CD1d-restricted T-cell subsets and dendritic cell function in autoimmunity

CD1-restricted T cells have been shown to play a critical role in host defence, tumour surveillance, and maintenance of tolerance. However, immunologic outcomes resulting from activation of CD1d-restricted T cells can be either beneficial or deleterious. A major mechanism by which CD1d-restricted T cells are thought to exert immunoregulatory control is via effects on dendritic cell (DC) differentiation and migration. Important functional subsets of CD1d-restricted T cells are also known to exist and the potential implications for preferential subset activations are discussed.     

Hsp70B' regulation and function

Heat shock protein (Hsp) 70B' is a human Hsp70 chaperone that is strictly inducible, having little or no basal expression levels in most cells. Using siRNAs to knockdown Hsp70B' and Hsp72 in HT-29, SW-480, and CRL-1807 human colon cell lines, we have found that the two are regulated coordinately in response to stress. We also have found that proteasome inhibition is a potent activator of hsp70B'. Flow cytometry was used to assay hsp70B' promoter activity in HT-29eGFP cells in this study. Knockdown of both Hsp70B' and Hsp72 sensitized cells to heat stress and increasing concentrations of proteasome inhibitor. These data support the conclusion that Hsp72 is the primary Hsp70 family responder to increasing levels of proteotoxic stress, and Hsp70B' is a secondary responder. Interestingly ZnSO4 induces Hsp70B' and not Hsp72 in CRL-1807 cells, suggesting a stressor-specific primary role for Hsp70B'. Both Hsp70B' and Hsp72 are important for maintaining viability under conditions that increase the accumulation of damaged proteins in HT-29 cells. These findings are likely to be important in pathological conditions in which Hsp70B' contributes to cell survival.     

Hsp70B' regulation and function

Heat shock protein (Hsp) 70B' is a human Hsp70 chaperone that is strictly inducible, having little or no basal expression levels in most cells. Using siRNAs to knockdown Hsp70B' and Hsp72 in HT-29, SW-480, and CRL-1807 human colon cell lines, we have found that the two are regulated coordinately in response to stress. We also have found that proteasome inhibition is a potent activator of Hsp70B'. Flow cytometry was used to assay Hsp70B' promoter activity in HT-29eGFP cells in this study. Knockdown of both Hsp70B'- and Hsp72-sensitized cells to heat stress and increasing concentrations of proteasome inhibitor. These data support the conclusion that Hsp72 is the primary Hsp70 family responder to increasing levels of proteotoxic stress, and Hsp70B' is a secondary responder. Interestingly ZnSO4 induces Hsp70B' and not Hsp72 in CRL-1807 cells, suggesting a stressor-specific primary role for Hsp70B'. Both Hsp70B' and Hsp72 are important for maintaining viability under conditions that increase the accumulation of damaged proteins in HT-29 cells. These findings are likely to be important in pathological conditions in which Hsp70B' contributes to cell survival.     

Hsp 70 expression and function during gametogenesis

The dramatic transformations in nuclear content and cellular organization that occur during gametogenesis require unique regulation and execution of the mitotic and meiotic cell cycle, apoptotic cell death, DNA recombination and repair, and cellular differentiation. These processes are accompained by the constitutive and developmentally regulated expression of a number of hsp70 genes encoding 70 kDa heat shock proteins (Hsp70), including several hsp70s whose expression is unique to male germ cells. Examining the expression and function of Hsp70s in germ cells has provided significant insights into mechanisms of hsp70 gene regulation and Hsp70 protein function, as well as the developmental processes of gametogenesis.     

