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)

A series of overlapping fragments spanning the entire coding sequence of the gH gene of murine cytomegalovirus (MCMV) were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST) using the pGEX expression system. A region of antibody-binding was mapped to the NH2-terminus of glycoprotein H (gH) between amino acid residues 26 and 90 on the basis of the reactivity of GST-gH fusion proteins with polyclonal antibodies to MCMV in Western blot analysis. Antibodies to gH were generated in mice immunized with the GST-gH fusion protein SK and shown to react with an 87-kDa polypeptide present in virion particles which was conserved in MCMV isolates obtained from diverse locations. They also recognized the gH protein in MCMV-infected cells, as well as gH expressed in Chinese Hamster Ovary cells. The antibodies to gH had a significant ELISA titer but no neutralizing activity in vitro. The antibody response to the GST-gH fusion protein did not modify the level of MCMV replication in the spleens of mice.
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PMID:Expression of the glycoprotein H of murine cytomegalovirus and identification of an N-terminal antibody-binding region. 752 76

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor, which is important for the normal development of hematopoietic cells, melanoblasts, and germ cells. Autophosphorylation of c-kit receptor on tyrosine creates binding sites for cellular src homology 2 (SH2)-containing signaling molecules. The discovery of phosphotyrosine phosphatases that contain SH2 domains suggests roles for these molecules in growth factor signaling pathways. We found that Syp, a phosphotyrosine phosphatase widely expressed in all the tissues in mammals, associates with c-kit receptor after activation with its ligand, steel factor, in the factor-dependent cell line, M07e. Both NH2-terminal and COOH-terminal SH2 domains of Syp, made as glutathione S-transferase fusion proteins, were able to bind to the activated c-kit receptor in vitro. Furthermore, Syp became marginally phosphorylated on tyrosine upon c-kit receptor activation, and tyrosine-phosphorylated Syp was found to be complexed with Grb2 in steel factor-stimulated M07e cells. Direct binding between Syp and Grb2 was also observed in vitro. Last, Ras and Raf interacts in vitro as a result of steel factor-stimulated Ras activation. These results suggest that Syp may be an important signaling component downstream of the c-kit receptor and involved in activation of the Ras signaling pathway in hematopoietic cells.
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PMID:The ubiquitously expressed Syp phosphatase interacts with c-kit and Grb2 in hematopoietic cells. 752 81

Engagement of the T cell antigen receptor (TCR) results in activation of several tyrosine kinases leading to tyrosine phosphorylation of protein substrates and activation of multiple biochemical pathways. TCR-mediated activation of the src-family kinases, Lck and Fyn, results in tyrosine phosphorylation of the TCR zeta and CD3 chains. The site of phosphorylation in these chains is the tyrosine-based activation motif (TAM), a 15-16 amino acid module containing two tyrosine residues. Tyrosine-phosphorylated TAMs serve as targets for binding of the zeta-associated protein (ZAP-70) tyrosine kinase via its tandem SH2 domains. This binding correlates with activation of ZAP-70, a critical event in T cell activation. To further define the structural requirements for ZAP-70 interaction with the TCR, we developed a binding assay using immobilized glutathione S-transferase fusion proteins containing the NH2- and/or COOH-terminal SH2 domains of ZAP-70, and soluble synthetic peptides with the sequence of the cytoplasmic region of the TCR zeta chain (TCR zeta cyt) or individual TCR zeta and CD3 epsilon TAM motifs. Direct binding studies demonstrated that the tandem ZAP-70 SH2 domains bind phosphorylated, but not nonphosphorylated, TCR zeta cyt. The NH2-terminal ZAP-70 SH2 domain also binds to TCR zeta cyt but with 100-fold lower affinity. No binding was observed with the COOH-terminal ZAP-70 SH2 domain. Similar studies demonstrated that the ZAP-70 tandem SH2 domain can bind a TCR zeta 3 TAM peptide in which both tyrosine residues are phosphorylated: Little or no binding was observed with peptides phosphorylated at only one tyrosine residue, or a nonphosphorylated peptide. Binding of the tandem SH2 domains to the other two TCR zeta TAM peptides and to a CD3 epsilon TAM peptide was also observed. All four doubly tyrosine phosphorylated TAM peptides cross-compete with each other for binding to the tandem SH2 domains of ZAP-70. The affinity of these peptides for the tandem SH2 construct demonstrated a hierarchy of TAM zeta 1 > or = TAM zeta 2 > TAM epsilon > or = TAM zeta 3. The results provide further evidence that the ZAP-70 interaction with the TCR requires prior phosphorylation of both tyrosine residues within a TAM motif. Binding of ZAP-70 to phospho-TAMs is notable for the high level of cooperativity between the two SH2 domains, which individually demonstrate low affinity interaction with the ligand. The cooperativity ensures higher affinity for the doubly phosphorylated ligand. Affinity differences of as much as 30-fold indicates a significant specificity of interaction of ZAP-70 SH2 domains for different phospho-TAMs.
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PMID:ZAP-70 binding specificity to T cell receptor tyrosine-based activation motifs: the tandem SH2 domains of ZAP-70 bind distinct tyrosine-based activation motifs with varying affinity. 752 72

