Saturation, competition, and specificity in interaction of heat shock proteins (hsp) gp96, hsp90, and hsp70 with CD11b+ cells

Heat shock proteins (hsp(s)) have been postulated to interact with APCs through specific receptors, although the receptors are yet to be identified. Specificity, saturation, and competition are the three defining attributes of a receptor-ligand interaction. We demonstrate here that the interaction of the heat shock proteins gp96 and hsp90 with CD11b+ cells is specific and saturable and that gp96 can compete with itself in gp96-macrophage interaction. Interestingly, the phylogenetically related hsp90 also competes quite effectively with gp96 for binding to macrophages, whereas the unrelated hsp70 does so relatively poorly, although it binds CD11b+ cells just as effectively. These data provide evidence that the heat shock proteins interact with APCs with specificity and for the existence of at least two distinct receptors, one for gp96 and hsp90 and the other for hsp70.     

Cytokine function of heat shock proteins

Extensive work in the last 10 years has suggested that heat shock proteins (HSPs) may be potent activators of the innate immune system. It has been reported that Hsp60, Hsp70, Hsp90, and gp96 are capable of inducing the production of proinflammatory cytokines by the monocyte-macrophage system and the activation and maturation of dendritic cells (antigen-presenting cells) in a manner similar to the effects of lipopolysaccharide (LPS) and bacterial lipoprotein, e.g., via CD14/Toll-like receptor2 (TLR2) and CD14/TLR4 receptor complex-mediated signal transduction pathways. However, recent evidence suggests that the reported cytokine effects of HSPs may be due to the contaminating LPS and LPS-associated molecules. The reasons for previous failure to recognize the contaminant(s) as being responsible for the reported HSP cytokine effects include failure to use highly purified, low-LPS preparations of HSPs; failure to recognize the heat sensitivity of LPS; and failure to consider contaminant(s) other than LPS. Thus it is essential that efforts should be directed to conclusively determine whether the reported HSP cytokine effects are due to HSPs or to contaminant(s) present in the HSP preparations before further exploring the implication and therapeutic potential of the putative cytokine function of HSPs. tumor necrosis factor-; lipopolysaccharide; macrophages; innate immune system     

Assembly of progesterone receptor with heat shock proteins and receptor activation are ATP mediated events.

To better understand assembly mechanisms of progesterone receptor (PR) complexes, we have developed a cell-free system for studying PR interactions with the 90- and 70-kDa heat shock proteins (hsp90 and hsp70), and we have used this system to examine requirements for hsp90 binding to PR. Purified chick PR, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: 1) absence of progesterone, 2) elevated temperature (30 degrees C), 3) presence of ATP, and 4) presence of Mg2+. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl2 and an ATP-regenerating system. ATP depletion of lysate by dialysis or by enzymatic means blocks hsp90 binding to PR; likewise, addition of EDTA to lysate blocks hsp90 binding, but binding is restored by the addition of excess Mg2+. Addition to lysate of monoclonal antibody against hsp70 inhibits hsp90 binding to PR and destabilizes preformed complexes. Stabilization of hsp90-receptor complexes also requires ATP, indicating that ATP and hsp70 are needed to form and to maintain hsp90 complexes. Hormone-dependent activation of reconstituted receptor complexes was also examined. The addition of progesterone to the reticulocyte lysate promotes dissociation of hsp90 and hsp70 from the receptor. This also appears to require ATP and dissociation is most efficient in the presence of an ATP-regenerating system. In conclusion, these studies indicate that PR-hsp90 complexes do not self-assemble; instead, assembly is probably a multistep process requiring ATP and other cellular factors.     

Heat shock proteins gp96 and hsp70 activate the release of nitric oxide by APCs

NO is a cytotoxic and immunomodulatory cytokine produced by macrophages and dendritic cells. We show that stimulation of murine and human macrophages with the heat shock proteins gp96 and hsp70 results in induction of inducible NO synthase and the production of NO. The release of NO by monocytes exposed to hsp60 has been documented previously. Immature, but not mature, dendritic cells respond in the same manner. The activity of heat shock proteins is relatively unaffected by an antagonist of LPS, and is abrogated by heat denaturation. Macrophages have been shown previously to produce NO in response to stimulation with IFN-gamma; stimulation of macrophages with mixtures of IFN-gamma and gp96 or hsp70 leads to a synergistic production of NO. The present observations extend the roles of these heat shock proteins in innate immune responses to another potent and highly conserved function of APC.     

