Biomedical subjects

James E Hopper

Publications and source records attributed to James E Hopper.

9 recordsLinked to original sources

PubMed2006

Breast cancer metastasis suppressor 1 (BRMS1) is stabilized by the Hsp90 chaperone.

Breast cancer metastasis suppressor 1 (BRMS1) is a member of the mSin3-HDAC transcription co-repressor complex. However, the proteins associated with BRMS1 have not been fully identified. Yeast two-hybrid screen, immuno-affinity chromatography, and co-immunoprecipitation experiments were performed to identify BRMS1 interacting proteins (BIPs). In addition to known core mSin3 transcriptional complex components RBBP1 and mSDS3, BRMS1 interacted with other proteins including three chaperones: DNAJB6 (MRJ), Hsp90, and Hsp70. Hsp90 is a known target of HDAC6 and reversible acetylation is one of the mechanisms that is implicated in regulation of Hsp90 chaperone complex activity. BRMS1 interacted with class II HDACs, HDAC 4, 5, and 6. We further found that BRMS1 is stabilized by Hsp90, and its turnover is proteasome dependent. The stability of BRMS1 protein may be important in maintaining the functional role of BRMS1 in metastasis suppression.

Animals↗

PubMed2005

Intragenic suppression of Gal3C interaction with Gal80 in the Saccharomyces cerevisiae GAL gene switch.

Gal4-mediated activation of GAL gene transcription in Saccharomyces cerevisiae requires the interaction of Gal3 with Gal80, the Gal4 inhibitor protein. While it is known that galactose and ATP activates Gal3 interaction with Gal80, neither the mechanism of activation nor the surface that binds to Gal80 is known. We addressed this through intragenic suppression of GAL3C alleles that cause galactose-independent Gal3-Gal80 interaction. We created a new allele, GAL3SOC, and showed that it suppressed a new GAL3C allele. We tested the effect of GAL3SOC on several newly isolated and existing GAL3C alleles that map throughout the gene. All except one GAL3C allele, D368V, were suppressible by GAL3SOC. GAL3SOC and all GAL3C alleles were localized on a Gal3 homology model that is based on the structure of the highly related Gal1 protein. These results provide evidence for allosterism in the galactose- and ATP-activation of Gal3 binding to Gal80. In addition, because D368V and residues corresponding to Gal80-nonbinder mutations colocalized to a domain that is absent in homologous proteins that do not bind to Gal80, we suggest that D368 is a part of the Gal80-binding surface.

Adenosine Triphosphate↗

PubMed2004

Alpha V integrin prolongs collagenase production through Jun activation binding protein 1.

Robust expression of alphav integrin and matrix metalloproteinase 1 (MMP1) plays an important role in cancer metastasis and wound healing. A patient with an abnormal scar that appeared stretched and thinned out was found to have fibroblasts that overexpressed alphav integrin; therefore, a relationship between alphav integrin expression and MMP1 production was sought. A yeast 2 hybrid screen revealed alphav integrin interacts with jun activation binding domain-1 (JAB1). Mesenchymal-derived cells were transfected with the alphav integrin gene and incorporated into collagen lattices. Transfected cells maximally contracted collagen lattices beginning on day 5, whereas control transfected cells did not contract lattices. Late-phase collagen lattice contraction was inhibited by a pan-MMP inhibitor, BB4. Overexpression of alphav correlated with enhanced MMP1 transcription, as determined by a luciferase assay (P < or = 0.05). Diminution of JAB1 with JAB1 antisense abolished alphav integrin up-regulation of MMP1. We conclude alphav integrin signals through JAB1 to prolong MMP1 production and that this signaling pathway in fibroblasts may lead to abnormal scarring.

COP9 Signalosome Complex↗

PubMed2003

Breast cancer metastasis suppressor 1 (BRMS1) forms complexes with retinoblastoma-binding protein 1 (RBP1) and the mSin3 histone deacetylase complex and represses transcription.

Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis of multiple human and murine cancer cells without inhibiting tumorigenicity. By yeast two-hybrid and co-immunoprecipitation, BRMS1 interacts with retinoblastoma binding protein 1 and at least seven members of the mSin3 histone deacetylase (HDAC) complex in human breast and melanoma cell lines. BRMS1 co-immunoprecipitates enzymatically active HDAC proteins and represses transcription when recruited to a Gal4 promoter in vivo. BRMS1 exists in large mSin3 complex(es) of approximately 1.4-1.9 MDa, but also forms smaller complexes with HDAC1. Deletion analyses show that the carboxyl-terminal 42 amino acids of BRMS1 are not critical for interaction with much of the mSin3 complex and that BRMS1 appears to have more than one binding point to the complex. These results further show that BRMS1 may participate in transcriptional regulation via interaction with the mSin3.HDAC complex and suggest a novel mechanism by which BRMS1 might suppress cancer metastasis.

Animals↗

PubMed2003

Self-association of the amino-terminal domain of the yeast TATA-binding protein.

The amino-terminal domain of yeast TATA-binding protein has been proposed to play a crucial role in the self-association mechanism(s) of the full-length protein. Here we tested the ability of this domain to self-associate under a variety of solution conditions. Escherichia coli two-hybrid assays, in vitro pull-down assays, and in vitro cross-linking provided qualitative evidence for a limited and specific self-association. Sedimentation equilibrium analysis using purified protein was consistent with a monomer-dimer equilibrium with an apparent dissociation constant of approximately 8.4 microM. Higher stoichiometry associations remain possible but could not be detected by any of these methods. These results demonstrate that the minimal structure necessary for amino-terminal domain self-association must be present even in the absence of carboxyl-terminal domain structures. On the basis of these results we propose that amino-terminal domain structures contribute to the oligomerization interface of the full-length yeast TATA-binding protein.

