Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Galectin 1 (Gal-1), a lactose-binding lectin, is a component of vascular extracellular matrix and secreted by human vascular smooth muscle cells (SMCs). The purpose of this study was to investigate a possible role of Gal-1 in controlling adhesion and migration of cultured human vascular SMCs. Gal-1 co-localised with laminin and cellular fibronectin in extracellular matrix (ECM) secreted by cultured human vascular SMCs. Recombinant glutathione S-transferase (GST)-Gal-1 fusion protein bound to laminin and cellular fibronectin in ELISA. GST-Gal-1 inhibited SMC attachment to laminin via interactions with both SMCs and laminin. GST-Gal-1 inhibited SMC spreading on plastic or on laminin, but not on cellular fibronectin. GST-Gal-1 modulated SMC migration on laminin and inhibited migration on cellular fibronectin. GST-Gal-1 bound to several 35S-labelled proteins in SMC extracts including laminin and alpha1beta1 integrin, identified by depletion of SMC protein extracts with respective antibodies. We conclude that Gal-1 is able to modulate SMC attachment, spreading and migration via interactions with ECM proteins and alpha1beta1 integrin.
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PMID:Galectin 1 modulates attachment, spreading and migration of cultured vascular smooth muscle cells via interactions with cellular receptors and components of extracellular matrix. 1005 73

Alternative splicing of the fibronectin gene transcript gives rise to a group of adhesive glycoproteins showing restricted spatial and temporal expression during embryonic development, tumor growth, and tissue repair. Alternative splicing occurs in three segments termed EIIIB, EIIIA, and V. The EIIIA (or ED-A) segment of fibronectin is expressed prominently but transiently in healing wounds coincident with fibroblast expression of an activation marker, smooth muscle cell alpha-actin. A monoclonal antibody (IST-9) to the EIIIA segment blocks transforming growth factor-beta-mediated smooth muscle cell alpha-actin expression by fibroblasts in culture. A second monoclonal antibody (DH1) blocks chondrocyte condensation in chicken embryos. We find that IST-9 and DH1 react with human, rat, and chicken but not with mouse or frog EIIIA, suggesting that His44 may be important for antibody binding. A series of deletion mutants of rat EIIIA, constructed as glutathione S-transferase fusion proteins, do not react with either IST-9, DH1, or a third monoclonal antibody (3E2). Mutations of pairs of amino acids to alanine have little effect, except for either (Val34Thr35) or (Tyr36Ser37), which are located in a beta strand upstream from His44. For these double mutants, the binding to all three monoclonal antibodies is markedly reduced. By contrast, single mutants at Thr35, Tyr36, or Ser37 retain full activity, suggesting that the epitope for these antibodies is determined in part by conformation. Alanine-scanning mutagenesis of rat EIIIA demonstrates the importance of Ile43 and His44 for binding. Mutation of frog EIIIA (normally Val43Lys44) to rat (Ile43His44) is sufficient to restore fully IST-9 binding and much of the activity of DH1 and 3E2. Our findings demonstrate that the function-blocking antibodies, IST-9 and DH1, bind to the Ile43 and His44 residues in a conformationally dependent fashion, implicating the loop region encompassing both residues as critical for mediating EIIIA function.
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PMID:Identification of two amino acids within the EIIIA (ED-A) segment of fibronectin constituting the epitope for two function-blocking monoclonal antibodies. 1036 33

RIM1 is a putative effector protein for Rab3s, synaptic GTP-binding proteins. RIM1 is localized close to the active zone at the synapse, where it interacts in a GTP-dependent manner with Rab3 located on synaptic vesicles. We now describe a second RIM protein, called RIM2, that is highly homologous to RIM1 and also expressed primarily in brain. Like RIM1, RIM2 contains an N-terminal zinc finger domain that binds to Rab3 as a function of GTP, a central PDZ domain, and two C-terminal C(2) domains that are separated by long alternatively spliced sequences. Unexpectedly, the 3'-end of the RIM2 gene produces an independent mRNA that encodes a smaller protein referred as NIM2. NIM2 is composed of a unique N-terminal sequence followed by the C-terminal part of RIM2. Data bank searches identified a third RIM/NIM-related gene, which encodes a NIM isoform referred to as NIM3; no RIM transcript from this gene was detected. To test if NIMs, like RIMs, may function in secretion, we investigated the effect of NIM3 on calcium-triggered exocytosis in PC12 cells. NIM3 induced a dramatic increase in calcium-evoked exocytosis (50%), with no significant effect on base-line release, suggesting that NIMs, like RIMs, regulate exocytosis The combination of conserved and variable sequences in RIMs and NIMs indicates that the individual domains of these proteins provide binding sites for interacting molecules during exocytosis, as shown for the zinc finger domain of RIM, which binds to GTP-bound Rab3s. To search for additional interacting proteins for RIMs, we employed yeast two-hybrid screens with the C-terminal half of RIM1. Two members of a new family of homologous brain proteins, referred to as RIM-binding proteins (RIM-BPs), were identified. RIM-BPs bind to RIM in yeast two-hybrid and GST pull-down assays, suggesting a specific interaction. In RIMs, the binding site for RIM-BPs consists of a conserved proline-rich sequence between the two C(2) domains, N-terminal to the beginning of NIMs. RIM-BPs are composed of multiple domains, including three fibronectin type III-domains and three Src homology 3 domains, of which the second Src homology 3 domain binds to RIMs. With the RIM-BPs, we have identified a partner for RIMs that may bind to RIMs at the synapse in addition to Rab3.
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PMID:The RIM/NIM family of neuronal C2 domain proteins. Interactions with Rab3 and a new class of Src homology 3 domain proteins. 1074 13

