A review of the role of 70 kDa heat shock proteins in protein translocation across membranes

Summary The compartmentalization of essential hsp70 proteins indicates that hsp70s carry out crucial functions in several compartments of the cell. The use of conditional mutants has allowed study of the cellular processes that require hsp70 function. For efficient translocation of proteins across membranes hsp70s are required in the cytoplasm, as well as in the matrix of mitochondria and in the lumen of the endoplasmic reticulum.     

The transport of proteins into the nucleus requires the 70-kilodalton heat shock protein or its cytosolic cognate.

The 70-kDa heat shock protein hsp70 and its constitutively expressed cognate, hsc70, are abundant proteins implicated in a number of cellular processes. When a permeabilized cell system for examining the transport of proteins into the nucleus is depleted of hsc70 and hsp70, either by affinity chromatography on ATP-agarose or with antibodies against these proteins, nuclear transport activity is lost. Full activity is restored by the addition of HeLa proteins that bind to ATP-agarose. hsc70 and hsp70 are the active factors, since activity is also fully restored by the addition of either recombinant hsc70 or hsp70 which has been bacterially expressed and highly purified. The restoration of activity is saturable. The transport system requires other cytosolic factors as well, including at least one protein that is sensitive to inactivation by N-ethylmaleimide, but neither hsc70 nor hsp70 is the sensitive protein.     

A novel hsp70-like protein (P70) is present in mouse spermatogenic cells.

Mouse spermatogenic cells contain relatively large amounts of a 70-kilodalton protein (P70) that is closely related to hsp70, the major inducible heat shock protein. When hsp70 from spermatogenic cells is heat induced, it migrates to the same location as does P70 on two-dimensional polyacrylamide gels, indicating that it has an apparently identical mass and isoelectric point. P70 reacts strongly and specifically with an anti-Drosophila hsp70 monoclonal antibody that is specific for products of the hsp70 gene family. Both P70 and hsp70 are also ATP-binding proteins and are purified by using ATP-affinity chromatography. However, P70 and hsp70 are unique proteins on the basis of peptide map analysis and are regulated differently in germ cells. P70 appears to be a novel heat shock protein of spermatogenic cells which is synthesized in association with germ cell differentiation.     

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.     

Characterization of proteins associated with heat shock protein hsp27 in the squamous cell carcinoma cell line A431.

Heat shock protein hsp27 is a molecular chaperone and identification of hsp27-binding proteins might help to elucidate its functional role in keratinocyte biology. In the present investigation we used a human epidermal cell carcinoma cell line (A431) transfected with hsp27 (A431/16) to study interference between hsp27 protein and other proteins. Immunoprecipitation experiments with anti-hsp27 antibody revealed a multicomponent complex when analysed by silver staining. By immunoblotting analysis we could demonstrate that hsp27 associates with actin, the mutant form of p53, hsp70 and hsp90. Immunofluorescence analysis showed a co-localization between hsp27 and p53, hsp70 and hsp90. To control for the specificity of the observed interactions, immuno-precipitations with antibodies to actin, p53, hsp70 and hsp90 respectively, were performed. All of the tested proteins demonstrated a coimmunoprecipitation with hsp27. We conclude that hsp27, like the other heat shock proteins, is part of a complex system of molecular chaperones in epidermal keratinocytes.     

Mammalian protein RAP46: an interaction partner and modulator of 70 kDa heat shock proteins.

A ubiquitously expressed nuclear receptor-associating protein of approximately 46 kDa (RAP46) was identified recently. Interaction experiments with in vitro-translated proteins and proteins contained in cell extracts revealed that a great variety of cellular regulators associate with RAP46. However, in direct interaction tests by the far-Western technique, only 70 kDa proteins showed up and were identified as members of the 70 kDa heat shock protein (hsp70) family. Interaction is specific since not all members of the hsp70 family bind to RAP46; interaction occurs through their ATP-binding domain. RAP46 forms complexes with hsp70 in mammalian cells and interacts with hsp70 in the yeast two-hybrid system. Consistent with the fact that hsp70 can bind a multitude of proteins, we identified heteromeric complexes of RAP46-hsp70 with some selected proteins, most notably c-Jun. Complex formation is increased significantly by pre-treatment with alkaline phosphatase, thus suggesting modulation of interactions by protein phosphorylation. We observed that RAP46 interferes with efficient refolding of thermally denatured luciferase. Moreover, ATP-dependent binding of misfolded proteins to hsp70 was greatly inhibited by RAP46. These data suggest that RAP46 functions as a regulator of hsp70 in higher eukaryotes.     