Cloning, Molecular↗

PubMed2002

Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein.

Galactose-inducible genes (GAL genes) in yeast Saccharomyces cerevisiae are efficiently transcribed only when the sequence-specific transcription activator Gal4p is activated. Activation of Gal4p requires the interaction between the Gal4p inhibitory protein Gal80p and the galactokinase paralog, Gal3p. It has been proposed that Gal3p binds to a Gal80p-Gal4p complex in the nucleus to activate Gal4p. Here, we present evidence that the Gal3p-Gal80p interaction occurs in the cytoplasm, and concurrently, Gal80p is removed from Gal4p at the GAL gene promoter. We also show that GAL gene expression can be activated by heterologous protein-protein interaction in the cytoplasm that is independent of galactose and Gal3p function. These results indicate that galactose-triggered Gal3p-Gal80p association in the cytoplasm activates Gal4p in the nucleus.

Base Sequence↗

PubMed2002

Gal80 confers specificity on HAT complex interactions with activators.

Several yeast transcription activators have been shown to interact with and recruit histone acetyltransferase complexes to promoters in chromatin. The promiscuity of activator/HAT interactions suggests that additional factors temporally regulate these interactions in response to signaling pathways. In this study, we demonstrate that the negative regulator, Gal80, blocks interactions between the SAGA and NuA4 HAT complexes and the Gal4 activator. By contrast, Gal80 did not inhibit SAGA and NuA4 interaction with another activator Gcn4. The function of Gal80 prevented Gal4 targeting of SAGA and displaced SAGA targeted by Gal4 to a promoter within a nucleosome array. In the same set of experiments, targeting of SAGA by Gcn4 was unaffected by Gal80. These studies demonstrate that the specificity of HAT/activator interactions can be dictated by cofactors that modulate activation domain function in response to cellular signals.

Acetyltransferases↗

PubMed2002

Overexpression of integrin alphav promotes human osteosarcoma cell populated collagen lattice contraction and cell migration.

Cells attach and interact with the extracellular matrix (ECM) through heterodimeric alphabeta integrin receptors. Specifically, the promiscuous alphavbeta3 integrin and the alpha2beta1 integrin receptors engage numerous matrix components to influence cell adhesion, cell motility, and matrix organization. However, the role of alphav integrin mediating cell-collagen interactions is not clear. In the in vitro cell populated collagen lattice (PCL), a model of cell-matrix interaction, integrin receptors play a role in lattice contraction. To elucidate alphav integrins' effects on cell-collagen interactions, human osteosarcoma (HOS) cells were transfected with alphav integrin (alphav-pcDNA 3.1+). Control HOS cells were transfected with pcDNA 3.1+ vector alone. HOS-alphav cell PCLs contracted to a greater degree than control HOS cell PCLs (P < or = 0.0001). RT-PCR revealed that HOS-alphav cells express both beta1 and beta3 integrins, indicating that alphav has the potential to form a partnership with either beta1 or beta3 integrin. The alphavbeta3 specific inhibitory antibody LM609 significantly retarded HOS-alphav cell PCL contraction (P < or = 0.001), suggesting that alphavbeta3 promotes enhanced HOS-alphav cell PCL contraction. When plated on plastic, control HOS cells show greater elongation compared to HOS-alphav cells. In addition, HOS-alphav cells migrated faster and to a greater degree than control HOS cells (P < or = 0.0001). The possibility that enhanced HOS-alphav cell migration and HOS-alphav cell PCL contraction was caused by increased myosin ATPase activity was examined. HOS-alphav cells showed less myosin ATPase activity than control HOS cells, by an ATP cell contraction bioassay. The enhancement of HOS-alphav cell migration and lattice contraction appears unrelated to increased myosin ATPase activity.

Antibodies, Monoclonal↗

PubMed2002

Activation of prophage eib genes for immunoglobulin-binding proteins by genes from the IbrAB genetic island of Escherichia coli ECOR-9.

Four distinct Escherichia coli immunoglobulin-binding (eib) genes, each of which encodes a surface-exposed protein that binds immunoglobulins in a nonimmune manner, are carried by separate prophages in E. coli reference (ECOR) strain ECOR-9. Each eib gene was transferred to test E. coli strains, both in the form of multicopy recombinant plasmids and as lysogenized prophage. The derived lysogens express little or no Eib protein, in sharp contrast to the parental lysogen, suggesting that ECOR-9 has an expression-enhancing activity that the derived lysogens lack. Supporting this hypothesis, we cloned from ECOR-9 overlapping genes, ibrA and ibrB (designation is derived from "immunoglobulin-binding regulator"), which together activated eib expression in the derived lysogens. The proteins encoded by ibrA and ibrB are very similar to uncharacterized proteins encoded by genes of Salmonella enterica serovar Typhi and E. coli O157:H7 (in a prophage-like element of the Sakai strain and in two O islands of strain EDL933). The genomic segment containing ibrA and ibrB has been designated the IbrAB island. It contains regions of homology to the Shiga toxin-converting prophage, Stx2, as well as genes homologous to phage antirepressor genes. The left boundary between the IbrAB island and the chromosomal framework is located near min 35.8 of the E. coli K-12 genome. Homology to IbrAB was found in certain other ECOR strains, including the other five eib-positive strains and most strains of the phylogenetic group B2. Sequencing of a 1.1-kb portion of ibrAB revealed that the other eib-positive strains diverge by </=0.1% from ECOR-9, whereas eib-negative ECOR-47 diverges by 16%.

Chromosome Mapping↗