induced in fish acclimated to 10 °C was between 20 and 25 °C. Actinomycin D blocked de novo Hsp70 synthesis, implying that synthesis of Hsp70 is regulated at the level of transcription in rainbow trout rbcs. Since trout rbcs rely heavily on aerobic metabolism, but may also experience very low oxygen levels within the circulation, we also examined the relative importance of (1) anoxia as a stimulus for Hsp70 synthesis and (2) oxygen as a requirement for protein synthesis under control and heat-shock conditions. We found that trout rbcs were capable of protein synthesis during 2 h of anoxia, but did not increase Hsp70 synthesis. Moreover, rbcs subjected to combined anoxia and heat shock exhibited increases in Hsp70 synthesis that were similar in magnitude to those in cells exposed to heat shock alone. The latter results suggest that rainbow trout rbcs are (1) able to synthesize non-stress proteins during anoxia, (2) capable of tolerating periods of reduced oxygen availability without increased synthesis of stress proteins and (3) able to maintain the integrity of their heat-shock response even during periods of anoxia.

scholarly journals Disruption of the three cytoskeletal networks in mammalian cells does not affect transcription, translation, or protein translocation changes induced by heat shock.

1985 ◽  
Vol 5 (7) ◽  
pp. 1571-1581 ◽  
Author(s):  
W J Welch ◽  
J R Feramisco
Keyword(s):  
Heat Shock ◽  
Heat Shock Response ◽  
Mammalian Cells ◽  
Stress Proteins ◽  
Protein Translocation ◽  
Shock Response ◽  
Cytochalasin E ◽  
Cytoskeletal Networks ◽  
Shock Proteins ◽  
Cytoskeletal Elements

Mammalian cells show a complex series of transcriptional and translational switching events in response to heat shock treatment which ultimately lead to the production and accumulation of a small number of proteins, the so-called heat shock (or stress) proteins. We investigated the heat shock response in both qualitative and quantitative ways in cells that were pretreated with drugs that specifically disrupt one or more of the three major cytoskeletal networks. (These drugs alone, cytochalasin E and colcemid, do not result in induction of the heat shock response.) Our results indicated that disruption of the actin microfilaments, the vimentin-containing intermediate filaments, or the microtubules in living cells does not hinder the ability of the cell to undergo an apparently normal heat shock response. Even when all three networks were simultaneously disrupted (resulting in a loose, baglike appearance of the cells), the cells still underwent a complete heat shock response as assayed by the appearance of the heat shock proteins. In addition, the major induced 72-kilodalton heat shock protein was efficiently translocated from the cytoplasm into its proper location in the nucleus and nucleolus irrespective of the condition of the three cytoskeletal elements.

scholarly journals Induction by prostaglandin A1 of haem oxygenase in myoblastic cells: an effect independent of expression of the 70 kDa heat shock protein

1995 ◽  
Vol 308 (2) ◽  
pp. 455-463 ◽  
Author(s):  
A Rossi ◽  
M G Santoro
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Present Report ◽  
Oxidant Stress ◽  
Stress Protein ◽  
Hsp70 Expression ◽  
Transcriptional Level ◽  
Protective Mechanisms ◽  
Haem Oxygenase ◽  
Prostaglandin A1

Prostaglandins of the A type (PGA) induce the synthesis of 70 kDa heat shock proteins (hsp70) in a large variety of mammalian cells. Induction of hsp70 has been associated with a cytoprotective effect of PGA1 after virus infection or thermal injury. In the present report we provide evidence that, in murine myoblasts, PGA1 is not able to induce hsp70 expression, whereas it increases the synthesis of the constitutive protein, hsc70, and dramatically induces the synthesis of a 32 kDa protein (p32). The p32 protein has been identified as haem oxygenase. PGA1 acts at the transcriptional level by inducing haem oxygenase mRNA synthesis, and the signal for induction appears to be associated with decreased intracellular GSH levels. Haem oxygenase, a low-molecular-mass stress protein induced in mammalian cells by oxidant stress, is known to be part of a general inducible antioxidant defence pathway. The fact that prostaglandin synthesis is stimulated in muscle during contraction and in the heart in response to ischaemia raises the possibility that induction of haem oxygenase by PGA in myoblasts could be part of a protective mechanisms in operation during stress and hypoxia.

Retinoic acid stimulates the synthesis of a novel heat shock protein in the regenerating forelimb of the newt

1993 ◽  
Vol 71 (1-2) ◽  
pp. 43-50 ◽  
Author(s):  
Robert L. Carlone ◽  
Robert P. Boulianne ◽  
K. Marion Vijh ◽  
Heather Karn ◽  
Gordon A. D. Fraser
Keyword(s):  
Retinoic Acid ◽  
Heat Shock ◽  
Stress Proteins ◽  
Limb Regeneration ◽  
Stress Protein ◽  
Cross Reactivity ◽  
Hsp 70 ◽  
Peptide Map ◽  
Shock Proteins