Induction of IL-10 and inhibition of experimental arthritis are specific features of microbial heat shock proteins that are absent for other evolutionarily conserved immunodominant proteins

Bacterial heat shock proteins (hsp) are evolutionary conserved immunodominant proteins that manifest amino acid homologies with hsp present in mammalian cells. Preimmunization with mycobacterial hsp65 has been found to protect against various forms of experimental arthritis. As these protective effects have previously been attributed to induction of self homologue cross-reactive T cell responses, the question was raised as to whether this protective effect could be extended to other highly conserved and immunodominant microbial Ags with mammalian homologues. Therefore, we immunized Lewis rats with conserved bacterial Ags (superoxide dismutase, aldolase, GAPDH, and hsp70). Although all Ags appeared highly immunogenic, we only found a protective effect in experimental arthritis after immunization with bacterial hsp70. The protective effect of hsp70 was accompanied with a switch in the subclasses of hsp70-specific Abs, suggesting the induction of Th2-like response. The most striking difference between immunization with hsp70 and all other immunodominant Ags was the expression of IL-10 found after immunization with hsp70. Even more, while immunization with hsp70 led to Ag-induced production of IL-10 and IL-4, immunization with aldolase led to increased production of IFN-gamma and TNF-alpha. Thus, the protective effect of conserved immunodominant proteins in experimental arthritis seems to be a specific feature of hsp. Therefore, hsp may offer unique possibilities for immunological intervention in inflammatory diseases.     

Differential distribution of the adenovirus E1A proteins and colocalization of E1A with the 70-kilodalton cellular heat shock protein in infected cells.

Five distinct localization patterns were observed for the adenovirus E1A proteins in the nuclei of infected HeLa cells: diffuse, reticular, nucleolar, punctate, and peripheral. The variable distribution of E1A was correlated with the time postinfection and the cell cycle stage of the host cell at the time of infection. All staining patterns, with the exception of peripheral E1A localization, were associated with the early phase of infection since only the diffuse, reticular, nucleolar, and punctate staining patterns were observed in the presence of hydroxyurea. Because the E1A proteins (12S and 13S) stimulate the expression of the cellular heat shock 70-kilodalton protein (hsp70), we examined the intracellular distribution of hsp70 in the adenovirus-infected cells. Whereas hsp70 was predominantly cytoplasmic in the cells before infection, after adenovirus infection most of the protein was now found within the nucleus. Specifically, hsp70 was found within the nucleoli as well as exhibiting reticular, diffuse, and punctate nuclear staining patterns, analogous to those observed for the E1A proteins. Double-label indirect immunofluorescence of E1A and hsp70 in infected cells demonstrated a colocalization of these proteins in the nucleus. Translocation of hsp70 to the nucleus was dependent upon both adenovirus infection and expression of the E1A proteins. The localization of hsp70 was unaltered by infection with an E1A 9S cDNA virus which does not synthesize a functional E1A gene product. Moreover, the discrete nuclear localization patterns of E1A and the colocalization of E1A with hsp70 were not observed in adenovirus-transformed 293 cells which constitutively express E1A and E1B. E1A displayed exclusively diffuse nuclear staining in 293 cells; however, localization of E1A into the discrete nuclear patterns occurred after adenovirus infection of 293 cells. Immunoprecipitation of labeled infected-cell extracts with a monoclonal antibody directed against the E1A proteins resulted in precipitation of small amounts of hsp70 along with E1A. These data indicate that the adenovirus E1A proteins colocalize with, and possibly form a physical complex with, cellular hsp70 in infected cells. The relevance of this association, with respect to the function of these proteins during infection and the association of other oncoproteins with hsp70, is discussed.     

A heat shock protein 90 inhibitor that modulates the immunophilins and regulates hormone receptors without inducing the heat shock response

When a cell encounters external stressors, such as lack of nutrients, elevated temperatures, changes in pH or other stressful environments, a key set of evolutionarily conserved proteins, the heat shock proteins (hsps), become overexpressed. Hsps are classified into six major families with the hsp90 family being the best understood; an increase in cell stress leads to increased levels of hsp90, which leads to cellular protection. A hallmark of hsp90 inhibitors is that they induce a cell rescue mechanism, the heat shock response. We define the unique molecular profile of a compound (SM145) that regulates hormone receptor protein levels through hsp90 inhibition without inducing the heat shock response. Modulation of the binding event between heat shock protein 90 and the immunophilins/homologs using SM145, leads to a decrease in hormone receptor protein levels. Unlike N-terminal hsp90 inhibitors, this hsp90 inhibitor does not induce a heat shock response. This work is proof of principle that controlling hormone receptor expression can occur by inhibiting hsp90 without inducing pro-survival protein heat shock protein 70 (hsp70) or other proteins associated with the heat shock response. Innovatively, we show that blocking the heat shock response, in addition to hsp90, is key to regulating hsp90-associated pathways.     

