HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression.

HIV-1 efficiently hijacks host cellular machinery and exploits a plethora of host-viral interactions for its successful survival. Identifying host factors that affect susceptibility or resistance to HIV-1 may offer a promising therapeutic strategy against HIV-1. Previously, we have reported that heat shock proteins, HSP40 and HSP70 reciprocally regulate HIV-1 gene-expression and replication. In the present study, we have identified HSP70 binding protein 1 (HspBP1) as a host-intrinsic inhibitor of HIV-1. HspBP1 level was found to be significantly down modulated during HIV-1 infection and virus production inversely co-related with HspBP1 expression. Our results further demonstrate that HspBP1 inhibits HIV-1 long terminal repeat (LTR) promoter activity. Gel shift and chromatin immunoprecipitation assays revealed that HspBP1 was recruited on HIV-1 LTR at NF-κB enhancer region (κB sites). The binding of HspBP1 to κB sites obliterates the binding of NF-κB hetero-dimer (p50/p65) to the same region, leading to repression in NF-κB mediated activation of LTR-driven gene-expression. HspBP1 also plays an inhibitory role in the reactivation of latently infected cells, corroborating its repressive effect on NF-κB pathway. Thus, our results clearly show that HspBP1 acts as an endogenous negative regulator of HIV-1 gene-expression and replication by suppressing NF-κB-mediated activation of viral transcription.

Extracellular HspBP1 inhibits formation of a cytotoxic Tag7-Hsp70 complex in vitro and in human serum.

Tag7 (PGRP-S) was described as an innate immunity protein. Earlier we have shown that Tag7 forms with Hsp70 a stable complex with cytotoxic and antitumor activity. The same complex is formed in and secreted by cytotoxic T-lymphocytes. We have also found that Hsp-binding protein HspBP1 incapacitates the Tag7-Hsp70 complex. Here we have studied the interaction of extracellular Tag7 and HspBP1. We have shown that HspBP1 binds Tag7 in the conditioned medium of tumor CSML0 cells, thereby preventing formation of the cytotoxic Tag7-Hsp70 complex. We have also found that Tag7, if present in serum (in every third donor on average), is always in complex with HspBP1. This may be a protective measure against indiscriminate attack of the cytotoxic complex on normal cells.

The heat shock-binding protein (HspBP1) protects cells against the cytotoxic action of the Tag7-Hsp70 complex.

Heat shock-binding protein HspBP1 is a member of the Hsp70 co-chaperone family. The interaction between HspBP1 and the ATPase domain of the major heat shock protein Hsp70 up-regulates nucleotide exchange and reduces the affinity between Hsp70 and the peptide in its peptide-binding site. Previously we have shown that Tag7 (also known as peptidoglycan recognition protein PGRP-S), an innate immunity protein, interacts with Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against some tumor cell lines. This complex can be produced in cytotoxic lymphocytes and released during interaction with tumor cells. Here the effect of HspBP1 on the cytotoxic activity of the Tag7-Hsp70 complex was examined. HspBP1 could bind not only to Hsp70, but also to Tag7. This interaction eliminated the cytotoxic activity of Tag7-Hsp70 complex and decreased the ATP concentration required to dissociate Tag7 from the peptide-binding site of Hsp70. Moreover, HspBP1 inhibited the cytotoxic activity of the Tag7-Hsp70 complex secreted by lymphocytes. HspBP1 was detected in cytotoxic CD8+ lymphocytes. This protein was released simultaneously with Tag7-Hsp70 during interaction of these lymphocytes with tumor cells. The simultaneous secretion of the cytotoxic complex with its inhibitor could be a mechanism protecting normal cells from the cytotoxic effect of this complex.

Heat shock protein 70-binding protein 1 is highly expressed in high-grade gliomas, interacts with multiple heat shock protein 70 family members, and specifically binds brain tumor cell surfaces.

Chaperone proteins and heat shock proteins (HSP) are essential components of cellular protein folding systems under normal conditions; their expression and activities are upregulated during stress. Chronically stressed tumors frequently exhibit high chaperone protein levels, exploiting their anti-apoptotic mechanisms and general proteome homeostasis amidst a background of genetic instability. Co-chaperones interact with chaperones as malleable regulatory components of protein folding activity and may represent a conduit for modification of chaperone activity to the detriment of the tumor. We have initially characterized one such co-chaperone, heat shock protein 70-binding protein (HspBP) 1 from human brain tumors, their xenografts grown in immune-compromised mice, and in syngeneic murine models in immune-competent mice. Immunohistochemical analyses show HspBP1 overexpression (with unusual subcellular localizations) in patient brain tumors relative to normal brain tissue. This holds true for the xenograft and syngeneic murine tumor models. In biochemical affinity chromatography assays, HspBP1 interacts with members of the HSP70 family from brain tumor lysates and from surface-derived samples, including HSP70, glucose regulated protein (GRP)75, GRP78, and HSP110. From normal brain lysates, only heat shock cognate (HSC)70, GRP75, and HSP110 bind to HspBP1. FACS analyses indicate that HspBP1 binds to brain tumor cell surfaces, possibly via HSP70 family members, and internalizes into cells. This has implications for HspBP1 biology as well as its utility as a tumor-targeting agent. Our results suggest that HspBP1 may play a role in tumor (dys)regulation of chaperone proteins, and that HspBP1 may have extracellular roles with therapeutic implications.

