The role of heat-shock proteins as molecular chaperones.

Recent studies have revealed that protein folding and assembly events in vivo require the participation of accessory components, now being referred to as 'molecular chaperones'. A number of chaperones have been identified as members of the heat-shock (or stress) protein family. This review discusses the roles of two classes of chaperones, the heat-shock protein 70 and groEL/ES families, in facilitating protein maturation, and describes how such events are perturbed in the cell subjected to metabolic stress.

Autoantibodies to the constitutive 73-kD member of the hsp70 family of heat shock proteins in systemic lupus erythematosus.

Serum from patients with systemic lupus erythematosus (SLE) frequently contain IgM and IgG autoantibodies to the constitutively expressed 73-kD/pI 5.5 member of the hsp70 family of heat shock proteins, as determined by one-dimensional (SDS-PAGE) and two-dimensional (IEF/SDS-PAGE) immunoblotting, and by solid-phase SLE Ig immunoprecipitation experiments using hsp70 protein-specific mAbs as probes. Autoantibodies to hsp70 also were detected in a minority of sera from patients with other rheumatic or viral diseases, but not in normal sera. These data may provide additional insight into etiologic and pathophysiologic mechanisms in this and related autoimmune disorders.

Activation primes human B lymphocytes to respond to heat shock.

Crosslinkage of the B cell antigen receptor by anti-mu beads or SAC results in the selective induction of hsp70. We have observed that activated cells, having enhanced expression of hsp70, survive lethal stimuli much better than their unactivated counterparts. These results are in accordance with the proposal that hsp70 is essential for cells to survive lethal environmental stresses. Moreover, the activation event itself primes B cells thereby enabling them to increase the expression of both hsp70 mRNA and protein. This is the first demonstration that triggering of B cells via crosslinkage of sIg is accompanied by the induction of thermotolerance without the need for a prior sublethal heat treatment.

Constitutive expression of a groEL-related protein on the surface of human gamma/delta cells.

Rabbit antibodies to hsp58 (P1), the human homologue of the Escherichia coli stress protein groEL, react specifically in indirect immunofluorescence and complement-dependent microcytoxicity experiments with a cell surface antigen expressed constitutively by T cell lines bearing gamma/delta receptors. This anti-hsp58-reactive antigen is not demonstrable on T cells that express alpha/beta receptors or on various cells that lack T cell receptors. Certain evidence was obtained to suggest that the target antigen on the surface of gamma/delta T cells is a approximately 77-kD protein distinct from intracellular hsp58 and known members of the hsp70 stress protein family. While the exact nature and significance of this anti-hsp58-reactive protein remain to be determined, these data may help to clarify the roles of groEL-related stress proteins and gamma/delta cells that recognize groEL homologous in immunologic defense against infection and in autoimmune disease.

Endothelial prostaglandin D2 opposes angiotensin II contractions in mouse isolated perfused intracerebral microarterioles.

HYPOTHESIS: A lack of contraction of cerebral microarterioles to Ang II ("resilience") depends on cyclooxygenase (COX) and lipocalin type prostaglandin D sythase L-PGDS producing PGD2 that activates prostaglandin D type 1 receptors (DP1Rs) and nitric oxide synthase (NOS). MATERIALS & METHODS: Contractions were assessed in isolated, perfused vessels and NO by fluorescence microscopy. RESULTS: The mRNAs of penetrating intraparenchymal cerebral microarterioles versus renal afferent arterioles were >3000-fold greater for L-PGDS and DP1R and 5-fold for NOS III and COX 2. Larger cerebral arteries contracted with Ang II. However, cerebral microarterioles were entirely unresponsive but contracted with endothelin 1 and perfusion pressure. Ang II contractions were evoked in cerebral microarterioles from COX1 -/- mice or after blockade of COX2, L-PGDS or NOS and in deendothelialized vessels but effects of deendothelialization were lost during COX blockade. NO generation with Ang II depended on COX and also was increased by DP1R activation. CONCLUSION: The resilience of cerebral arterioles to Ang II contractions is specific for intraparenchymal microarterioles and depends on endothelial COX1 and two products that are metabolized by L-PGDS to generate PGD2 that signals via DP1Rs and NO.

Cellular and biochemical events in mammalian cells during and after recovery from physiological stress.