Hsp70 expression and function during embryogenesis

This review focuses on the expression and function of 70-kDa heat shock proteins (Hsp70s) during mammalian embryogenesis, though many features of embryogenesis and the developmental expression of Hsp70s are conserved between mammals and other vertebrates. A variety of Hsp70s are expressed from the point of zygotic gene activation in cleavage-stage embryos, through blastulation, implantation, gastrulation, neurulation, organogenesis, and on throughout fetal maturation. The regulation and patterns of hsp70 gene expression and the known and putative Hsp70 protein functions vary from constitutive and metabolic housekeeping to stress-inducible and embryo-protective roles. Understanding the genetic regulation and molecular function of Hsp70s has been pursued by developmental biologists interested in the control of gene expression in early embryos as well as reproductive toxicologists and teratologists interested in how Hsp70s protect embryos from the adverse effects of environmental exposures. These efforts have also been joined by those interested in the chaperone functions of Hsp70s, and this confluence of effort has yielded many advances in our understanding of Hsp70s during critical phases of embryonic development and cellular differentiation.     

CpG-B oligodeoxynucleotide promotes cell survival via up-regulation of Hsp70 to increase Bcl-xL and to decrease apoptosis-inducing factor translocation

Unmethylated CpG oligodeoxynucleotides (ODNs) activate immune responses in a TLR9-dependent manner. In this study, stimulation of mouse macrophages with CpG-B ODN increased cellular Hsp70 expression and prevented apoptosis induced by serum starvation or staurosporine treatment. CpG-B ODN-induced Hsp70 expression depended on TLR9, MyD88, and phosphatidylinositol 3-kinase. Inhibition of Hsp70 synthesis by an inhibitor (quercetin) or antisense hsp70 attenuated not only Hsp70 expression but also the anti-apoptotic capacity of CpG-B ODN. Ectopic expression of Hsp70 rescued the inhibitory effect of quercetin on CpG-B ODN-induced anti-apoptosis. Additional experiments demonstrated that quercetin and anti-sense hsp70 modulated CpG-B ODN-induced anti-apoptosis via a caspase-3-independent pathway by down-regulating the survival gene bcl-x(L) and by increasing translocation of apoptosis-inducing factor. These findings suggest that CpG-B ODN may up-regulate Hsp70 via a TLR9/MyD88/phosphatidylinositol 3-kinase pathway to increase Bcl-x(L) and to decrease apoptosis-inducing factor nuclear translocation, resulting in anti-apoptosis.     

Structure and in vivo function of Hsp90

Until recently, Hsp90 was one of the least well understood of the molecular chaperones, but considerable progress is now being made in unravelling its biochemistry. Hsp90 has now been shown to possess an inherent ATPase that is essential for the activation of authentic 'client' proteins in vivo and in vitro. The molecular detail of Hsp90's interactions with co-chaperones is also becoming clearer and the identification of key roles in assembling regulatory and signalling pathways has made it a target for anticancer drug development. Despite this, a clear understanding of how Hsp90 contributes to the folding and/or activation of its client proteins remains some way off.     

Beta cell function, peripheral sensitivity to insulin and islet cell autoimmunity in cystic fibrosis patients with normal glucose tolerance

Beta cell function, peripheral sensitivity to insulin and specific pancreatic autoimmunity were studied in 30 youngsters with cystic fibrosis (CF) accurately selected in order to fulfill the criteria for normal glucose tolerance. With respect to weight-matched controls, patients with CF exhibited a significantly lower glucose tolerance and a globally preserved, although delayed, insulin response to oral glucose tolerance test, while first-phase insulin secretion after i.v. glucose was blunted. Peripheral sensitivity to insulin, assessed in vivo by both the euglycemic clamp technique and the number of insulin receptors, directly measured in circulating monocytes, was superimposable in patients and controls. Serum islet-cell antibodies were not found in any of the patients. In conclusion, disorders of beta cell function may be observed in CF patients even when glucose tolerance is within the normal range. Such abnormalities are not associated with changes in peripheral sensitivity to insulin and do not seem to depend on specific autoimmune events.     

