Endoplasmic reticulum proteins SDF2 and SDF2L1 act as components of the BiP chaperone cycle to prevent protein aggregation

The folding of newly synthesized proteins in the endoplasmic reticulum (ER) is assisted by ER‐resident chaperone proteins. BiP (immunoglobulin heavy‐chain‐binding protein), a member of the HSP70 family, plays a central role in protein quality control. The chaperone function of BiP is regulated by its intrinsic ATPase activity, which is stimulated by ER‐resident proteins of the HSP40/DnaJ family, including ERdj3. Here, we report that two closely related proteins, SDF2 and SDF2L1, regulate the BiP chaperone cycle. Both are ER‐resident, but SDF2 is constitutively expressed, whereas SDF2L1 expression is induced by ER stress. Both luminal proteins formed a stable complex with ERdj3 and potently inhibited the aggregation of different types of misfolded ER cargo. These proteins associated with non‐native proteins, thus promoting the BiP–substrate interaction cycle. A dominant‐negative ERdj3 mutant that inhibits the interaction between ERdj3 and BiP prevented the dissociation of misfolded cargo from the ERdj3–SDF2L1 complex. Our findings indicate that SDF2 and SDF2L1 associate with ERdj3 and act as components in the BiP chaperone cycle to prevent the aggregation of misfolded proteins, partly explaining the broad folding capabilities of the ER under various physiological conditions.     

Chaperonin GroEL and its mutant D87K protect from ischemia in vivo and in vitro

Protein aggregation and misfolding are central mechanisms of both acute and chronic neurodegeneration. Overexpression of chaperone Hsp70 protects from stroke in animal and cell culture models. Although it is accepted that chaperones protect cells, the mechanism of protection by chaperones in ischemic injury is poorly understood. In particular, the relative importance of preventing protein aggregation compared to facilitating correct protein folding during ischemia and recovery is not known. To test the importance of protein folding and minimize interaction with co-chaperones we studied the bacterial chaperonin GroEL (HSPD1) and a folding-deficient mutant D87K. Both molecules protected cells from ischemia-like injury, and reduced infarct volume and improved neurological outcome after middle cerebral artery occlusion in rats. Protection was associated with reduced protein aggregation, assessed by ubiquitin immunohistochemistry. Marked neuroprotection by the folding-deficient chaperonin demonstrates that inhibition of aggregation is sufficient to protect the brain from ischemia. This suggests that strategies to maintain protein solubility and inhibit aggregation in the face of acute insults such as stroke may be a useful protective strategy.     

Emerging picture of host chaperone and cyclophilin roles in RNA virus replication

Many plus-strand (+)RNA viruses co-opt protein chaperones from the host cell to assist the synthesis, localization and folding of abundant viral proteins, to regulate viral replication via activation of replication proteins and to interfere with host antiviral responses. The most frequently subverted host chaperones are heat shock protein 70 (Hsp70), Hsp90 and the J-domain co-chaperones. The various roles of these host chaperones in RNA virus replication are presented to illustrate the astonishing repertoire of host chaperone functions that are subverted by RNA viruses. This review also discusses the emerging roles of cyclophilins, which are peptidyl-prolyl isomerases with chaperone functions, in replication of selected (+)RNA viruses.     

Heat-shock proteins as powerful weapons in vaccine development

Heat-shock proteins (HSPs) have been known as multifunctional proteins. They facilitate the folding and unfolding of proteins, participate in vesicular transport processes, prevent protein aggregation in the densely packed cytosol and are involved in signaling processes. HSPs have been involved in different fields, including autoimmunity, immunity to infections and tumor immunology. Although there are many different kinds of HSPs, only some HSPs, including HSP70 and Gp96, have immunological properties. HSP molecules have been applied into DNA- or protein (peptide)-based vaccines as antigens, chaperones or adjuvants. HSP-based vaccines have been shown to immunize against cancer and infectious diseases in both prophylactic and therapeutic protocols. The immunogenicity of HSPs results from two different properties: a peptide-dependent capacity to chaperone and elicit adaptive cytotoxic T-lymphocyte responses against antigenic peptides and a peptide-independent immunomodulatory capacity. Furthermore, HSPs could be immunoregulatory agents with potent and widely applicable therapeutic uses. Accordingly, certain HSPs, such as HSP70 and Gp96, are highly effective carrier molecules for cross-presentation. Their ability in eliciting immune responses against different pathogens (parasite and virus) and their role in cancer immunity will be discussed in this review.     

