Heat shock proteins HSP70 and GP96: structural insights

Several heat shock proteins (HSPs) act as potent adjuvants for eliciting anti-tumor immunity. HSP-based tumor vaccine strategies have been highly successful in animal models and are undergoing testing in clinical trials. It is generally accepted that HSPs, functioning as chaperones for tumor antigens, elicit tumor-specific adaptive immune responses. HSPs also appear to induce innate immune responses in an antigen-independent fashion. Innate responses generated by HSPs may contribute to anti-tumor immunity. Immunologically active chaperones with anti-tumor activity are referred to as “immunochaperones”. Here, we review the studies that address the role of structural domains or regions of the immunochaperones HSP70 and GP96 that may be involved in the induction of adaptive or innate immune responses. This article forms part of the Symposium in Writing “Thermal stress-related modulation of tumor cell physiology and immune responses”, edited by Elfriede Noessner.     

The therapeutic implications of clinically applied modifiers of heat shock protein 70 (Hsp70) expression by tumor cells

Evidence that membrane-bound and extracellular heat shock proteins (HSPs) with molecular weights of 70 and 90 kDa are potent stimulators of the immune responses has accumulated over the last decade. In this review, we discuss the modulation of Hsp70 expression, a major stress-inducible member of the HSP70 family, in the cytoplasm and on the plasma membrane of tumor cells by clinically applied interventions such as radio- and chemotherapy.     

Hsp70 promotes antigen-presenting cell function and converts T-cell tolerance to autoimmunity in vivo

Pathogens or pathogen-associated molecular patterns can signal to cells of the innate immune system and trigger effective adaptive immunity. However, relatively little is known about how the innate immune system detects tissue injury or necrosis. Evidence suggests that the release of heat-shock proteins (HSPs) may provide adjuvant-like signals, but the ability of HSPs to promote activation or tolerance in vivo has not been addressed. In this study we show that Hsp70 promotes dendritic cell (DC) function and, together with antigen, triggers autoimmune disease in vivo.     

Binding of Silurus asotus lectin to Gb3 on Raji cells causes disappearance of membrane-bound form of HSP70

Heat shock proteins (HSPs) are divided into stress-inducible and constitutive types. Generally, HSP70 (stress inducible) and HSC70 (constitutive) are representative of their types, respectively. From the results of immunocytochemical analysis, both HSP70 and HSC70 were constitutively expressed in globotriaosylceramide (Gb3)-expressing Raji cells as well as Gb3-negative K562 cells. Furthermore, the membrane-bound form of HSP70 was present on the surfaces of two cell lines as patch and cap-like structures, and was recovered in the cholesterol rich microdomains (CRM) prepared from them. On the other hand, HSP70 was partially co-localized with Gb3 on the surface of Raji cells. This result suggested that HSP70 was not associated with all of Gb3 molecules but with Gb3 specifically located in the particular environment. The effect of Silurus asotus lectin (SAL), which is one of the rhamnose-binding lectins and specifically binds to Gb3, on the disappearance of membrane-bound HSP70 was dependent on whether Gb3 was present or not. These results suggested that the disappearance of membrane-bound HSP70 was caused by SAL binding to Gb3, that the reduction of membrane-bound HSP70 might result in the decrease in cell volume observed, and that the mechanism of SAL-induced HSP70 expression may differ from that of heat shock in Raji cells.     

Heat shock proteins as endogenous adjuvants in sterile and septic inflammation

Heat shock proteins (HSPs) have been reported to stimulate the immune system via innate receptors. However, the role of HSPs as endogenous adjuvants has been challenged by reports claiming that pure HSPs are not innate ligands; it is only the bacterial molecules trapped by the HSPs that can signal the innate immune system. In this review, we discuss data suggesting that both views, in essence, are correct; pure HSPs are indeed innate immunostimulators, but HSPs can also function as transducers of pathogen signals. In other words, HSPs perform diverse functions in two alternative modes of inflammation: sterile inflammation, which results from endogenous stimuli and is necessary for body maintenance, and septic inflammation, which protects us from environmental pathogens. Endogenous HSPs are key players in the modulation of these two modes of inflammation, and as such, they are potential targets for new and more efficient therapies for cancer, infections, and autoimmunity.     