The chaperone function of hsp70 is required for protection against stress-induced apoptosis

Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochrome c-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.     

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.     

Intracellular distribution of mammalian stress proteins. Effects of cytoskeletal-specific agents

Following a brief period of heat stress, the two highly conserved mammalian stress proteins, hsp68 and 70, were examined with respect to their intracellular locations. In four independent cell lines, hsp68 and 70 were found to partition into both Triton X-100-soluble and insoluble fractions as assessed by two-dimensional gel analysis of newly synthesized polypeptides, whereas a fifth cell line showed these proteins only in the Triton X-100-insoluble fraction. In addition, a previously described cell fractionation technique was utilized to gain information regarding the segregation of the two major mammalian stress proteins, hsp68 and 70, into distinct biochemically and morphologically characterized subcellular compartments of PtK2-epithelial cells. Two cytoskeletal-specific agents, taxol and colchicine, were also probed for their effects on the disposition of these polypeptides. Under our conditions of acute heat exposure, hsp68, 70 and their isoforms were globally distributed in all subcellular fractions examined, with a few notable exceptions in drug-treated cells. Colchicine, a microtubule-depolymerizing drug, inhibited the association of hsp68 and its variants with the double-detergent-extractable labile "cytoskeleton," whereas taxol, a microtubule-stabilizing agent, in some manner, facilitated the transit of hsp68 and its isovariants from a cytoplasmic to nuclear domain. Degree of cell density is a factor which influences the synthesis of various cytoskeletal proteins; therefore, we studied the effect of cell confluency on the disposition of mammalian stress proteins hsp68 and 70 in human FS-4 fibroblasts. In confluent cultures, where cell-cell contact was maximal, we observed the appearance of a previously undetected polypeptide which was not found in sparsely populated cultures. This protein may represent a post-translationally modified isoform of a preexisting heat shock protein, or perhaps, a novel stress protein.     

Expression and localization of Drosophila melanogaster hsp70 cognate proteins.

Monoclonal antibodies have been used to identify three proteins in Drosophila melanogaster that share antigenic determinants with the major heat shock proteins hsp70 and hsp68. While two of the proteins are major proteins at all developmental stages, one heat shock cognate protein, hsc70, is especially enriched in embryos. hsc70 is shown to be the product of a previously identified gene, Hsc4. We have examined the levels of hsp70-related proteins in adult flies and larvae during heat shock and recovery. At maximal induction in vivo, hsp70 and hsp68 never reach the basal levels of the major heat shock cognate proteins. Monoclonal antibodies to hsc70 have been used to localize it to a meshwork of cytoplasmic fibers that are heavily concentrated around the nucleus.     

Human humoral immunity to hsp70 during Trypanosoma cruzi infection

Immunologic screening of cDNA expression libraries has been widely used for the identification of DNA sequences encoding the immunologically relevant proteins of many pathogenic microorganisms. For reasons that are not entirely clear, sequences encoding 70-kDa heat shock and related proteins (hsp70), which are among the most highly conserved proteins known, have routinely been identified by this approach. Consequently, hsp70 proteins have been proposed to be involved in the autoimmune processes thought responsible for the pathogenesis of the diseases caused by some of these organisms, e.g., chronic Trypanosoma cruzi infection (Chagas' disease). Therefore, we investigated whether hsp70 might be a specific target of the human humoral immune response to T. cruzi infection, and, if so, whether humoral autoimmunity to hsp70 might play a role in pathogenesis. We found that hsp70 is indeed a major polypeptide Ag in Chagas' disease, but that the antibodies to T. cruzi hsp70 do not react with human hsp70--even though the proteins display 73% amino acid sequence identify. These results indicate that self-tolerance to hsp70 is maintained during chronic T. cruzi infection and strongly argue against a role for humoral autoimmunity to hsp70 in the pathogenesis of Chagas' disease.     

The heat shock response in hydra: immunological relationship of hsp60, the major heat shock protein of Hydra vulgaris,to the ubiquitous hsp70 family

The major heat shock protein (hsp) of Hydra vulgaris has recently been found to be a 60 kDa protein. Since in all organisms studied so far, the major heat shock protein is a 70 kDa protein, we have analyzed the relationship of hydra hsp60 to the highly conserved 70 kDa heat shock protein family. Genes and proteins related to the 70 kDa class of stress proteins are present in hydra. However, antibodies known to cross-react with hsp70 proteins in several different organisms do not cross-react with hydra hsp60 suggesting that hsp60 is not related to the conserved hsp70 proteins.     