In vivo bipartite interaction between the Hsp40 Sis1 and Hsp70 in Saccharomyces cerevisiae

The essential Hsp40, Sis1, is a J-protein cochaperone for the Ssa class of Hsp70's of Saccharomyces cerevisiae. Sis1 is required for the maintenance of the prion [RNQ(+)], as Sis1 lacking its 55-amino-acid glycine-rich region (G/F) does not maintain [RNQ(+)]. We report that overexpression of Sis1DeltaG/F in an otherwise wild-type strain had a negative effect on both cell growth and [RNQ(+)] maintenance, while overexpression of wild-type Sis1 did not. Overexpression of the related Hsp40 Ydj1 lacking its G/F region did not cause inhibition of growth, indicating that this dominant effect of Sis1DeltaG/F is not a characteristic shared by all Hsp40's. Analysis of small deletions within the SIS1 G/F region indicated that the observed dominant effects were caused by the absence of sequences known to be important for Sis1's unique cellular functions. These inhibitory effects of Sis1DeltaG/F were obviated by alterations in the N-terminal J-domain of Sis1 that affect interaction with Ssa's ATPase domain. In addition, a genetic screen designed to isolate additional mutations that relieved these inhibitory effects identified two residues in Sis1's carboxy-terminal domain. These alterations disrupted the interaction of Sis1 with the 10-kD carboxy-terminal regulatory domain of Ssa1, indicating that Sis1 has a bipartite interaction with Ssa in vivo.     

Administration of Hsp70 in vivo inhibits motor and sensory neuron degeneration

The induction of heat shock proteins (Hsps) serves not only as a marker for cellular stress but also as a promoter of cell survival, which is especially important in the nervous system. We examined the regulation of the constitutive and stress-induced 70-kD Hsps (Hsc70 and Hsp70, respectively) after sciatic nerve (SN) axotomy in the neonatal mouse. Additionally, the prevention of axotomy-induced SN cell death by administration of several preparations of exogenous Hsc70 and Hsp70 was tested. Immunohistochemistry and Western blot analyses showed that endogenous levels of Hsc70 and Hsp70 did not increase significantly in lumbar motor neurons or dorsal root ganglion sensory neurons up to 24 hours after axotomy. When a variety of Hsc70 and Hsp70 preparations at doses ranging from 5 to 75 microg were applied to the SN stump after axotomy, the survival of both motor and sensory neurons was significantly improved. Thus, it appears that motor and sensory neurons in the neonatal mouse do not initiate a typical Hsp70 response after traumatic injury and that administration of exogenous Hsc/Hsp70 can remedy that deficit and reduce the subsequent loss of neurons by apoptosis.     

Distinguishing integral and receptor-bound heat shock protein 70 (Hsp70) on the cell surface by Hsp70-specific antibodies

Cell Stress & Chaperones journal has become a major outlet for papers and review articles about anti-heat shock protein (HSP) antibodies. In the last decade, it became evident that apart from their intracellular localization, members of the heat shock protein 90 (Hsp90; HSPC) and Hsp70 (HSPA) family are also found on the cell surface. In this review, we will focus on Hsp70 (HSPA1A), the major stress-inducible member of the human Hsp70 family. Depending on the cell type, the membrane association of Hsp70 comes in two forms. In tumor cells, Hsp70 appears to be integrated within the plasma membrane, whereas in non-malignantly transformed (herein termed normal) cells, Hsp70 is associated with cell surface receptors. This observation raises the question whether or not these two surface forms of Hsp70 in tumor and normal cells can be distinguished using Hsp70 specific antibodies. Presently a number of Hsp70 specific antibodies are commercially available. These antibodies were generated by immunizing mice either with recombinant or HeLa-derived human Hsp70 protein, parts of the Hsp70 protein, or with synthetic peptides. This review aims to characterize the binding of different anti-human Hsp70 antibodies and their capacity to distinguish between integrated and receptor-bound Hsp70 in tumor and normal cells.     