Expression of 65- and 67-kilodalton heat-regulated proteins and a 70-kilodalton heat shock cognate protein of Leishmania donovani in macrophages.

Heat shock protein (HSP) expression was examined in murine bone marrow-derived macrophages infected with stationary-phase promastigotes of Leishmania donovani. Immunoblotting performed with a rabbit polyclonal antiserum raised against HSP60 from Heliothis virescens (moth) revealed the de novo appearance of 65- and 67-kDa proteins in leishmania-infected macrophages. A third protein of 60 kDa, which represented murine HSP60, was also detected, and its expression did not change in response to infection. In contrast, expression of the novel 65- and 67-kDa proteins in infected cells was coordinately regulated and, at 24 h of infection, reached maximal levels of 52 to 100% increases above initial levels determined at 3 h. Proteins which had identical electrophoretic mobilities and were similarly regulated in response to heat were also detected in promastigotes. The appearance of these proteins in macrophages was specific to leishmania infection in that neither protein was detected in noninfected cells either in the basal state or following several treatments, including (i) infection with Yersinia pseudotuberculosis, (ii) phagocytosis of Staphylococcus aureus, (iii) NaAsO2 treatment, and (iv) heat shock. Expression of the 65- and 67-kDa heat-regulated Leishmania proteins was also observed to be selective, in that as their concentration was increasing, the abundance of the Leishmania surface protease gp63 in infected cells was noted to decrease. Murine HSP60 but not the Leishmania heat-regulated proteins was also recognized by a distinct rabbit antiserum raised against human HSP60, suggesting the presence of specific determinants within these Leishmania proteins. A monoclonal antibody that recognizes both mammalian HSP70 and HSP70 from plasmodia detected single isoforms of both Leishmania and murine HSP70 in infected cells, and the level of neither protein changed during infection. Moreover, although a murine HSP of 73 kDa was induced in response to both heat shock and NaAsO2 treatment, it was not induced to detectable levels by infection. The rapid and relatively high level of expression of inducible HSP60-related proteins of L. donovani and Leishmania HSP70 in infected macrophages suggests that these proteins are involved in pathogenesis and may be important targets of the immune response.     

Legionella pneumophila htpAB heat shock operon: nucleotide sequence and expression of the 60-kilodalton antigen in L. pneumophila-infected HeLa cells

A 60-kilodalton (kDa) immunodominant antigen of Legionella pneumophila is a heat shock protein (HSP) of the GroEL class of HSPs. The gene (htpB) coding the 60-kDa protein was localized to a 3.2-kilobase DNA fragment of L. pneumophila cloned into pUC19 (pSH16) (P. S. Hoffman, C. A. Butler, and F. D. Quinn, Infect. Immun. 57:1731-1739, 1989). The nucleotide sequence of the DNA fragment cloned into M13 confirmed two open reading frames, htpA and htpB, that code for proteins of 96 and 548 amino acids, respectively. A consensus heat shock promoter sequence upstream of the start of htpA was identified, and no obvious promoter sequences were detected upstream of htpB. Amino acid sequence comparison studies revealed that the L. pneumophila HtpB protein exhibited 76% homology with the 65-kDa protein of Mycobacterium tuberculosis and 85% homology with both GroEL of Escherichia coli and HtpB of Coxiella burnetii. A comparison of the amino acid sequences among these proteins revealed several regions of nearly absolute sequence conservation, with the variable regions occurring in common areas. The purified L. pneumophila 60-kDa protein was antigenic for human T lymphocytes. Indirect fluorescent antibody studies indicated that the 60-kDa protein may be located in the periplasm or expressed on the surface by intracellular bacteria, suggesting that a stress-related mechanism may be involved in the expression of this immunodominant antigen.     