Extracellular HspBP1 and Hsp72 synergistically activate epidermal growth factor receptor.

BACKGROUND INFORMATION: Heat-inducible Hsp72 is the founding member of the Hsp70 (heat shock proteins of 70 kDa) family of molecular chaperones. It is localized primarily in cytoplasm and nucleus but is also found extracellularly. The source of e-Hsp72 (extracellular Hsp72) is not precisely identified and may not be the same in every situation. A number of studies demonstrated that e-Hsp72 plays an important role in cell survival, tumour rejection and immune response. However, currently little is known about regulation of e-Hsp72 function. In cells, Hsp72 is controlled by co-chaperones. An abundant co-chaperone, HspBP1 (Hsp72-binding protein 1) was found extracellularly in the serum. In the present study we analysed the secretion and function of e-HspBP1 (extracellular HspBP1). RESULTS: A431 human squamous carcinoma cells accumulated Hsp72 and HspBP1 in chromogranin A-positive granules following heat stress or in the presence of U73122, an inhibitor of phospholipase C. Following these treatments, A431 cells also increased the secretion of both proteins into the culture medium. The secreted e-Hsp72 and e-HspBP1 were co-immunoprecipitated from the conditioned medium. Purified recombinant HspBP1 augmented e-Hsp72-mediated phosphorylation of EGFR (epidermal growth factor receptor) and its down-stream targets, ERK1 (extracellular signal-regulated kinase 1) and ERK2 in a concentration-dependent manner. Finally, a HspBP1 N-terminal domain deletion mutant and boiled recombinant HspBP1 did not affect the e-Hsp72-mediated activity. CONCLUSIONS: Heat stress and PLC (phospholipase C) inhibition result in the enhanced secretion of both Hsp72 and HspBP1. In an extracellular environment, the two chaperones interact both physically and functionally, leading to the activation of th EGFR-ERK1/2 signalling pathway. However, the magnitude of EGFR activation depends on the e-HspBP1/e-Hsp72 ratio in the medium. Extracellular chaperone-mediated activation of EGFR can provide a survival advantage to cells under heat shock and other stresses.

Increased expression of heat shock protein-binding protein 1 and heat shock protein 70 in human hepatocellular carcinoma tissues.

Heat shock protein-binding protein 1 (HspBP1) is a co-chaperone that inhibits heat shock 70-kDa protein (Hsp70) activity. In mouse neuroblastomas and lung tumors, the protein levels of HspBP1 and Hsp70 are elevated by a similar amount compared to non-tumor tissues. However, no studies have been reported regarding the levels of HspBP1 in human cancer tissues. Our previous proteomic study demonstrated that the expression of Hsp70 was increased in human hepatitis C virus-related hepatocellular carcinoma (HCV-HCC) tissues. Here, we investigated the expression of HspBP1 in human HCV-HCC. Immunoblotting analysis of HspBP1 and Hsp70 was performed in human HCV-HCC tissues from 20 patients. In 80% of the patients, Hsp70 increased an average of 3.55-fold, and in 50% of the patients, HspBP1 increased an average of 2.02-fold. Comparison and analysis of expression and clinical data revealed a significant difference between moderately-differentiated HCC and non-tumor tissues. In addition, there was a significant difference between the ratio of HspBP1 to Hsp70 levels and tumor size (<3 cm vs. ≥ 3cm) with larger tumors having a lower ratio. This ratio was significantly lower in moderately-differentiated HCC tissues than in non-tumor HCC tissues. In conclusion, HspBP1 was up-regulated in human HCV-HCC, an increase which correlated with the increase of Hsp70 levels. The ratio of HspBP1 to Hsp70 in HCC may provide novel information concerning the characterization of tumors, tumor progression and resistance to treatment.

Human serum contains detectable levels of the Hsp70 cochaperone HspBP1 and antibodies bound to HspBP1.