We have examined and compared a number of cellular and biochemical events associated with the recovery process of rat fibroblasts placed under stress by different agents. Metabolic pulse-labeling studies of cells recovering from either heat-shock treatment, exposure to sodium arsenite, or exposure to an amino acid analogue of proline, L-azetidine 2-carboxylic acid, revealed interesting differences with respect to the individual stress proteins produced, their kinetics of induction, as well as the decay in their synthesis during the recovery period. In the initial periods of recovery, the major stress-induced 72-kD protein accumulates within the altered nucleoli in close association with the pre-ribosomal-containing granular region. During the later times of recovery from stress, the nucleoli begin to regain a normal morphology, show a corresponding loss of the 72-kD protein, and the majority of the protein now begins to accumulate within the cytoplasm in three distinct locales: the perinuclear region, along the perimeter of the cells, and finally in association with large phase-dense structures. These latter structures appear to consist of large aggregates of phase-dense material with no obvious encapsulating membrane. More interestingly we show, using double-label indirect immunofluorescence analysis, that much of the perinuclear and cell perimeter-distributed 72-kD protein coincides with the distribution of the cytoplasmic ribosomes. We discuss the possible implications of the presence of the 72-kD stress proteins within the pre-ribosomal-containing granular region of the nucleolus as well as its subsequent colocalization with cytoplasmic ribosomes in terms of the translational changes which occur in cells both during and after recovery from physiological stress.

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

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.

Examining the function and regulation of hsp 70 in cells subjected to metabolic stress.

Members of the heat-shock protein (hsp) 70 family, distributed within various cellular compartments, have been implicated in facilitating protein maturation events. In particular, related hsp 70 family members appear to bind nascent polypeptides which are in the course of synthesis and/or translocation into organelles. We previously reported that in normal, unstressed cells, cytosolic hsp 70 (hsp 72/73) interacted transiently with nascent polypeptides. We suspect that such interactions function to prevent or slow down the folding of the nascent polypeptide chain. Once synthesis is complete, and now with all of the information for folding present, the newly synthesized protein appears to commence along its folding pathway, accompanied by the ATP-dependent release of hsp 72/73. Herein, we examined how these events occur in cells subjected to different types of metabolic stress. In cells exposed to either an amino acid analog or sodium arsenite, two potent inducers of the stress response, newly synthesized proteins bind to but are not released from hsp 70. Under these conditions of metabolic stress, we suspect that the newly synthesized proteins are unable to commence proper folding and consequently remain bound to their hsp 70 chaperone. In cells subjected to heat shock, a large number of both newly synthesized as well as mature proteins are rendered insoluble. Within this insoluble material are appreciable amounts of hsp 72/73. Finally, we show that in cells depleted of ATP, the release of hsp 70 from maturing proteins is inhibited. Thus, in cells experiencing metabolic stress, newly synthesized proteins unable to properly fold, as will as mature proteins which begin to unfold become stably bound to hsp 72/73. As a consequence and over time, the free or available levels of pre-existing hsp 72/73 are reduced. We propose that this reduction in the available levels of hsp 72/73 is the trigger by which the stress response is initiated.

Heat shock gene regulation by nascent polypeptides and denatured proteins: hsp70 as a potential autoregulatory factor.

Heat shock genes encode proteins (hsp's) that play important structural roles under normal circumstances and are essential to the cells' ability to survive environmental insults. Evidence is presented herein that transcriptional regulation of hsp gene expression is linked with the regulation of overall protein synthesis as well as with the accumulation of proteins denatured by stressful events. The factor that connects the three processes appears to be one of the hsp's, presumably a member(s) of the hsp70 family. Biochemical experiments demonstrate that complexes containing hsp70 and heat shock transcription factor, the specific regulator of hsp gene activity, are formed in the cells.

Characterization of the thermotolerant cell. II. Effects on the intracellular distribution of heat-shock protein 70, intermediate filaments, and small nuclear ribonucleoprotein complexes.