Chaperone-related immune dysfunction: an emergent property of distorted chaperone networks

Molecular chaperones (heat shock proteins) are important components of cellular networks, such as protein-protein and gene regulatory networks. Chaperones participate in the folding of immunologically important proteins, presentation of antigens and activation of the immune system. Here, we propose that chaperone-related immune dysfunction might be more general than was previously thought. Mutations and polymorphism of chaperones and the regulators of their synthesis, heat shock factor-1, chaperone diseases, sick chaperones and chaperone overload might all affect (mostly impairing) immune responses.     

感光受体外周蛋白结合蛋白对虫荧光素酶体外折叠的促进作用

探讨感光受体外周蛋白结合(PBP)在体外是否有促进变性蛋白质复性的作用。虫荧光素酶用盐酸胍变性，然后在PBP，HSP70，HSP60存在下进行复性，用虫荧光素酶分析系统检测该酶的复性程度。结果显示PBP对虫荧光素酶的复性能力很低，当与HSP70同时作用时，酶活力明显高于BP或HSP70组，而PBP与HSP60组合对酶的复性能力较HSP60组未见有明显差异；当PBP，HSP70，HSP60三者同时存在时，酶活力恢复率最高。去除复性缓冲液中的ATP及其再生系统，酶活力的复性率明显下降。结果表明PBP具有协助HSP70，在ATP依赖的情况下促进变性虫荧光酶体外复性的分子伴侣功能。     

A 77-kilodalton protein of Cryptococcus neoformans, a member of the heat shock protein 70 family, is a major antigen detected in the sera of mice with pulmonary cryptococcosis.

Heat shock proteins (HSPs) from several pathogenic microbes have been shown to be target molecules of humoral responses as well as cellular immune responses. However, little is known about target molecules in pulmonary cryptococcosis. Western blotting analysis revealed that experimentally induced pulmonary cryptococcosis in (BALB/c x DBA/2)F1 mice was associated with the appearance of serum antibodies to a 77-kDa protein derived from Cryptococcus neoformans as well as to 18-, 22-, 25-, 36-, and 94-kDa proteins. Since the 77-kDa band also reacted with rabbit polyclonal antibodies against 70-kDa HSP (HSP70) family members, the protein was predicted to be a member of the HSP70 family. We also purified HSP70 directly from a C. neoformans cell extract by Mono Q fast protein liquid chromatography and ATP-agarose affinity column chromatography and showed that it was positive in immunoblot analysis using either serum from C. neoformans-infected mice or rabbit anti-HSP70 antibodies. N-terminal amino acid sequencing of this purified protein confirmed that the 77-kDa protein was a member of the HSP70 protein family. A 66-kDa protein, which coincidentally purified with the HSP70 protein and was identified as a member of the HSP60 family by N-terminal amino acid sequencing, was not reactive with sera from C. neoformans-infected mice. Thus, a protein associated with the HSP70 family and derived from C. neoformans was a major target molecule of the humoral response in murine pulmonary cryptococcosis.     

Involvement of the ubiquitin-proteasome pathway and molecular chaperones in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominant muscular dystrophy that results from small expansions of a polyalanine tract in the PABPN1 gene. Intranuclear inclusions are the pathological hallmark of OPMD. The mechanism by which protein aggregation in OPMD might relate to a toxic gain-of-function has so far remained elusive. Whether protein aggregates themselves are pathogenic or are the consequence of an unidentified underlying molecular mechanism is still unclear. Here, we report that protein aggregation in a cell model of OPMD directly impaires the function of the ubiquitin-proteasome pathway (UPP) as well as molecular chaperone functions. The proteasome inhibitor lactacystin causes significant increase of protein aggregation and toxicity. Moreover, overexpression of molecular chaperones (HSP40 and HSP70) suppressed protein aggregation and toxicity. We also provide evidence that mPABPN1-ala17 protein aggregation proportionally correlates with toxicity. Furthermore, we show that co-expression of chaperones in our OPMD cell model increases the solubility of mPABPN1-ala17 and transfected cell survival rate. Our studies suggest that molecular regulators of polyalanine protein solubility and degradation may provide insights into new mechanisms in OPMD pathogenesis. Further analysis of the cellular and molecular mechanisms by which UPP and molecular chaperones influence the degradation of misfolded proteins could provide novel concepts and targets for the treatment and understanding of the pathogenesis of OPMD and neurodegenerative diseases.     

