Biomedical subjects

Vince Guerriero

Publications and source records attributed to Vince Guerriero.

8 recordsLinked to original sources

PubMed2006

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.

Actins↗

PubMed2006

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.

Adaptor Proteins, Signal Transducing↗

PubMed2006

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.

Adaptor Proteins, Signal Transducing↗

PubMed2005

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.

Adaptor Proteins, Signal Transducing↗

PubMed2005

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.

Adaptor Proteins, Signal Transducing↗

PubMed2003

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.

Adaptor Proteins, Signal Transducing↗

PubMed2002

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.

Adaptor Proteins, Signal Transducing↗

PubMedDate not supplied

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.

Adaptor Proteins, Signal Transducing↗