Here we further characterize a number of properties inherent to the thermotolerant cell. In the preceding paper, we showed that the acquisition of the thermotolerant state (by a prior induction of the heat-shock proteins) renders cells translationally tolerant to a subsequent severe heat-shock treatment and thereby results in faster kinetics of both the synthesis and subsequent repression of the stress proteins. Because of the apparent integral role of the 70-kD stress proteins in the acquisition of tolerance, we compared the intracellular distribution of these proteins in both tolerant and nontolerant cells before and after a severe 45 degrees C/30-min shock. In both HeLa and rat embryo fibroblasts, the synthesis and migration of the major stress-induced 72-kD protein into the nucleolus and its subsequent exit was markedly faster in the tolerant cells as compared with the nontolerant cells. Migration of preexisting 72-kD into the nucleolus was shown to be dependent upon heat-shock treatment and independent of active heat-shock protein synthesis. Using both microinjection and immunological techniques, we observed that the constitutive and abundant 73-kD stress protein similarly showed a redistribution from the cytoplasm and nucleus into the nucleolus as a function of heat-shock treatment. We show also that other lesions that occur in cells after heat shock can be prevented or at least minimized if the cells are first made tolerant. Specifically, the heat-induced collapse of the intermediate filament cytoskeleton did not occur in cells rendered thermotolerant. Similarly, the disruption of intranuclear staining patterns of the small nuclear ribonucleoprotein complexes after heat-shock treatment was less apparent in tolerant cells exposed to a subsequent heat-shock treatment.

Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments in rat fibroblasts after heat-shock treatment.

Using both electron microscopy and immunological methods, we have characterized a number of changes occurring in rat fibroblasts after heat-shock treatment. Incubation of the cells for 3 h at 42 degrees-43 degrees C resulted in a number of changes within the cytoplasm including: a disruption and fragmentation of the Golgi complex; a modest swelling of the mitochondria and subtle alterations in the packing of the cristae; and alterations in cytoskeletal elements, specifically a collapse and aggregation of the vimentin-containing intermediate filaments around the nucleus. A number of striking changes were also found within the nuclei of the heat-treated cells: (a) We observed the appearance of rod-shaped bodies consisting of densely packed filaments. Using biochemical and immunological methods, these nuclear inclusion bodies were shown to be comprised of actin filaments. (b) Considerable alterations in the integrity of the nucleoli were observed after the heat-shock treatment. Specifically, there appeared to be a general relaxation in the condensation state of the nucleoli, changes in both the number and size of the granular ribonucleoprotein components, and finally a reorganization of the nucleolar fibrillar reticulum. These morphological changes in the integrity of the nucleoli are of significant interest since previous work as well as studies presented here show that two of the mammalian stress proteins, the major stress-induced 72-kD protein and the 110-kD protein, localize within the nucleoli of the cells after heat-shock treatment. We discuss these morphological changes with regards to the known biological and biochemical events that occur in cells after induction of the stress response.

Prefoldin-nascent chain complexes in the folding of cytoskeletal proteins.

In vitro transcription/translation of actin cDNA and analysis of the translation products by native-PAGE was used to study the maturation pathway of actin. During the course of actin synthesis, several distinct actin-containing species were observed and the composition of each determined by immunological procedures. After synthesis of the first approximately 145 amino acids, the nascent ribosome-associated actin chain binds to the recently identified heteromeric chaperone protein, prefoldin (PFD). PFD remains bound to the relatively unfolded actin polypeptide until its posttranslational delivery to cytosolic chaperonin (CCT). We show that alpha- and beta-tubulin follow a similar maturation pathway, but to date find no evidence for an interaction between PFD and several noncytoskeletal proteins. We conclude that PFD functions by selectively targeting nascent actin and tubulin chains pending their transfer to CCT for final folding and/or assembly.

Characterization and inducibility of hsp 70 proteins in the male mouse germ line.

The properties and inducibility of the heat shock protein 70 (hsp 70) gene products were examined during differentiation of mouse testicular cells by one and two-dimensional gel electrophoresis and immunoblotting. Low levels of the 72- and 73-kD heat shock proteins normally found in mouse cell lines were detected in the mouse testis. A novel isoform with a relative molecular mass of 73 kD (called 73T) was also observed, in the presence or absence of heat shock. 73T was shown to be produced by germ cells since it was not detected in testes from mutant mice devoid of germ cells. Furthermore, 73T was found only in adult mouse testicular cells, not in testes from animals that lack meiotic germ cells. 73T was synthesized in enriched cell populations of both meiotic prophase and postmeiotic cells, but was not inducible by in vitro heat shock. In the adult testis, low levels of the bona fide 72-kD heat-inducible (hsp72) were induced in response to elevated temperatures. In contrast, in testes from animals in which only somatic cells and premeiotic germ cells were present, there was a substantial induction of hsp 72. It is suggested that hsp 72 is inducible in the somatic compartment and possibly in the premeiotic germ cells, but not in germ cells which have entered meiosis and which are expressing members of the hsp 70 gene family in a developmentally regulated fashion.

Cell cycle-dependent association of HSP70 with specific cellular proteins.