Binding of heat shock proteins to the avian progesterone receptor.

The protein composition of the avian progesterone receptor was analyzed by immune isolation of receptor complexes and gel electrophoresis of the isolated proteins. Nonactivated cytosol receptor was isolated in association with the 90-kilodalton (kDa) heat shock protein, hsp90, as has been described previously. A 70-kDa protein was also observed and was shown by Western immunoblotting to react with an antibody specific to the 70-kDa heat shock protein. Thus, two progesterone receptor-associated proteins are identical, or closely related, to heat shock proteins. When the two progesterone receptor species, A and B, were isolated separately in the absence of hormone, both were obtained in association with hsp90 and the 70-kDa protein. However, activated receptor isolated from oviduct nuclear extracts was associated with the 70-kDa protein, but not with hsp90. A hormone-dependent dissociation of hsp90 from the cytosolic form of the receptor complex was observed within the first hour of in vivo progesterone treatment, which could explain the lack of hsp90 in nuclear receptor complexes. In a cell-free system, hsp90 binding to receptor was stabilized by molybdate but disrupted by high salt. These treatments, however, did not alter the binding of the 70-kDa protein to receptor. Association of the 70-kDa protein with the receptor could be disrupted by the addition of ATP at elevated temperatures (23 degrees C). The receptor-associated 70-kDa protein is an ATP-binding protein, as demonstrated by its affinity labeling with azido[32P]ATP. These results indicate that the two receptor-associated proteins interact with the progesterone receptor by different mechanisms and that they are likely to affect the structure or function of the receptor in different ways.     

Reconstitution of progesterone receptor with heat shock proteins

Nonactivated chick progesterone receptor from hypotonic tissue extracts exists in a large complex containing the heat shock proteins hsp90 and hsp70 plus additional smaller proteins; activation of receptor to a DNA-binding form involves the dissociation of proteins from the complex. Whereas numerous attempts to reversibly bind components to the activated receptor have been unsuccessful, we now report conditions that promote the reassociation of hsp90 and hsp70 to progesterone receptor. Cytosolic receptor was dissociated from hsp90 and hsp70 by treatment with 0.5 M KCl and 10 mM ATP in the absence of progesterone. It was then purified by binding to immunoaffinity resins. After wash steps, the receptor-resin complex was incubated in rabbit reticulocyte lysate at 30 C, rewashed, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Saturable binding of rabbit hsp90 and hsp70 to chick receptor was found after incubation with reticulocyte lysate; hsp binding was temperature dependent, but not dependent on exogenous ATP. Incubation of dissolved receptor with oviduct cytosol, from which receptor was obtained, or with purified hsp did not result in hsp binding. Furthermore, mixing oviduct cytosol with lysate inhibited hsp reconstitution, suggesting negative factors for hsp binding in oviduct cytosol. The steroid-binding domain of the receptor was required, since no hsp binding was observed in the reconstitution system using a receptor mutant lacking this domain. When the receptor was isolated in the presence of progesterone, reconstitution with hsp90 and hsp70 did not occur. This is consistent with the in vivo effects of progesterone in promoting hsp dissociation.     

Characterization of a receptor for heat shock protein 70 on macrophages and monocytes

Heat shock proteins (Hsps) and molecular chaperones isolated from tumors or virally infected cells elicit an efficient CD8+ T cell response against bound antigenic peptides. This immune response is mediated by presentation of the peptides on MHC class I complexes of antigen-presenting cells (APCs), but the cellular mechanism of this presentation process is not yet understood. Here we provide evidence for the existence of a proteinaceous receptor on the surface of APCs that is specific for mammalian Hsp70. Using a flow cytometry-based assay, saturable binding of Hsp70 to the cell surface of macrophages and peripheral blood monocytes, but not of lymphocytes, can be demonstrated. The affinity of the receptor is in the sub-micromolar range (Kd < 100 nM). Only mammalian Hsc70/Hsp70, but not bacterial Hsp70, is bound with high affinity. Subsequent to binding, Hsp70 is taken up by endocytosis, resulting in an intracellular localization. Our results suggest that receptor-mediated endocytosis forms the basis for the demonstrated efficacy of Hsp70-peptide complexes as anti-tumor vaccines.     

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.     