The integrin alpha4beta1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via alpha4beta1 using GST fusion proteins. We show that alpha4beta1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the alpha4beta1 binding region in OPN lies between amino acid residues 125 and 168 (aa125-168). This region contains the central RGD motif of OPN, which also interacts with integrins alphavbeta3, alphavbeta5, alphavbeta1, alpha8beta1, and alpha5beta1. Mutating the RGD motif to RAD had no effect on the interaction with alpha4beta1. To define the binding site the region incorporating aa125-168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via alpha4beta1, aa132-146, and aa153-168; of these only a synthetic peptide, SVVYGLR (aa162-168), derived from aa153-168 was able to inhibit alpha4beta1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of alpha4beta1, and the primary alpha4beta1 binding site within OPN.
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PMID:Analysis of the alpha4beta1 integrin-osteopontin interaction. 1089 85

The use of so-called protein scaffolds has recently attracted considerable attention in biochemistry in the context of generating novel types of ligand receptors for various applications in research and medicine. This development started with the notion that immunoglobulins owe their function to the composition of a conserved framework region and a spatially well-defined antigen-binding site made of peptide segments that are hypervariable both in sequence and in conformation. After the application of antibody engineering methods along with library techniques had resulted in first successes in the selection of functional antibody fragments, several laboratories began to exploit other types of protein architectures for the construction of practically useful binding proteins. Properties like small size of the receptor protein, stability and ease of production were the focus of this work. Hence, among others, single domains of antibodies or of the immunoglobulin superfamily, protease inhibitors, helix-bundle proteins, disulphide-knotted peptides and lipocalins were investigated. Recently, the scaffold concept has even been adopted for the construction of enzymes. However, it appears that not all kinds of polypeptide fold which may appear attractive for the engineering of loop regions at a first glance will indeed permit the construction of independent ligand-binding sites with high affinities and specificities. This review will therefore concentrate on the critical description of the structural properties of experimentally tested protein scaffolds and of the novel functions that have been achieved on their basis, rather than on the methodology of how to best select a particular mutant with a certain activity. An overview will be provided about the current approaches, and some emerging trends will be identified. (c) 2000 John Wiley & Sons, Ltd. Abbreviations used: ABD albumin-binding domain of protein G APPI Alzheimer's amyloid beta-protein precursor inhibitor BBP bilin-binding protein BPTI bovine (or basic) pancreatic trypsin inhibitor BSA bovine serum albumin CBD cellulose-binding domain of cellobiohydrolase I CD circular dichroism Cdk2 human cyclin-dependent kinase 2 CDR complementarity-determining region CTLA-4 human cytotoxic T-lymphocyte associated protein-4 FN3 fibronectin type III domain GSH glutathione GST glutathione S-transferase hIL-6 human interleukin-6 HSA human serum albumin IC(50) half-maximal inhibitory concentration Ig immunoglobulin IMAC immobilized metal affinity chromatography K(D) equilibrium constant of dissociation K(i) equilibrium dissociation constant of enzyme inhibitor LACI-D1 human lipoprotein-associated coagulation inhibitor pIII gene III minor coat protein from filamentous bacteriophage f1 PCR polymerase-chain reaction PDB Protein Data Bank PSTI human pancreatic secretory trypsin inhibitor RBP retinol-binding protein SPR surface plasmon resonance TrxA E. coli thioredoxin
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PMID:Engineered protein scaffolds for molecular recognition. 1093 55