Roles of heat-shock proteins in innate and adaptive immunity

Heat-shock proteins (HSPs) are the most abundant and ubiquitous soluble intracellular proteins. In single-cell organisms, invertebrates and vertebrates, they perform a multitude of housekeeping functions that are essential for cellular survival. In higher vertebrates, their ability to interact with a wide range of proteins and peptides--a property that is shared by major histocompatibility complex molecules--has made the HSPs uniquely suited to an important role in organismal survival by their participation in innate and adaptive immune responses. The immunological properties of HSPs enable them to be used in new immunotherapies of cancers and infections.     

Parasite heat-shock proteins

Many parasites, including most of those of medical or veterinary importance, experience a major increase in ambient temperature at some stage during their life cycle. This occurs when a cyst or free-living larval form is ingested by a warm-blooded host, when a poikilotherm-infecting parasite is transmitted to a homeotherm, or when a transiently free-living invasive larva penetrates the skin of a mammal. This sudden change in temperature could be expected to stress the intruder, as it should dramatically alter rates of metabolic reactions and of denaturation of proteins. This would especially affect the function of near-equilibrium, regulatory, and membrane-bound enzymes (changes in temperature affect membrane fluidity). In this article George Newport, Janice Culpepper and Nina Agabian consider how parasites cope with this problem, emphasizing the possible role of heat-shock proteins (HSPs), how the expression of these molecules is regulate, and how HSPs interact with the host immune system.     

果蝇热激蛋白的研究进展

热休克蛋白(heat shock proteins,HSPs)是生物体受到应激刺激时诱导产生的一组保守性蛋白,普遍存在于各种生物体中。近年来,果蝇Drosophila作为生命科学与人类疾病研究的重要模式生物,其热激蛋白的研究取得了许多新的进展。文章对果蝇热激蛋白的类别、热激蛋白基因的表达调控机制、热激蛋白的分子伴侣功能、调节细胞存亡和影响发育及寿命等相关生物学功能进行综述,并对热激蛋白在神经退行性疾病治疗中的应用前景作展望。     

Purification of recombinant and endogenous HSP70s

Heat shock proteins (HSPs) are powerful immunogens against the antigenic peptides they chaperone. The antigenic peptides are MHC I-binding peptides and their elongated precursors derived from tumor antigens, viral antigens, minor histocompatibility antigens, or model antigens. HSP-peptide complexes can immunize against tumors and pathogen-infected cells. Remarkably, HSPs do not immunize after elution of the peptides they chaperone, demonstrating that HSPs are not immunogenic per se, whereas HSP-peptide complexes are. Additionally, HSPs activate professional antigen presenting cells (APC) through specific receptor(s) to stimulate secretion of pro-inflammatory cytokines, up-regulation of co-stimulatory molecules and activation of dendritic cells. The mechanistic exploration of the role of the HSPs on the innate and adaptive component of the immune system requires their isolation in large quantity. On one hand, isolation of naturally formed HSP-peptide complexes is key to study their specific immunogenicity. On the other hand, purification of HSPs free of endotoxin contamination is an absolute requirement for the analysis of their ability to activate APC in vitro. This chapter describes a convenient and fast method of purification of endogenous and recombinant HSP of 70 kDa (HSP70) that addresses these two considerations.     

Hsp70, a messenger from hyperthermia for the immune system

Heat shock proteins (Hsps) hold a dual role depending on their location. Inside cells, they fulfill essential survival functions as molecular chaperones forming complexes with intracellular polypeptides (self or foreign) to help in protein folding, the resolution of protein aggregates and intracellular protein transport. Released from the cell, they act as messengers communicating the cells’ interior protein composition to the immune system for initiation of immune responses against intracellular proteins. Here we describe the mechanisms by which Hsp70, the heat-inducible Hsp70 family member, crosstalks with the immune system. Further, we discuss that clinical hyperthermia could be a way to initiate the immunologic activity of Hsp70 by upregulating its expression and facilitating release through local necrosis.     