Heat shock protein 70 and ATP as partners in cell homeostasis (Review).

Heat shock proteins (HSP) are molecular chaperones, involved in many cellular functions such as protein folding, transport, maturation and degradation. Since they control the quality of newly synthesized proteins, HSP take part in cellular homeostasis. The Hsp70 family in particular exerts these functions in an adenosine triphosphate (ATP)-dependent manner. ATP is the main energy source used by cells to assume fundamental functions (respiration, proliferation, differentiation, apoptosis). Therefore, ATP levels have to be adapted to the requirements of the cells and ATP generation must constantly compensate ATP consumption. Nevertheless, under particular stress conditions, ATP levels decrease, threatening cell homeostasis and integrity. Cells have developed adaptive and protective mechanisms, among which Hsp70 synthesis and overexpression. In this review, we focus on the mechanisms which regulate Hsp70 expression under ATP depletion, using ischaemia as a paradigmatic model for ATP depletion in vivo, and analyze the molecular targets for Hsp70-mediated protection against ATP depletion. We also consider how these Hsp70-mediated protective effects could be applied in the therapeutical approaches of human diseases associated with cellular ATP depletion.     

Chaperoning a cellular upheaval in malaria: heat shock proteins in Plasmodium falciparum

In addition to their ability to help newly synthesized proteins to fold, molecular chaperones are also recognized for their participation in cellular processes ranging from protein trafficking, signal transduction, differentiation and development. Novel roles for this group of proteins have come to light through studies on important human pathogens like Leishmania, Trypanosoma as well as Plasmodia species. This review analyzes our current state of knowledge on molecular chaperones in human malarial parasite Plasmodium falciparum. In addition to a comparative analysis of their structures, complexes, client proteins and functions, a discussion on their potential as vaccine candidates as well as drug targets is also presented. The major chaperone classes of Hsp90, Hsp70, Hsp60 and Hsp40 family are well represented in the malarial parasite. Genomic cataloguing of all the parasite chaperone homologs indicates that about 2% of the total number of genes are dedicated to this function. While Hsp90 and Hsp70 are the most abundantly expressed, the Hsp40 class appears to be the best represented among the 92 chaperones encoded by the parasite genome. Importantly PfHsp70 is considered a potential vaccine candidate and PfHsp90 has been implicated as a drug target against the parasite. Available information suggests fascinating roles for chaperones in the life cycle of the parasite. In addition to their value as therapeutic targets, the study of chaperones in parasitic systems may likely reveal new principles of chaperone function in biology.     

Characterization of the heat shock response and identification of heat shock protein antigens of Borrelia burgdorferi.

The heat shock response of Borrelia burgdorferi B31 cells was characterized with regard to the heat shock proteins (Hsps) produced. Five to seven Hsps were detected by sodium dodecyl sulfate-gel electrophoresis and fluorography of proteins from cells labeled with [35S]methionine after shifts from 33 degrees C to 37 or 40 degrees C or from 20 degrees C to 33, 37, or 40 degrees C. Analysis of [35S]methionine-labeled Hsps by two-dimensional electrophoresis and autoradiography revealed 12 Hsps. Western immunoblot analysis with antisera to highly conserved Escherichia coli and Mycobacterium tuberculosis Hsps revealed a single 72-kilodalton (kDa) protein band that reacted with antibodies to E. coli DnaK and with antibodies to the M. tuberculosis 71-kDa Hsp homolog of E. coli DnaK. Two proteins with apparent molecular masses of 66 and 60 kDa reacted with antibodies against the M. tuberculosis 65-kDa Hsp homolog of E. coli GroEL. Human immune sera collected from patients with Lyme disease reacted with both the 66-kDa Hsp and the 60-kDa Hsp but failed to react with the 72-kDa Hsp. These data are discussed with regard to the possibility that host recognition of highly conserved epitopes of GroEL homologs of B. burgdorferi may result in autoimmune reactions causing arthritis and other pathologies.     