Autologous transplantation of bone marrow cells (BMCs) transduced with the multidrug resistance 1 (MDR1) gene or dihydrofolate reductase (DHFR) gene has already been applied in clinical chemoprotection trials. However, anticancer drugs frequently used in high-dose chemotherapy (HDC), such as alkylating agents, are not relevant to MDR1 or DHFR gene products. In this context, we have previously reported that glutathione S-transferase-pi (GST-pi) gene-transduced human CD34(+) cells showed resistance in vitro against 4-hydroperoxicyclophosphamide, an active form of cyclophosphamide (CY). In the present study, a subsequent attempt was made in a murine model to evaluate the effectiveness of transplantation of GST-pi-transduced BMCs to protect bone marrow against high-dose CY. The gene transfection was carried out retrovirally, employing a recombinant fibronectin fragment. Transfection efficiency into CFU-GM was 30%. After the transplantation, recipient mice (GST-pi mice) received three sequential courses of high-dose CY. As the chemotherapy courses advanced, both shortening of recovery period from WBC nadir and shallowing of WBC nadir were observed. In contrast to the fact that three of seven control mice died, possibly due to chemotoxicity, all seven GST-pi mice were alive after the third course, at which point the vector GST-pi gene was detected in 50% of CFU-GM derived from their BMCs and peripheral blood mononuclear cells. When BMCs obtained from these seven mice were retransplanted into secondary recipient mice, 20% of CFU-GM from BMCs showed positive signals for vector GST-pi DNA after 6 months. These data indicate that the GST-pi gene can confer resistance to bone marrow against CY by being transduced into long-term repopulating cells.
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PMID:GST-pi gene-transduced hematopoietic progenitor cell transplantation overcomes the bone marrow toxicity of cyclophosphamide in mice. 1095 1

The secreted phosphoprotein osteopontin (OPN), when immobilized on a surface, supports cell adhesion, prevents apoptosis of endothelial cells, and is a ligand for the alpha(v)beta(3) integrin, which is important in endothelial cell biology and neovascularization. OPN synthesized by tumor cells stimulates tumor growth, but the mechanism by which the protein acts remains unclear. One possibility, therefore, is that OPN may exert its effects on tumor growth by enhancing angiogenesis. While OPN is found at high levels in bone, where it is a component of the mineralized matrix, we have asked here whether OPN present in tumors is similarly extracellular matrix associated. We have shown that OPN is detectable in tumor extracts and in serum of tumor-bearing mice, and that the protein in tumors and in serum can be synthesized by both tumor and the host cells. Biochemical fractionation of tumor tissue confirmed that there is little if any association of OPN with the insoluble fraction. Immunochemical analysis of murine mammary tumors shows no co-localization of OPN with the extracellular matrix, identified by laminin staining. Ras-transformed cells in culture produce abundant OPN, however, the protein was found to be associated with the cell fraction but not with the matrix fraction. An enzyme-linked immunosorbent assay was used to demonstrate that OPN in conditioned medium from these cells fails to associate with extracellular matrix components, including laminin and fibronectin, in vitro. Recombinant OPN (GST-OPN) when coated onto a plastic surface can support human umbilical vein endothelial cell adhesion, suppressing apoptosis and allowing cell cycle progression, at concentrations from 1 to 50 microg/ml. Soluble GST-OPN in the same concentration range has no effect on HUVECs held in suspension. Thus, we conclude that OPN associated with tumors is primarily soluble, and that soluble OPN can neither support endothelial cell proliferation nor prevent apoptosis of these cells in the absence of adhesion.
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PMID:Tumor-derived osteopontin is soluble, not matrix associated. 1174 94

The interaction of cells with the extracellular matrix (ECM) form of fibronectin (FN) triggers changes in growth, migration, and cytoskeletal organization that differ from those generated by soluble FN. As cells deposit and remodel their FN matrix, the exposure of new epitopes may serve to initiate responses unique to matrix FN. To determine whether a matricryptic site within the III1 module of FN modulates cell growth or cytoskeletal organization, a recombinant FN with properties of matrix FN was constructed by directly linking the cryptic, heparin-binding COOH-terminal fragment of III1 (III1H) to the integrin-binding III8-10 modules (glutathione-S-transferase [GST]-III1H,8-10). GST-III1H,8-10 specifically stimulated increases in cell growth and contractility; integrin ligation alone was ineffective. A construct lacking the integrin-binding domain (GST-III1H,2-4) retained the ability to stimulate cell contraction, but was unable to stimulate cell growth. Both GST-III1H,2-4 and matrix FN colocalized with caveolin and fractionated with low-density membrane complexes by a mechanism that required heparan sulfate proteoglycans. Disruption of caveolae inhibited the FN- and III1H-mediated increases in cell contraction and growth. These data suggest that a portion of ECM FN partitions into lipid rafts and differentially regulates cytoskeletal organization and growth, in part, through the exposure of a neoepitope within the conformationally labile III1 module.
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PMID:A cryptic fragment from fibronectin's III1 module localizes to lipid rafts and stimulates cell growth and contractility. 1210 89