The identification of the proteins that comprise the serum proteome is a current major research goal that will provide useful information for the diagnosis and treatment of various diseases. It is well established that Hsp70 and Hsp70 antibodies are present in human serum. This study reports on the development of an ELISA assay for the Hsp70 co-chaperone, HspBP1. HspBP1 is present in human serum at concentrations ranging between 0.74 to 3.98 ng/mL. No gender or age differences in the HspBP1 levels were identified. It was also found that human serum contained antibodies to HspBP1, and there were no gender or age differences in these levels. In addition, there was no correlation between the level of HspBP1 in a sample and the antibody titer. Finally, we found that HspBP1 in serum is complexed to anti-HspBP1 antibodies. This report provides initial baseline data on HspBP1 in human serum and provides the methods for future studies to determine if these levels are altered in response to disease.

Expression of the cochaperone HspBP1 is not coordinately regulated with Hsp70 expression.

Intracellular levels of the heat stress protein Hsp70 are elevated following exposure to elevated temperature. The cochaperone HspBP1 is an intracellular protein that is known to bind to and regulate Hsp70 activity. The purpose of this study was to determine if HspBP1 levels changed when Hsp70 levels were altered. Heat stress resulted in an increase in Hsp70 levels but no change in HspBP1 levels. Treatment of cells with the apoptosis inducing drug camptothecin lowered Hsp70 levels but again had no effect on HspBP1 levels. Cells treated with camptothecin plus heat stress did not exhibit an increase in Hsp70 levels. Over-expression in cells stably transfected with HspBP1 cDNA resulted in a 290% increase in HspBP1 levels without a similar change in Hsp70 levels. These results demonstrate that Hsp70 and HspBP1 are not coordinately regulated but provide evidence that an increase in the ratio of HspBP1 to Hsp70 correlates with apoptosis, in a similar way to reducing the amount of Hsp70.

Development of a sensitive assay for the measurement of antibodies against heat shock protein binding protein 1 (HspBP1): increased levels of anti-HspBP1 IgG are prevalent in HIV infected subjects.

The 70 kDa heat shock protein (Hsp70) is generally considered to be an intracellular protein, however, there is evidence that Hsp70 can be found in the extracellular environment. Hsp70 and antibodies against Hsp70 have been reported in human serum. Recent evidence has shown that Hsp70 antibodies are elevated in HIV infected individuals. This study reports on the antibody levels against a co-chaperone, HspBP1, that regulates Hsp70 activity. We have developed a solid-phase enzyme linked assay for the determination of anti-HspBP1 IgG antibodies. We report here that HspBP1 antibodies are present in human serum and the levels are elevated approximately twofold in HIV infected patients. There was no correlation between HspBP1 antibody levels and clinical parameters nor was there a relation between anti-Hsp70 levels and anti-HspBP1 levels. The presence of HspBP1 antibodies in human serum suggests that the protein may also be present in the serum. The increased level of HspBP1 antibodies in HIV infected individuals suggests a relationship directly to the virus or indirectly to secondary consequences of HIV infection.

A proteomic study of resistance to deoxycholate-induced apoptosis.

The development of apoptosis resistance appears to be an important factor in colon carcinogenesis. To gain an understanding of the molecular pathways altered during the development of apoptosis resistance, we selected three cell lines for resistance to induction of apoptosis by deoxycholate, an important etiologic agent in colon cancer. We then evaluated gene expression levels for 825 proteins in these resistant lines, compared with a parallel control line not subject to selection. Eighty-two proteins were identified as either over-expressed or under-expressed in at least two of the resistant lines, compared with the control. Thirty-five of the 82 proteins (43%) proved to have a known role in apoptosis. Of these 35 proteins, 21 were over-expressed and 14 were under-expressed. Of those that were over-expressed 18 of 21 (86%) are anti-apoptotic in some circumstances, of those that were under-expressed 11 of 14 (79%) are pro-apoptotic in some circumstances. This finding suggests that apoptosis resistance during selection among cultured cells, and possibly in the colon during progression to cancer, may arise by constitutive over-expression of multiple anti-apoptotic proteins and under-expression of multiple pro-apoptotic proteins. The major functional groups in which altered expression levels were found are post-translational modification (19 proteins), cell structure (cytoskeleton, microtubule, actin, etc.) (17 proteins), regulatory processes (11 proteins) and DNA repair and cell cycle checkpoint mechanisms (10 proteins). Our findings, overall, bear on mechanisms by which apoptosis resistance arises during progression to colon cancer and suggest potential targets for cancer treatment. In addition, assays of normal-appearing mucosa of colon cancer patients, for over- or under-expression of genes found to be altered in our resistant cell lines, may allow identification of early biomarkers of colon cancer risk.

Expression of a unique drug-resistant Hsp90 ortholog by the nematode Caenorhabditis elegans.