Morphogenetic effects of retinoic acid (RA) on the urodele amphibian limb regenerate pattern have been well documented, but little is known regarding the mechanism of this action of RA at the molecular level. Since exogenous RA, at concentrations sufficient to cause proximalization, represents a significant stress to newts and has been shown previously to elicit increased synthesis of heat shock proteins (HSPs) in mouse embryo limb buds, we investigated the effects of this putative morphogen on the synthesis of members of the 70-kilodalton (70-kDa) stress protein family in amputated forelimbs of the newt Notophthalmus viridescens. Injection (i.p.) of RA in dimethyl sulfoxide (DMSO), at a dose sufficient to cause significant proximal–distal reduplication of the pattern in 50% of animals treated, resulted in increased synthesis and accumulation of a 73-kDa protein with a pi of approximately 6.75. The synthesis of this same protein is increased in limb tissues as a result of a brief 35 °C heat shock. This protein is electrophoretically distinct from the newt HSP 70 family members, displays a different partial peptide map, and shows no immunological cross-reactivity with an anti-human HSP 70 monoclonal antibody. It may be a member of a separate family of 70- to 73-kDa HSPs. Interestingly, the synthesis of this protein is increased and it is more abundant in control, proximal moderate-early bud stage regenerates at 6 days after i.p. injection of DMSO than in similarly treated distal regenerates. This protein is, in addition, increased in distal regenerates to proximal levels by a prior injection of RA. The significance of these findings with regard to the possible role of stress proteins in the morphogenetic processes underlying limb regeneration is discussed.Key words: heat shock, limb regeneration, retinoic acid, pattern formation, newt.

scholarly journals Extracellular cell stress (heat shock) proteins—immune responses and disease: an overview

2017 ◽  
Vol 373 (1738) ◽  
pp. 20160522 ◽  
Author(s):  
A. Graham Pockley ◽  
Brian Henderson
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Stress Proteins ◽  
Biological Fluids ◽  
Stress Protein ◽  
Cell Stress ◽  
Immune Reactivity ◽  
Protein Levels ◽  
Shock Proteins ◽  
Adaptive Immune Systems

Extracellular cell stress proteins are highly conserved phylogenetically and have been shown to act as powerful signalling agonists and receptors for selected ligands in several different settings. They also act as immunostimulatory ‘danger signals’ for the innate and adaptive immune systems. Other studies have shown that cell stress proteins and the induction of immune reactivity to self-cell stress proteins can attenuate disease processes. Some proteins (e.g. Hsp60, Hsp70, gp96) exhibit both inflammatory and anti-inflammatory properties, depending on the context in which they encounter responding immune cells. The burgeoning literature reporting the presence of stress proteins in a range of biological fluids in healthy individuals/non-diseased settings, the association of extracellular stress protein levels with a plethora of clinical and pathological conditions and the selective expression of a membrane form of Hsp70 on cancer cells now supports the concept that extracellular cell stress proteins are involved in maintaining/regulating organismal homeostasis and in disease processes and phenotype. Cell stress proteins, therefore, form a biologically complex extracellular cell stress protein network having diverse biological, homeostatic and immunomodulatory properties, the understanding of which offers exciting opportunities for delivering novel approaches to predict, identify, diagnose, manage and treat disease. This article is part of the theme issue ‘Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective’.

Exercising mammals synthesize stress proteins

1990 ◽  
Vol 258 (4) ◽  
pp. C723-C729 ◽  
Author(s):  
M. Locke ◽  
E. G. Noble ◽  
B. G. Atkinson
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Stress Proteins ◽  
Sprague Dawley ◽  
Spleen Cells ◽  
Sprague Dawley Rats ◽  
Electrophoretic Separations ◽  
Shock Proteins ◽  
Sufficient Stimulus

Spleen cells, peripheral lymphocytes, and soleus muscles were removed from male Sprague-Dawley rats that had been run on a treadmill (24 m/min) for either 20, 40, or 60 min or to exhaustion (86 +/- 41 min) and were labeled in vitro with [35S]methionine at 37 degrees C. Similar tissues from nonrunning control rats were labeled in vitro at either 37 or 43 degrees C (heat shock). Fluorographic analyses of one- and two-dimensional polyacrylamide gel electrophoretic separations of the proteins from cells and tissues of exercised rats demonstrate the new or enhanced synthesis of proteins of approximately 65, 72, 90, and 100 kDa. Although synthesis of these proteins is low or not detectable in tissues from control rats labeled at 37 degrees C, they are prominent products of similar tissues labeled under heat-shock conditions (43 degrees C) and, in fact, correspond in Mr and pI with the so-called heat-shock proteins. These results suggest that exercise is a sufficient stimulus to induce or enhance the synthesis of heat shock and/or stress proteins in mammalian cells and tissues.

Analysis of the temperature-dependent temporal pattern of heat-shock-protein synthesis in fish cells

1983 ◽  
Vol 3 (7) ◽  
pp. 647-658 ◽  
Author(s):  
Lashitew Gedamu ◽  
Beverly Culham ◽  
John J. Heikkila
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Temporal Pattern ◽  
Stress Proteins ◽  
Incubation Temperature ◽  
Stress Protein ◽  
In Vitro Translation ◽  
Continuous Exposure ◽  
Temperature Dependent

Continuous exposure of Chinook salmon embryo cells to an elevated incubation temperature of 24°C induces the transient expression of a set of heat-shock or stress proteins whereas maintenance of the cells at a higher incubation temperature of 28°C produces a continuous synthesis of these stress proteins. In vitro translation studies suggest that the temperature-dependent temporal pattern of stress-protein synthesis is correlated with the levels of stress-protein mRNA. This was verified using a recombinant-DNA probe complementary to the 70K heat-shock-protein mRNA. A transient increase in the level of the fish heat-shock 70K mRNA was observed in RNA samples isolated from cells continuously exposed at 24°C However, a constant increase in the level of this specific mRNA was found in RNA preparations obtained from cells maintained at 28°C Therefore, the temperature-dependent pattern of fish heat-shockprotein synthesis appears to be directly related to the level of heat-shock-protein mRNA.

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