In asynchronous populations of HeLa cells maintained at control or heat shock temperatures, HSP70 levels and its subcellular distribution exhibit substantial heterogeneity as demonstrated by indirect immunofluorescence with HSP70-specific monoclonal antibodies. Of particular interest is a subpopulation of cells in which the characteristic nuclear accumulation and nucleolar association of HSP70 is not detected after heat shock treatment. This apparent variation in the heat shock response is not observed when synchronized cells are examined. In this study, we demonstrate that three monoclonal antibodies to HSP70, in particular, do not detect nucleolar-localized HSP70 in heat-shocked G2 cells. This is not due to an inability of G2 cells to respond to heat shock as measured by increased HSP70 mRNA and protein synthesis, or due to a lack of accumulation of HSP70 after heat shock in G2. Rather the epitopes recognized by the various antibodies appear to be inaccessible, perhaps due to the association of HSP70 with other proteins. Non-denaturing immunoprecipitations with these HSP70-specific antibodies suggest that HSP70 may interact with other cellular proteins in a cell cycle-dependent manner.

Modulation of vimentin containing intermediate filament distribution and phosphorylation in living fibroblasts by the cAMP-dependent protein kinase.

Microinjection of the purified catalytic subunit of the cAMP-dependent protein kinase (A-kinase) into living rat embryo fibroblasts leads to dramatic changes in vimentin intermediate filament (IF) organization, involving the collapse of the filaments into tight bundles. In some cell types, this rearrangement of the IF proceeds further, leading to an apparent loss of filament integrity, resulting in a punctate staining pattern throughout the cytoplasm. Both these types of IF rearrangement are fully reversible, and similar to structural changes previously described for IF during mitosis. As shown by electron microscopy, in rat embryo fibroblasts these changes in IF structure do not involve the loss of the 10-nM filament structure but instead correspond to the bundling together of 25 or more individual filaments. Metabolic pulse labeling of injected cells reveals that accompanying these changes in IF organization is a dramatic increase in vimentin phosphorylation which appears maximal when the IF are fully rearranged. However, this increase in IF phosphorylation is not accompanied by any significant increase in soluble vimentin. Analysis of the sites of phosphorylation on vimentin from injected cells by either V8 protease cleavage, or two-dimensional tryptic peptide mapping, revealed increased de novo phosphorylation of two vimentin phosphopeptides after microinjection of A-kinase. These data strongly suggest that the site-specific phosphorylation of vimentin by A-kinase is responsible for the dynamic changes in IF organization observed after injection of the kinase into living cells, and may be involved in similar rearrangement of the IF previously described during mitosis or after heat shock.

Involvement of 70-kD heat-shock proteins in peroxisomal import.

This report describes the involvement of 70-kD heat-shock proteins (hsp70) in the import of proteins into mammalian peroxisomes. Employing a microinjection-based assay (Walton, P. A., S. J. Gould, J. R. Feramisco, and S. Subramani. 1992. Mol. Cell Biol. 12:531-541), we demonstrate that proteins of the hsp70 family were associated with proteins being imported into the peroxisomal matrix. Import of peroxisomal proteins could be inhibited by coinjection of antibodies directed against the constitutive hsp70 proteins (hsp73). In a permeabilized-cell assay (Wendland and Subramani. 1993. J. Cell Biol. 120:675-685), antibodies directed against hsp70 proteins were shown to inhibit peroxisomal protein import. Inhibition could be overcome by the addition of exogenous hsp70 proteins. Purified rat liver peroxisomes were shown to have associated hsp70 proteins. The amount of associated hsp70 was increased under conditions of peroxisomal proliferation. Furthermore, proteinase protection assays indicated that the hsp70 molecules were located on the outside of the peroxisomal membrane. Finally, the process of heat-shocking cells resulted in a considerable delay in the import of peroxisomal proteins. Taken together, these results indicate that heat-shock proteins of the cytoplasmic hsp70 family are involved in the import of peroxisomal proteins.

Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light chain kinase.

Microinjection of the catalytic subunit of cAMP-dependent protein kinase (A-kinase) into living fibroblasts or the treatment of these cells with agents that elevate the intracellular cAMP level caused marked alterations in cell morphology including a rounded phenotype and a complete loss of actin microfilament bundles. These effects were transient and fully reversible. Two-dimensional gel electrophoresis was used to analyze the changes in phosphoproteins from cells injected with A-kinase. These experiments showed that accompanying the disassembly of actin microfilaments, phosphorylation of myosin light chain kinase (MLCK) increased and concomitantly, the phosphorylation of myosin P-light chain decreased. Moreover, inhibiting MLCK activity via microinjection of affinity-purified antibodies specific to native MLCK caused a complete loss of microfilament bundle integrity and a decrease in myosin P-light chain phosphorylation, similar to that seen after injection of A-kinase. These data support the idea that A-kinase may regulate microfilament integrity through the phosphorylation and inhibition of MLCK activity in nonmuscle cells.

