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.

HspBP1 levels are elevated in breast tumor tissue and inversely related to tumor aggressiveness.

HspBP1 is a co-chaperone that binds to and regulates the chaperone Hsp70 (Hsp70 is used to refer to HSPA1A and HSPA1B). Hsp70 is known to be elevated in breast tumor tissue, therefore the purpose of these studies was to quantify the expression of HspBP1 in primary breast tumors and in serum of these patients with a follow-up analysis after 6 to 7 years. Levels of HspBP1, Hsp70, and anti-HspBP1 antibodies in sera of breast cancer patients and healthy individuals were measured by enzyme-linked immunosorbent assay. Expression of HspBP1 was quantified from biopsies of tumor and normal breast tissue by Western blot analysis. The data obtained were analyzed for association with tumor aggressiveness markers and with patient outcome. The levels of HspBP1 and Hsp70 were significantly higher in sera of patients compared to sera of healthy individuals. HspBP1 antibodies did not differ significantly between groups. HspBP1 levels were significantly higher in tumor (14.46 ng/microg protein, n = 51) compared to normal adjacent tissue (3.17 ng/microg protein, n = 41, p < 0.001). Expression of HspBP1 was significantly lower in patients with lymph node metastasis and positive for estrogen receptors. HspBP1 levels were also significantly lower in patients with a higher incidence of metastasis and death following a 6 to 7-year follow-up. The HspBP1/Hsp70 molar ratio was not associated with the prognostic markers analyzed. Our results indicate that low HspBP1 expression could be a candidate tumor aggressiveness marker.

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.

Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure.

Epithelial tight junctions form a barrier against passive paracellular flux. This barrier is regulated by complex physiologic and pathophysiologic signals that acutely fine-tune tight junction permeability. Although actomyosin contraction and myosin light chain phosphorylation are clearly involved in some forms of tight junction regulation, the contributions of other signaling events and the role of myosin light chain phosphorylation in this response are poorly understood. Here we ask if activation of myosin light chain kinase alone is sufficient to induce downstream tight junction regulation. We use a confluent polarized intestinal epithelial cell model system in which constitutively active myosin light chain kinase, tMLCK, is expressed using an inducible promoter. tMLCK expression increases myosin light chain phosphorylation, reorganizes perijunctional F-actin, and increases tight junction permeability. TJ proteins ZO-1 and occludin are markedly redistributed, morphologically and biochemically, but effects on claudin-1 and claudin-2 are limited. tMLCK inhibition prevents changes in barrier function and tight junction organization induced by tMLCK expression, suggesting that these events both require myosin light chain phosphorylation. We conclude that myosin light chain phosphorylation alone is sufficient to induce tight junction regulation and provide new insights into the molecular mechanisms that mediate this regulation.

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.

Regulation of Hsp70 function by HspBP1: structural analysis reveals an alternate mechanism for Hsp70 nucleotide exchange.

HspBP1 belongs to a family of eukaryotic proteins recently identified as nucleotide exchange factors for Hsp70. We show that the S. cerevisiae ortholog of HspBP1, Fes1p, is required for efficient protein folding in the cytosol at 37 degrees C. The crystal structure of HspBP1, alone and complexed with part of the Hsp70 ATPase domain, reveals a mechanism for its function distinct from that of BAG-1 or GrpE, previously characterized nucleotide exchange factors of Hsp70. HspBP1 has a curved, all alpha-helical fold containing four armadillo-like repeats unlike the other nucleotide exchange factors. The concave face of HspBP1 embraces lobe II of the ATPase domain, and a steric conflict displaces lobe I, reducing the affinity for nucleotide. In contrast, BAG-1 and GrpE trigger a conserved conformational change in lobe II of the ATPase domain. Thus, nucleotide exchange on eukaryotic Hsp70 occurs through two distinct mechanisms.

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.