Calcium signaling in dendritic cells by human or mycobacterial Hsp70 is caused by contamination and is not required for Hsp70-mediated enhancement of cross-presentation

Extracellular heat shock proteins (HSPs) can stimulate antigen-specific immune responses. Using recombinant human (rhu)Hsp70, we previously demonstrated that through complex formation with exogenous antigenic peptides, rhuHsp70 can enhance cross-presentation by antigen-presenting cells (APCs) resulting in stronger T cell stimulation. T cell stimulatory activity has also been described for mycobacterial (myc)Hsp70. MycHsp70-assisted T cell activation has been reported to act through the binding of mycHsp70 to chemokine receptor 5 (CCR5), calcium signaling, phenotypic maturation, and cytokine secretion by dendritic cells (DCs). We report that highly purified rhuHsp70 and mycHsp70 proteins both strongly enhance cross-presentation of exogenous antigens. Augmentation of cross-presentation was seen for different APCs, irrespective of CCR5 expression. Moreover, neither of the purified Hsp70 proteins induced calcium signals in APCs. Instead, calcium signaling activity was found to be caused by contaminating nucleotides present in Hsp70 protein preparations. These results refute the hypothesis that mycHsp70 proteins require CCR5 expression and calcium signaling by APCs for enhanced antigen cross-presentation for T cell stimulation.     

热休克蛋白HSP70和gp96在抗病毒感染中的作用

热休克蛋白(HSP)是一组在进化上高度保守、具有重要生理功能的蛋白质家族,是生物在应激条件下产生的一种非特异性防御产物,在调节免疫应答和抗病毒反应中起重要作用。现简要介绍HSP70、gp96(HSP96,GRP94)这两种HSP与病毒感染的关系及在抗病毒感染中的作用。     

Anticancer drugs cause release of exosomes with heat shock proteins from human hepatocellular carcinoma cells that elicit effective natural killer cell antitumor responses in vitro

Failure of immune surveillance related to inadequate host antitumor immune responses has been suggested as a possible cause of the high incidence of recurrence and poor overall survival outcome of hepatocellular carcinoma. The stress-induced heat shock proteins (HSPs) are known to act as endogenous "danger signals" that can improve tumor immunogenicity and induce natural killer (NK) cell responses. Exosome is a novel secretory pathway for HSPs. In our experiments, the immune regulatory effect of the HSP-bearing exosomes secreted by human hepatocellular carcinoma cells under stress conditions on NK cells was studied. ELISA results showed that the production of HSP60, HSP70, and HSP90 was up-regulated in both cell lines in a stress-specific manner. After exposure to hepatocellular carcinoma cell-resistant or sensitive anticancer drugs (hereafter referred to as "resistant" or "sensitive" anticancer drug), the membrane microvesicles were actively released by hepatocellular carcinoma cells, differing in their ability to present HSPs on the cell surface, which were characterized as exosomes. Acting as a decoy, the HSP-bearing exosomes efficiently stimulated NK cell cytotoxicity and granzyme B production, up-regulated the expression of inhibitory receptor CD94, and down-regulated the expression of activating receptors CD69, NKG2D, and NKp44. Notably, resistant anticancer drugs enhanced exosome release and generated more exosome-carried HSPs, which augmented the activation of the cytotoxic response. In summary, our findings demonstrated that exosomes derived from resistant anticancer drug-treated HepG2 cells conferred superior immunogenicity in inducing HSP-specific NK cell responses, which provided a clue for finding an efficient vaccine for hepatocellular carcinoma immunotherapy.     

Heat shock protein 60 reactive T cells in juvenile idiopathic arthritis: what is new?

Juvenile idiopathic arthritis (JIA) is a disease characterized by chronic joint inflammation, caused by a deregulated immune response. In patients with JIA, heat shock proteins (HSPs) are highly expressed in the synovial lining tissues of inflamed joints. HSPs are endogenous proteins that are expressed upon cellular stress and are able to modulate immune responses. In this review, we concentrate on the role of HSPs, especially HSP60, in modulating immune responses in both experimental and human arthritis, with a focus on JIA. We will mainly discuss the tolerogenic immune responses induced by HSPs, which could have a beneficial effect in JIA. Overall, we will discuss the immune modulatory capacity of HSPs, and the underlying mechanisms of HSP60-mediated immune regulation in JIA, and how this can be translated into therapy.     