The constitutive and stress inducible forms of hsp 70 exhibit functional similarities and interact with one another in an ATP-dependent fashion.

Mammalian cells constitutively express a cytosolic and nuclear form of heat shock protein (hsp) 70, referred to here as hsp 73. In response to heat shock or other metabolic insults, increased expression of another cytosolic and nuclear form of hsp 70, hsp 72, is observed. The constitutively expressed hsp 73, and stress-inducible hsp 72, are highly related proteins. Still unclear, however, is exactly why most eukaryotic cells, in contrast to prokaryotic cells, express a novel form of hsp 70 (i.e., hsp 72) after experiencing stress. To address this question, we prepared antibodies specific to either hsp 72 or hsp 73 and have compared a number of biological properties of the two proteins, both in vivo and in vitro. Using metabolic pulse-chase labeling and immunoprecipitation analysis, both the hsp 72 and hsp 73 specific antibodies were found to coprecipitate a significant number of newly synthesized proteins. Such interactions appeared transient and sensitive to ATP. Consequently, we suspect that both hsp 72 and hsp 73 function as molecular chaperones, interacting transiently with nascent polypeptides. During the course of these studies, we routinely observed that antibodies specific to hsp 73 resulted in the coprecipitation of hsp 72. Similarly, antibodies specific to hsp 72 were capable of coprecipitating hsp 73. Using a number of different approaches, we show that the constitutively expressed, pre-existing hsp 73 rapidly forms a stable complex with the newly synthesized stress inducible hsp 72. As is demonstrated by double-label indirect immunofluorescence, both proteins exhibit a coincident locale within the cell. Moreover, injection of antibodies specific to hsp 73 into living cells effectively blocks the ability of both hsp 73 and hsp 72 to redistribute from the cytoplasm into the nucleus and nucleolus after heat shock. These results are discussed as they relate to the possible structure and function of the constitutive (hsp 73) and highly stress inducible (hsp 72) forms of hsp 70, both within the normal cell as well as in the cell experiencing stress.

A eukaryotic cytosolic chaperonin is associated with a high molecular weight intermediate in the assembly of hepatitis B virus capsid, a multimeric particle.

We have established a system for assembly of hepatitis B virus capsid, a homomultimer of the viral core polypeptide, using cell-free transcription-linked translation. The mature particles that are produced are indistinguishable from authentic viral capsids by four criteria: velocity sedimentation, buoyant density, protease resistance, and electron microscopic appearance. Production of unassembled core polypeptides can be uncoupled from production of capsid particles by decreasing core mRNA concentration. Addition of excess unlabeled core polypeptides allows the chase of the unassembled polypeptides into mature capsids. Using this cell-free system, we demonstrate that assembly of capsids proceeds by way of a novel high molecular weight intermediate. Upon isolation, the high molecular weight intermediate is productive of mature capsids when energy substrates are manipulated. A 60-kD protein related to the chaperonin t-complex polypeptide 1 (TCP-1) is found in association with core polypeptides in two different assembly intermediates, but is not associated with either the initial unassembled polypeptides or with the final mature capsid product. These findings implicate TCP-1 or a related chaperonin in viral assembly and raise the possibility that eukaryotic cytosolic chaperonins may play a distinctive role in multimer assembly apart from their involvement in assisting monomer folding.

Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70.

Synthesis of a small group of highly conserved proteins in response to elevated temperature and other agents that induce stress is a universal feature of prokaryotic and eukaryotic cells. Although correlative evidence suggests that these proteins play a role in enhancing survival during and after stress, there is no direct evidence to support this in mammalian cells. To assess the role of the most highly conserved heat shock protein (hsp) family during heat shock, affinity-purified monoclonal antibodies to hsp70 were introduced into fibroblasts by needle microinjection. In addition to impairing the heat-induced translocation of hsp70 proteins into the nucleus after mild heat shock treatment, injected cells were unable to survive a brief incubation at 45 degrees C. Cells injected with control antibodies survived a similar heat shock. These results indicate that functional hsp70 is required for survival of these cells during and after thermal stress.