Members of the Rho GTPase family are key regulatory molecules that link surface receptors to the organization of the actin cytoskeleton. It is now well established that these small GTPases are also crucial for neuronal morphogenesis and connectivity. Moreover, mutations in ARHGEF6 (also known as alphaPIX or Cool-2 ), encoding a Rac1/Cdc42-specific guanine nucleotide exchange factor, have been implicated in X-linked mental retardation. In an attempt to get insight into the biological function of ARHGEF6 and the upstream signaling cascades leading to its activation, we used the full-length coding region of ARHGEF6 as bait in yeast-two hybrid screens and identified PARVB (beta-parvin or affixin) as a novel binding partner. The interaction was confirmed by co-immunoprecipitation and GST pull-down. We showed by immunofluorescence that ARHGEF6 and PARVB co-localize at the cell periphery to lamellipodia and ruffles in well-spread and actively spreading cells adhered to fibronectin. In addition, interaction of ARHGEF6 to ARHGEF7 (betaPIX or Cool-1), a close homolog of ARHGEF6, was confirmed. In in vivo assays, two ARHGEF6 mutations identified previously in patients with X-linked non-specific mental retardation, ARHGEF6 deltaaa56-83 and deltaaa396-776, abolished interaction of ARHGEF6 to PARVB. Binding between ARHGEF6 and ARHGEF7 was not affected by ARHGEF6 deltaaa56-83 but did not occur with ARHGEF6 deltaaa396-776. These data suggest that both the N-terminal calponin homology (CH) and C-terminal coiled-coil domains are necessary for the ARHGEF6-PARVB binding. In contrast, it seems that only the coiled-coil domain is required for the interaction and heterodimerization of ARHGEF6 and ARHGEF7. PARVB is known to interact with integrin-linked kinase (ILK) and is involved in the early stage of cell-substrate interaction through integrins. The identification of PARVB as an ARHGEF6 interacting partner together with the co-localization of ARHGEF6 and ILK in spreading cells suggest that ARHGEF6 is involved in integrin-mediated signaling leading to activation of the GTPases Rac1 and/or Cdc42.
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PMID:Interaction of alphaPIX (ARHGEF6) with beta-parvin (PARVB) suggests an involvement of alphaPIX in integrin-mediated signaling. 1249 96

CLCA (chloride channel, calcium-activated) proteins are novel pulmonary vascular addresses for blood-borne, lung-metastatic cancer cells. They facilitate vascular arrest of cancer cells via adhesion to beta4 integrin and promote early, intravascular, metastatic growth. Here we identify the interacting binding domains of endothelial CLCA proteins (e.g. hCLCA2, mCLCA5, mCLCA1, and bCLCA2) and beta4 integrin. Endothelial CLCAs share a common beta4-binding motif (beta4BM) in their 90- and 35-kDa subunits of the sequence F(S/N)R(I/L/V)(S/T)S, which is located in the second extracellular domain of the 90-kDa CLCA and near the N terminus of the 35-kDa CLCA, respectively. Using enzyme-linked immunosorbent, pull-down, and adhesion assays, we showed that glutathione S-transferase fusion proteins of beta4BMs from the 90- and 35-kDa CLCA subunits bind to the beta4 integrin in a metal ion-dependent manner. Fusion proteins from fibronectin and the integrins beta1 and beta3 served as negative controls. beta4BM fusion proteins competitively blocked the beta4/CLCA adhesion and prevented lung colonization of MDA-MB-231 breast cancer cells. A disrupted beta4BM in hCLCA1, which is not expressed in endothelia, failed to interact with beta4 integrin. The corresponding CLCA-binding domain of the beta4 integrin is localized to the specific determining loop (SDL). Again enzyme-linked immunosorbent, pull-down, and adhesion assays were used to confirm the interaction with CLCA proteins using a glutathione S-transferase fusion protein representing the C-terminal two-thirds of beta4 SDL (amino acids 184-203). A chimeric beta4 integrin in which the indicated SDL sequence had been replaced with the corresponding sequence from the beta1 integrin failed to bind hCLCA2. The dominance of the CLCA ligand in beta4 activation and outside-in signaling is discussed in reference to our previous report that beta4/CLCA ligation elicits selective signaling via focal adhesion kinase to promote metastatic growth.
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PMID:The interacting binding domains of the beta(4) integrin and calcium-activated chloride channels (CLCAs) in metastasis. 1451 19


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