Expression analysis of heat shock genes in the skin, spleen and blood of common carp (Cyprinus carpio) after cadmium exposure and hypothermia

Heat shock proteins are chaperones that play a pivotal role in controling multiple regulatory pathways such as stress defense, hormone signaling, cell cycle control, cell proliferation and differentiation, and apoptosis. In this study, the expression patterns of four well-known heat shock genes (hsp70, hsc70-1, hsc70-2 and hsp90α) were characterized in the skin, spleen and blood cells of the common carp, under unstressed conditions and after Cd2+ treatment or hypothermia. The examined genes were expressed in a tissue-specific manner: hsc70-2 was expressed constitutively, and was at best only slightly inducible; hsp90α exhibited a high basic expression in all three tissues, whereas hsc70-1 did so only in the blood cells, the expression of hsp70 proved to be below the level of detection in unstressed fish. Cold shock induced the expression of hsp genes in the spleen (hsp90α) and blood cells (hsp70, hsc70-1 and hsp90α), while Cd2+ treatment has no effect on the expression pattern. The highest inducibilities were detected in the skin: for hsp70 an induction of at least 20-fold after cadmium exposure, for hsc70-1 of at least 30-fold and for hsp90α of 3-fold after hypothermia.     

hsp70 is localized to the centrosome of dividing HeLa cells

Monoclonal antibodies specific for inducible and constitutive members of the hsp70 family have been used to investigate the distribution of these proteins during the cell cycle of HeLa cells with special reference to mitosis. Indirect immunofluorescence studies illustrate that a portion of the constitutive form, hsp73, is localized to the centrosome during cell division. In addition a subset of the inducible form, hsp72, collects at the centrosome of dividing cells following heat shock. These observations suggest that members of the hsp70 family are cell cycle specific components of the centrosome in HeLa cells and may play an important role in the function of this microtubule organizing center.     

Field validation of hsp70 stress proteins as biomarkers in Asian clam (Potamocorbula amurensis): is downregulation an indicator of stress?

The focus of this paper is to consider the applicability of the hsp70 stress protein response as a biomarker in field studies. Stress proteins (or heat shock proteins, hsp) of the hsp70 family are induced by sublethal concentrations of a variety of environmental pollutants. However, few studies have applied these proteins as biomarkers of environmental stress under field conditions. Our laboratory is investigating hsp70 proteins and other responses of Asian clam (Potamocorbula amurensis) as potential biomarkers in laboratory and field studies. Our efforts include two studies presently being conducted in northern San Francisco Bay: (1) monthly collection of clams from four sites along a cadmium contamination gradient; (2) 7 day in situ exposure of clams at two selected sites at Mare Island Naval Shipyard. Here we present results on hsp70 proteins in P. amurensis in field-collected and outplanted clams. Both field projects are ongoing, therefore the results presented here do not represent completed studies; rather, they illustrate a portion of our experience. For this workshop, we illustrate weaknesses and strengths of these proteins as biomarkers, and we underscore where additional work is needed. In field-collected clams (study no. 1), site-specific differences in levels of two hsp70 proteins, hsp70 and hsp76, were measured in May and June 1997. Although an inverse correlation exists between cadmium tissue concentrations and hsp70 protein levels, differences detected may be reflective of a salinity gradient. Results from recent laboratory exposures to cadmium and a range of salinities are discussed. After in situ exposure for 7 days (study no. 2), both hsp70 and hsp76 levels were significantly reduced in clams from site R. However, given a brief heat-shock in the laboratory, hsp70 protein levels were significantly higher in clams from this site than in controls. Results indicate that downregulation as well as upregulation of hsp70 proteins may be indicators of stress in P. amurensis.     

Overexpression of a cytosolic chaperone to improve solubility and secretion of a recombinant IgG protein in insect cells

The secretion of heterologous IgG proteins in the baculovirus-insect cell expression system is accompanied by substantial insoluble immunoglobulin in the infected cells. The accumulation of these insoluble forms suggests a limitation in the processing and secretory pathway of the infected cells. As a result, cytosolic hsp70 chaperones, which are known to associate and prevent aggregation of polypeptides in vitro, have been coexpressed in the infected cells. The hsp70 protein coprecipitated with the immunoglobulin to indicate the formation of a specific hsp70-immunoglobulin complex in vivo. Immunoblot and pulse chase studies indicated that coexpression of hsp70 increased intracellular immunoglobulin solubility. Metabolic labeling experiments revealed that hsp70 increased secreted immunoglobulin levels after several days infection as compared to infection with control baculoviruses. Pulse chase studies indicated that hsp70 increases the solubility of immunoglobulin precursors that are then processed and assembled into the complete antibody oligomer. A comparison of the action of cytosolic hsp70 chaperone to the endoplasmic reticulum chaperone BiP suggests sequential action in which hsp70 increases the solubility of preprocessed immunoglobulin, while BiP enhances the solubility of processed immunoglobulin chains.