Flagellin contamination of recombinant heat shock protein 70 is responsible for its activity on T cells

Heat shock proteins (Hsp) 60 and 70 have been intensively studied for their ability to activate innate immunity. Heat shock proteins had been shown to induce the activation of dendritic cells, T cells, and B cells. However, the possible contamination of endotoxin in heat shock protein preparations makes their function as an activator of immune system ambiguous. Here, we examined the ability of bacterial Hsp60 and Hsp70 to activate Jurkat T cells and primary T cells. We found that Burkholderia pseudomallei Hsp70 and Mycobacterium tuberculosis Hsp70 could costimulate Jurkat T cells to make IL-2 and signal through TLR5. This costimulatory activity is not due to endotoxin or contaminants signaling via TLR2 nor TLR4. However, recombinant Hsp70 expressed in Escherichia coli DeltafliC strain completely lost its ability to costimulate T cells. Thus, the activation of T cells by recombinant Hsp70 is ascribed to flagellin contamination.     

Extracellular heat shock proteins in cell signaling

Extracellular stress proteins including heat shock proteins (Hsp) and glucose regulated proteins (Grp) are emerging as important mediators of intercellular signaling and transport. Release of such proteins from cells is triggered by physical trauma and behavioral stress as well as exposure to immunological "danger signals". Stress protein release occurs both through physiological secretion mechanisms and during cell death by necrosis. After release into the extracellular fluid, Hsp or Grp may then bind to the surfaces of adjacent cells and initiate signal transduction cascades as well as the transport of cargo molecules such as antigenic peptides. In addition Hsp60 and hsp70 are able to enter the bloodstream and may possess the ability to act at distant sites in the body. Many of the effects of extracellular stress proteins are mediated through cell surface receptors. Such receptors include Toll Like Receptors 2 and 4, CD40, CD91, CCR5 and members of the scavenger receptor family such as LOX-1 and SREC-1. The possession of a wide range of receptors for the Hsp and Grp family permits binding to a diverse range of cells and the performance of complex multicellular functions particularly in immune cells and neurones.     

Specific immunogenicity of heat shock protein gp96 derives from chaperoned antigenic peptides and not from contaminating proteins

The peptide-binding property of MHC is central to adaptive immunological functions. A similar property of heat shock proteins (HSPs) hsp70 and hsp90 has been implicated in Ag presentation by MHC and in cross-priming. The peptide-binding pocket of hsp70 has been characterized structurally and functionally and a peptide-binding site in gp96 (of hsp90 family) has been defined. Nonetheless, questions persist whether the specific immunogenicity of HSP preparations derives from the peptides chaperoned by the HSPs or by proteins contaminating the HSP preparations. Because absolute purity of a protein preparation is a metaphysical concept, other approaches are necessary to address the question. In this study, we demonstrate that the specific immunogenicity of gp96 preparations isolated from cells expressing beta-galactosidase derives from the MHC I epitope precursors associated with the gp96 and not from contaminating beta-galactosidase protein nor unassociated fragments derived from it. Although the observations here are limited to a single HSP and antigenic peptides chaperoned by it, they can be extended broadly.     

Characterization of progesterone receptor binding to the 90- and 70-kDa heat shock proteins

In this study a model system for expression of the chicken progesterone receptor in cultured cells was developed using a quail fibroblast cell line, QT6. The chicken progesterone receptor form A expressed in QT6 cells was evaluated and determined to have a number of similarities to receptor isolated from chicken oviduct. These include hormone binding, sedimentation profile, phosphorylation pattern, heat shock protein (hsp) 70 and hsp90 associations and the ability to stimulate a reporter gene construct. Therefore, the receptor expressed in this system functioned adequately for further evaluation of the particular region (or regions) involved in hsp70 and hsp90 binding. Several receptor deletion mutants were tested for hsp70/hsp90 binding; only the d369-659 mutant, which has the entire steroid-binding domain deleted, was unable to bind hsp90 and hsp70. Three separate regions of the steroid-binding domain were found to partially restore hsp90 and hsp70 binding to the d369-659 mutant protein. However, hsp binding was not abolished when these or other regions of the steroid binding domain were deleted individually. These findings indicate that hsp90 and hsp70 both bind to the steroid-binding domain of the receptor through interactions at multiple locations or through some structural quality that is distributed throughout this region of the protein.     