In all species studied to date, the function of heat shock protein 90 (Hsp90), a ubiquitous and evolutionarily conserved molecular chaperone, is inhibited selectively by the natural product drugs geldanamycin (GA) and radicicol. Crystal structures of the N-terminal region of yeast and human Hsp90 have revealed that these compounds interact with the chaperone in a Bergerat-type adenine nucleotide-binding fold shared throughout the gyrase, Hsp90, histidine kinase mutL (GHKL) superfamily of adenosine triphosphatases. To better understand the consequences of disrupting Hsp90 function in a genetically tractable multicellular organism, we exposed the soil-dwelling nematode Caenorhabditis elegans to GA under a variety of conditions designed to optimize drug uptake. Mutations in the gene encoding C elegans Hsp90 affect larval viability, dauer development, fertility, and life span. However, exposure of worms to GA produced no discernable phenotypes, although the amino acid sequence of worm Hsp90 is 85% homologous to that of human Hsp90. Consistent with this observation, we found that solid phase-immobilized GA failed to bind worm Hsp90 from worm protein extracts or when translated in a rabbit reticulocyte lysate system. Further, affinity precipitation studies using chimeric worm-vertebrate fusion proteins or worm C-terminal truncations expressed in reticulocyte lysate revealed that the conserved nucleotide-binding fold of worm Hsp90 exhibits the novel ability to bind adenosine triphosphate but not GA. Despite its unusual GA resistance, worm Hsp90 appeared fully functional when expressed in a vertebrate background. It heterodimerized with its vertebrate counterpart and showed no evidence of compromising its essential cellular functions. Heterologous expression of worm Hsp90 in tumor cells, however, did not render them GA resistant. These findings provide new insights into the nature of unusual N-terminal nucleotide-binding fold of Hsp90 and suggest that target-related drug resistance is unlikely to emerge in patients receiving GA-like chemotherapeutic agents.

HspBP1, an Hsp70 cochaperone, has two structural domains and is capable of altering the conformation of the Hsp70 ATPase domain.

We present here the first structural information for HspBP1, an Hsp70 cochaperone. Using circular dichroism, HspBP1 was determined to be 35% helical. Although HspBP1 is encoded by seven exons, limited proteolysis shows that it has only two structural domains. Domain I, amino acids 1-83, is largely unstructured. Domain II, amino acids 84-359, is predicted to be 43% helical using circular dichroism. Using limited proteolysis we have also shown that HspBP1 association changes the conformation of the ATPase domain of Hsp70. Only domain II of HspBP1 is required to bring about this conformational change. Truncation mutants of HspBP1 were tested for their ability to inhibit the renaturation of luciferase and bind to Hsp70 in reticulocyte lysate. A carboxyl terminal truncation mutant that was slightly longer than domain I was inactive in these assays, but domain II was sufficient to perform both functions. Domain II was less active than full-length HspBP1 in these assays, and addition of amino acids from domain I improved both functions. These studies show that HspBP1 domain II can bind Hsp70, change the conformation of the ATPase domain, and inhibit Hsp70-associated protein folding.

Increased expression of the Hsp70 cochaperone HspBP1 in tumors.

Hsp70 levels are elevated in a number of different tumors. The Hsp70 cochaperone heat shock protein-binding protein 1 (HspBP1) has been shown to bind to Hsp70, inhibit its activity and promote dissociation of nucleotide from the Hsp70 ATPase domain. The purpose of this study was to determine if the levels of HspBP1 are altered in tumor cells. In this report, we show that HspBP1 levels are elevated in two mouse tumor models, 3LL cells (Lewis Lung carcinoma) and neuroblastoma tumors. The amounts of HspBP1 and Hsp70 in selected tissues, tumors and a rabbit reticulocyte lysate were determined using Western blots. It was found that the molar ratio of these two proteins was within a small range (0.21-0.42) in the normal and tumor tissues examined. This ratio was considerably below the HspBP1 to Hsp70 ratio of 4.0 needed for 50% inhibition of Hsp70-mediated refolding of a partially denatured protein in rabbit reticulocyte lysate. The ratio of HspBP1 to Hsp70 in these tissues is too low to inhibit Hsp70 globally in the cell, but is high enough to provide a pool of HspBP1 that could inhibit Hsp70 in a localized fashion. These studies have shown that HspBP1 is elevated in the tumors examined and therefore could be a new cancer marker.

HspBP1, a homologue of the yeast Fes1 and Sls1 proteins, is an Hsc70 nucleotide exchange factor.

The yeast FES1 and SLS1 genes encode conserved nucleotide exchange factors that act on the cytoplasmic and endoplasmic reticulum luminal Hsp70s, Ssa1p and BiP, respectively. We report here that mammalian HspBP1 is homologous to Fes1p and that HspBP1 promotes nucleotide dissociation from both Ssa1p and mammalian Hsc70. In contrast, Fes1p inefficiently strips nucleotide from mammalian Hsc70, and unlike HspBP1 does not inhibit chaperone-mediated protein refolding in vitro. Together, our data indicate that HspBP1 is a member of this new class of nucleotide exchange factors that exhibit varying degrees of compartment and species specificity.