Molecular analysis of Hsp70 mechanisms in plants and their function in response to stress

Studying the strategies of improving abiotic stress tolerance is quite imperative and research under this field will increase our understanding of response mechanisms to abiotic stress such as heat. The Hsp70 is an essential regulator of protein having the tendency to maintain internal cell stability like proper folding protein and breakdown of unfolded proteins. Hsp70 holds together protein substrates to help in movement, regulation, and prevent aggregation under physical and or chemical pressure. However, this review reports the molecular mechanism of heat shock protein 70 kDa (Hsp70) action and its structural and functional analysis, research progress on the interaction of Hsp70 with other proteins and their interaction mechanisms as well as the involvement of Hsp70 in abiotic stress responses as an adaptive defense mechanism.     

Mge1, a nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70 function

Despite the growing evidence of the role of oxidative stress in disease, its molecular mechanism of action remains poorly understood. The yeast Saccharomyces cerevisiae provides a valuable model system in which to elucidate the effects of oxidative stress on mitochondria in higher eukaryotes. Dimeric yeast Mge1, the cochaperone of heat shock protein 70 (Hsp70), is essential for exchanging ATP for ADP on Hsp70 and thus for recycling of Hsp70 for mitochondrial protein import and folding. Here we show an oxidative stress–dependent decrease in Mge1 dimer formation accompanied by a concomitant decrease in Mge1–Hsp70 complex formation in vitro. The Mge1-M155L substitution mutant stabilizes both Mge1 dimer and Mge1–Hsp70 complex formation. Most important, the Mge1-M155L mutant rescues the slow-growth phenomenon associated with the wild-type Mge1 strain in the presence of H₂O₂ in vivo, stimulation of the ATPase activity of Hsp70, and the protein import defect during oxidative stress in vitro. Furthermore, cross-linking studies reveal that Mge1–Hsp70 complex formation in mitochondria isolated from wild-type Mge1 cells is more susceptible to reactive oxygen species compared with mitochondria from Mge1-M155L cells. This novel oxidative sensor capability of yeast Mge1 might represent an evolutionarily conserved function, given that human recombinant dimeric Mge1 is also sensitive to H₂O₂.     

Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70

The role of cytosolic factors in protein targeting to mitochondria is poorly understood. Here, we show that in mammals, the cytosolic chaperones Hsp90 and Hsp70 dock onto a specialized TPR domain in the import receptor Tom70 at the outer mitochondrial membrane. This interaction serves to deliver a set of preproteins to the receptor for subsequent membrane translocation dependent on the Hsp90 ATPase. Disruption of the chaperone/Tom70 recognition inhibits the import of these preproteins into mitochondria. In yeast, Hsp70 rather than Hsp90 is used in import, and Hsp70 docking is required for the formation of a productive preprotein/Tom70 complex. We outline a novel mechanism in which chaperones are recruited for a specific targeting event by a membrane-bound receptor.     

Binding of the maize cytosolic Hsp70 to calmodulin, and identification of calmodulin-binding site in Hsp70

Using a gel-overlay technique of biotinylated calmodulin (CaM), we showed that maize cytosolic Hsp70 protein could bind to CaM in the presence of 1 mM CaCl2. The purified maize cytosolic Hsp70 inhibited the activity of CaM-dependent NADK in a concentration-dependent manner. A synthetic peptide, which possesses the 21 amino acid sequence, PRALRRLRTACERAKRTLSST, at positions 261-281 in maize cytosolic Hsp70, could associate with CaM in the presence of 1 mM calcium. The synthetic peptide inhibited CaM-dependent NADK activity and PDE activity. This indicates that the 21-amino acid sequence at positions 261-281 is the CaM-binding site. The binding of CaM to Hsp70 inhibited the ATPase activity of Hsp70. The possible regulator function of Hsp70 in cell signaling events in response to heat stress is discussed.     