Heat shock protein 70 and glycoprotein 96 are differentially expressed on the surface of malignant and nonmalignant breast cells

Heat shock proteins (HSPs), which are important for a number of different intracellular functions, are occasionally found on the surface of cells. The function of heat shock protein on the cell surface is not understood, although it has been shown to be greater in some tumor cells and some virally infected cells. Surface expression of both glycoprotein 96 (gp96) and Hsp70 occurs on tumor cells, and this expression correlates with natural killer cell killing of the cells. We examined the surface expression of gp96 and Hsp70 on human breast cell lines MCF7, MCF10A, AU565, and HS578, and in primary human mammary epithelial cells by immunofluorescence microscopy and flow cytometry. The nonmalignant cell lines HS578, MCF10A, and HMEC showed no surface expression of gp96, whereas malignant cell lines MCF7 and AU565 were positive for gp96 surface expression. All of the breast cell lines examined showed Hsp70 surface expression. These results also confirm previous studies, demonstrating that Hsp70 is on the plasma membrane of tumor cell lines. Given the involvement of heat shock proteins, gp96 and Hsp70, in innate and adaptive immunity, these observations may be important in the immune response to tumor cells.     

A model of glucocorticoid receptor unfolding and stabilization by a heat shock protein complex

It has recently been reported that incubation of avian progesterone receptors, mouse glucocorticoid receptors, or the viral tyrosine kinase pp60^src with rabbit reticulocyte lysate reconstitutes their association with the 90 kDa heat shock protein, hsp90. The reassociation is thought to require unfolding of the steroid receptor or pp60^src before hsp90 can bind. The unfoldase activity may be provided by hsp70, which is also present in the reconstituted receptor heterocomplex. In this paper we review evidence that hsp70 and hsp90 are associated in cytosolic heterocomplexes that contain a limited number of other proteins. From an analysis of known receptor-hsp interactions and a predicted direct interaction between hsp90 and hsp70 we have developed an admittedly very speculative model of glucocorticoid receptor unfolding and stabilization. One important feature of the model is that the receptor becomes attached to a heat shock protein heterocomplex rather than undergoing independent unfolding and stabilization events. The model requires that hsp70 and hsp90 bind directly to the receptor at independent sites. Importantly, the model accomodates the stoichiometry of 2 hsp90 per 1 molecule of receptor that has been assayed in the untransformed GR heterocomplex in cytosols prepared from hormone-free cells.     

Human heat shock protein 70 (Hsp70) as a peripheral membrane protein

While a significant fraction of heat shock protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-?SBD and Hsp70-?NBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD.     

Blind cavefish and heat shock protein chaperones: a novel role for hsp90alpha in lens apoptosis

Lens apoptosis plays a central role in cavefish eye degeneration. Heat shock proteins (hsps) can regulate apoptosis; therefore, we examined the relationship between constitutive hsp70 and hsp90 expression and lens apoptosis. The model system is Astyanax mexicanus, a teleost species consisting of an eyed surface-dwelling (surface fish) form and numerous blind cave-dwelling (cavefish) forms. Optic primordia are formed in the cavefish embryo but they subsequently undergo lens apoptosis, arrest in development and degenerate. Astyanax hsp90 and hsp70 DNAs were isolated to use as probes to compare gene expression during surface fish and cavefish development. Hsp90beta, which encodes one of two hsp90 isoforms, was not expressed in the surface fish or cavefish lens, whereas hsp70 was expressed in the lens of both forms, suggesting that neither is directly involved in lens apoptosis. In contrast, hsp90alpha, the other hsp90 isoform, was expressed in the cavefish but not the surface fish lens. Hsp90alpha expression peaked shortly before the beginning of lens apoptosis in three convergent cavefish populations, suggesting a close relationship with lens apoptosis. The absence of hsp90beta in the lens allowed us to use geldanamycin and radicicol, specific inhibitors of hsp90 chaperone function, to determine whether lens cell death requires hsp90alpha expression. Both inhibitors blocked TUNEL labeling in the cavefish lens, suggesting that hsp90alpha is required for apoptosis. In contrast to their effects on the lens, these inhibitors induced TUNEL labeling in the surface epidermis, presumably due to effects on hsp90beta function, implying that the two-hsp90 isoforms may have contrasting roles in cell survival. We conclude that hsp90alpha plays a novel role in lens apoptosis and cavefish eye degeneration.