Developmental expression of heat shock proteins 60, 70, 90, and A2 in rabbit testis

Currently, no reports exist concerning the expression patterns and developmental changes of heat shock proteins (HSPs) in the reproductive system of the male rabbit. In the present study, the testes of rabbits were collected at post-natal months 1, 2, 3, 4, 5, and 40. HSP60, HSC70, HSP90, and HSPA2 were detected by both Western blot and immunohistochemical methods. The expression levels of HSP60 and HSC70 showed no apparent change during the developmental progress. HSP90 increased at the second month; prior to the third month, HSPA2 was expressed at a low level. Immunohistochemistry localized HSP60 in the cytoplasm of all of the cell types in the testis and in the apical pole of the spermatids. The distribution pattern of HSC70 and HSP90 was similar, both being mainly located in the spermatids of stage VII-VIII and in the cytoplasm of the spermatogonium. HSPA2 staining was mainly observed in the cytoplasm of pachytene spermatocytes and spermatids in testes of 3-, 4-, 5-, and 40-month-old rabbits. These results provide a basic reference point for studying the functions of HSPs in the male rabbit reproductive system and should be beneficial for the future determination of the mechanisms of heat shock on male rabbit fertility.     

In vitro reconstitution of heat shock protein-peptide complexes for generating peptide-specific vaccines against cancers and infectious diseases

Known commonly as molecular chaperones for proteins, heat shock proteins (HSPs) have also been found to chaperone small molecular weight cellular peptides. HSP-peptide complexes can prime T cell immunity specific against the peptides bound to HSPs, but not against HSPs per se. This immunomodulatory functions of HSPs are based on two intrinsic properties. One, HSPs are excellent adjuvants due to their ability to activate dendritic cells (DCs). Two, HSPs can bind directly to their receptors on DCs to then channel HSP-associated peptides to associate with MHC molecules. When a specific antigenic peptide is defined, this peptide can also be complexed with either tissue derived or recombinant HSPs in vitro to generate HSP-peptide complexes as peptide-specific vaccines. This article focuses on the methods commonly used to reconstitute HSP-peptide complexes, and discusses assays to verify the efficiency of complexing for immunotherapy against cancers and infectious diseases.     

Target molecules of molecular chaperone (HSP70 family) in injured gastric mucosa in vivo

AimsSeveral recent studies, including ours, have indicated the importance of heat shock proteins (HSPs) in cytoprotection against cytotoxic agents and environmental stresses mediated by the chaperone function of HSPs (molecular chaperones). However, the target molecule that is recognized by HSPs in damaged cells currently remains unknown. As HSPs rapidly recognize and bind to degenerated protein in cells, target molecules of HSPs might be key molecules for the initiation and pathogenesis of cellular damage. In the present study, gastric mucosal proteins that specifically bind to the HSP70 family (HSC70) were analyzed using HSC70-affinity chromatography.Main methodsThe gastric mucosa was removed from Sprague–Dawley rats after exposure to water immersion-stress for 0, 1, 3 or 5 h. Soluble fractions of each gastric mucosa were applied to the HSC70-affinity column separately. After washing off non-specific binding proteins, specific binding proteins were eluted by ATP-containing buffer. Binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. In addition, the amino acid sequence of purified proteins was also analyzed.Key findingsSpecific HSC70-binding proteins with a molecular weight of 200-kDa and 45-kDa were eluted from an affinity column when gastric mucosal homogenate of 1-h stress exposure was applied. The amino acid sequencing showed that these binding proteins were cytoskeletal myosin (heavy chain) and actin, respectively.SignificanceDuring the pathogenesis of stress-induced gastric mucosal damage, structurally degenerated cytoskeletal myosin (heavy chain) and actin may be key or initiation molecules which structural changes were firstly recognized by molecular chaperone.