热休克蛋白60的研究进展

The family of HSP60 belongs to heat shock proteins with highly species conservatism and some important biologic functions. It can help other proteins for their assembling, folding and translocating, and plays a role in protecting cells against injuries and other types of stress. In addition, HSP60 is frequently recognized by the immune system as predominant antigens during infections and the progression of certain autoimmune diseases and might provide a novel strategy for the development of immunotherapeutics. This review focuses on distribution, molecular chaperone mechanism, function and gene expression regulation of HSP60.     

Expression of heat shock protein 70 in leukocytes of patients with sepsis

The heat shock proteins are known to protect cells against diverse injuries such as cytotoxicity by TNFalpha acting mainly as chaperones for denatured proteins. Lipopolysaccharide stimulates the production and the release of numerous endogenous mediators of sepsis: tumor necrosis factor alpha, interleukin-1, and interleukin-6 that induce fever production. Moreover, temperature at 40 degrees C is sufficient to induce heat shock and attenuate both TNFalpha and IL-1 expression. We demonstrate a distinct profile in gene expression of HSP 70 family in leukocytes obtained from different phases of septic patients. Our findings strongly suggest that HSP 70 may play a role in the outcome of septic shock patients.     

Message in a bottle: role of the 70-kDa heat shock protein family in anti-tumor immunity

Extracellular heat shock protein 70 (HSP70) is a potent agent for tumor immunotherapy, which can break tolerance to tumor-associated antigens and cause specific tumor cell killing by cytotoxic CD8+ T cells. The pro-immune effects of extracellular HSP70 are, to some extent, extensions of its molecular properties as an intracellular stress protein. The HSP70 are characterized by massive inducibility after stress, preventing cell death by inhibiting aggregation of cell proteins and directly antagonizing multiple cell death pathways. HSP70 family members possess a domain in the C terminus that chaperones unfolded proteins and peptides, and a N-terminal ATPase domain that controls the opening and closing of the peptide binding domain. These properties not only enable intracellular HSP70 to inhibit tumor apoptosis, but also promote formation of stable complexes with cytoplasmic tumor antigens that can then escape intact from dying cells to interact with antigen-processing cells (APC) and stimulate anti-tumor immunity. HSP70 may be released from tumors undergoing therapy at high local extracellular concentrations, and send a danger signal to the host leading to APC activation. Extracellular HSP70 bind to high-affinity receptors on APC, leading to activation of maturation and re-presentation of the peptide antigen cargo of HSP70 by the APC. The ability of HSP70-peptide complexes (HSP70-PC) to break tolerance and cause tumor regression employs these dual properties as signaling ligand and antigen transporter. HSP70-PC thus coordinately activate innate immune responses and deliver antigens for re-presentation by MHC class I and II molecules on the APC cell surface, leading to specific anti-tumor immunity.     

Co-translational protein aggregation after transient cerebral ischemia

Transient cerebral ischemia leads to irreversible translational inhibition which has been considered as a hallmark of delayed neuronal death after ischemia. This study utilized a rat transient cerebral ischemia model to investigate whether irreversible translational inhibition is due to abnormal aggregation of translational complex, i.e. the ribosomes and their associated nascent polypeptides, initiation factors, translational chaperones and degradation enzymes after ischemia. Translational complex aggregation was studied by electron microscopy, as well as by biochemical analyses. A duration of 15 or 20 min of cerebral ischemia induced severe translational complex aggregation starting from 30 min of reperfusion and lasting until the onset of delayed neuronal death at 48 h of reperfusion. Under electron microscopy, most rosette-shaped polyribosomes were relatively evenly distributed in the cytoplasm of sham-operated control neurons. After ischemia, most ribosomes were clumped into large abnormal aggregates in neurons destined to die. Translational complex components consisting of small ribosomal subunit protein 6, large subunit protein 28, eukaryotic initiation factor-3eta, co-translational chaperone heat shock cognate protein 70 and co-chaperone HSP40-Hdj1, as well as co-translational ubiquitin ligase c-terminus of hsp70-interacting protein were all irreversibly clumped into large abnormal protein aggregates after ischemia. Translational components were also highly ubiquitinated. To our knowledge, irreversible aggregation of translational components has not been reported after brain ischemia. This study clearly indicates that ischemia damages co-translational chaperone and degradation machinery, resulting in irreversible destruction of protein synthesis machinery by protein aggregation after ischemia.