Lamina-associated polypeptide 2alpha forms complexes with heat shock proteins Hsp70 and Hsc70 in vivo

Lamina-associated polypeptide 2α (LAP2α), one of the alternatively spliced isoforms of the LAP2 gene, is a nucleoplasmic protein which forms oligomers and presumably associates to chromosomes via the LEM- and LEM-like regions. To characterize components of the LAP2α-containing complexes, we have expressed the α-specific C-terminal domain of LAP2α in HeLa cells and, after immunopurification, found that the heat shock proteins Hsp70 and Hsc70 reproducibly co-purified with this domain. Association between endogenous LAP2α and Hsp70 in non-transfected cells was confirmed by co-immunoprecipitation. The association was not mediated by the retinoblastoma protein.     

Quantitative aspects of T-cell recognition: from within the antigen-presenting cell to within the T cell

T lymphocytes circulate continually throughout the peripheral lymphoid organs, where they scrutinize the surface of cells to detect the presence of nonself protein fragments. During the last years, many facets of T-cell function have been unravelled. After being bound by major histocompatibility complex (MHC) molecules, peptides derived from nonself as well as from self proteins are delivered to the cell surface. A few copies of a nonself peptide “presented” at the cell surface in the context of an MHC molecule can be detected by specific T cells, and suffice to trigger T-cell activation. This paper reviews the requirements imposed on T cells to fulfill this exquisite sensitivity. BioEssays 20 :412–422, 1998.© 1998 John Wiley & Sons Inc.     

Hsp70 alters tau function and aggregation in an isoform specific manner

Tauopathies are characterized by abnormal aggregation of the microtubule associated protein tau. This aggregation is thought to occur when tau undergoes shifts from its native conformation to one that exposes hydrophobic areas on separate monomers, allowing contact and subsequent association into oligomers and filaments. Molecular chaperones normally function by binding to exposed hydrophobic stretches on proteins and assisting in their refolding. Chaperones of the heat shock protein 70 (Hsp70) family have been implicated in the prevention of abnormal tau aggregation in adult neurons. Tau exists as six alternatively spliced isoforms, and all six isoforms appear capable of forming the pathological aggregates seen in Alzheimer's disease. Because tau isoforms differ in primary sequence, we sought to determine whether Hsp70 would differentially affect the aggregation and microtubule assembly characteristics of the various tau isoforms. We found that Hsp70 inhibits tau aggregation directly and not through inducer-mediated effects. We also determined that Hsp70 inhibits the aggregation of each individual tau isoform and was more effective at inhibiting the three repeat isoforms. Finally, all tau isoforms robustly induced microtubule formation while in the presence of Hsp70. The results presented herein indicate that Hsp70 affects tau isoform dysfunction while having very little impact on the normal function of tau to mediate microtubule assembly. This indicates that targeting Hsp70 to tau may provide a therapeutic approach for the treatment of tauopathies that avoids disruption of normal tau function.     

Expression of Hsp90, Hsp70 and Hsp60 in Trichinella species exposed to oxidative shock

Stress response and phosphorylation of heat shock proteins (HSPs) 60, 70 and 90 were studied in Trichinella nativa, T. nelsoni, T. pseudospiralis and T. spiralis larvae at 30-min intervals following exposure to 20, 100 and 200 mM H2O2. There was a time- and dose-dependent differential survival for the infective stage larvae (L1) of these four Trichinella species. Immunoblotting analysis revealed that constitutive Hsp60 and Hsp70, but not Hsp90, from test Trichinella species are constitutively phosphorylated on serine/threonine residues as they converted to forms with increased sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) mobility by treatment with alkaline phosphatase. After exposure to H2O2, while there was a time-related occurrence of the three HSPs with decreased SDS-PAGE mobility, these HSPs were insensitive to alkaline phosphatase except in the case of exposure to 20 mM H2O2 for Hsp60 from all Trichinella species and Hsp70 from T. spiralis and T. nelsoni. The synthesis of HSPs forms with decreased SDS-PAGE mobility is a susceptibility signal because the lower concentration of peroxide (20 mM) did not cause a decrease on HSPs SDS-PAGE mobility in T. spiralis and T. nelsoni, the two more resistant selected Trichinella species.