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid precursors. The phosphorylation of proteins at tyrosine residues is critical in the growth signaling induced by Epo. This mechanism is regulated by the activities of both protein-tyrosine kinases and protein tyrosine phosphatases. The discovery of phosphotyrosine phosphatases that contain SH2 domains suggests roles for these molecules in growth factor signaling pathways. We found that Syp, a phosphotyrosine phosphatase, widely expressed in all tissues in mammals became phosphorylated on tyrosine after stimulation with Epo in M07ER cells engineered to express high levels of human EpoR. Syp was complexed with Grb2 in Epo-stimulated M07ER cells. Direct binding between Syp and Grb2 was also observed in vitro. Furthermore, Syp appeared to bind directly to tyrosine-phosphorylated EpoR in M07ER cells. Both NH2-terminal and COOH-terminal SH2 domains of Syp, made as glutathione S-transferase fusion proteins, were able to bind to the tyrosine-phosphorylated EpoR in vitro. These results suggest that Syp may be an important signaling component downstream of the EpoR and may regulate the proliferation and differentiation of hematopoietic cells.
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PMID:Involvement of SH2-containing phosphotyrosine phosphatase Syp in erythropoietin receptor signal transduction pathways. 753 99

The retinoblastoma protein, Rb, is detected in extracts of monkey CV-1 cells complexed with Pur alpha, a sequence-specific single-stranded DNA-binding protein implicated in control of gene transcription and DNA replication. These complexes can be immunoextracted from cell lysates using monoclonal antibodies to either Pur alpha or Rb. The Pur alpha-Rb complexes contain a form of Pur alpha with extensive post-synthetic modification, as demonstrated following expression of Pur alpha cDNA fused to a 9-amino acid epitope tag. Human Pur alpha, expressed as a glutathione S-transferase fusion protein, specifically binds to the hypophosphorylated form of Rb with an affinity as high as that of SV40 large T-antigen. In the absence of DNA, glutathione S-transferase-Pur alpha binds to p56RB, an NH2-terminal-truncated Rb protein purified from Escherichia coli, containing the T-antigen binding domain, to form multimeric complexes. The single-stranded DNA Pur alpha recognition element disrupts these complexes. Conversely, high concentrations of p56RB prevent Pur alpha binding to DNA. Through use of a series of deletion mutants, the DNA binding activity of Pur alpha is localized to a series of modular amino acid repeats. Rb binding involves a Pur alpha region with limited homology to the Rb-binding region of SV40 large T-antigen. Binding of Pur alpha to p56RB, the COOH-terminal portion of Rb, is inhibited by a synthetic peptide containing the T-antigen Rb-binding motif.
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PMID:Association of human Pur alpha with the retinoblastoma protein, Rb, regulates binding to the single-stranded DNA Pur alpha recognition element. 759 47

The Bcl-2 protein is a suppressor of programmed cell death that homodimerizes with itself and forms heterodimers with a homologous protein Bax, a promoter of cell death. Expression of Bax in Saccharomyces cerevisiae as a membrane-bound fusion protein results in a lethal phenotype that is suppressible by co-expression of Bcl-2. Functional analysis of deletion mutants of human Bcl-2 in yeast demonstrated the presence of at least three conserved domains that are required to suppress Bax-mediated cytotoxicity, termed domains A (amino acids 11-33), B (amino acids 138-154), and C (amino acids 188-196). In vitro binding experiments using GST-Bcl-2 fusion proteins demonstrated that Bcl-2(delta B) and Bcl-2(delta C) deletion mutants had a markedly impaired ability to heterodimerize with Bax but retained the ability to homodimerize with wild-type Bcl-2. In contrast, Bcl-2(delta A) and an NH2-terminal deletion mutant Bcl-2(delta 1-82) retained Bax binding activity in vitro but failed to suppress Bax-mediated cytotoxicity in yeast. Sequences downstream of domain C in the region 197-218 also were shown to be required for Bax-binding in vitro and anti-death function in yeast. Analysis of Bcl-2/Bcl-2 homodimerization using both in vitro binding assays as well as a yeast two-hybrid method provided evidence in support of a head-to-tail model for Bcl-2/Bcl-2 homodimerization and revealed that sequences within the NH2-terminal A domain interact with a structure that requires the presence of both the carboxyl B and C domains in combination. In addition to further delineating structural features within Bcl-2 that are required for homo-dimerization, the findings reported here support the hypothesis that Bcl-2 promotes cell survival by binding directly to Bax but suggest that ability to bind Bax can be insufficient for anti-cell death function.
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PMID:Structure-function analysis of Bcl-2 protein. Identification of conserved domains important for homodimerization with Bcl-2 and heterodimerization with Bax. 774 46

A 24-kDa protein isolated by preparative gel electrophoresis from rat testes was reported by us as an active immunogen in rats. Anti-24-kDa antibodies inhibited murine sperm-oocyte binding in vitro. Here, we show similarity at the NH2 terminus shared by this protein purified on Sephadex G-75 followed by anion exchange high performance liquid chromatography with glutathione S-transferase (GST)-mu subunits. This protein purified by glutathione affinity chromatography also demonstrated similarity to GST-mu NH2 terminus in a 30-amino-acid overlap. Both proteins showed activity toward the GST substrate 1-chloro-2,4-dinitrobenzene (Km of 33 microM and 50 microM) which was inhibited by 17 beta-estradiol 3-sulfate. Antisera against both proteins recognized liver GST-mu on Western blots and sperm acrosome of multiple species immunocytochemically. Both antisera significantly inhibited in vitro fertilization of goat oocytes by sperm preincubated with them while anti-liver GST sera did not. GST activity was localized on rat sperm, seminiferous tubular fluid, and Sertoli cells. Seminiferous tubular fluid 24-kDa protein shared similarity to the NH2 terminus of GST-mu subunits in a 20-amino-acid overlap. Time-dependent accumulation of GST was detected in the spent culture medium of seminiferous tubules from rats of different ages suggesting secretion.
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PMID:A testicular protein important for fertility has glutathione S-transferase activity and is localized extracellularly in the seminiferous tubules. 779 68

The dihydrolipoyl acetyltransferase (E2) component of the mammalian pyruvate dehydrogenase complex forms a 60-subunit core in which E2's inner domain forms a dodecahedron shaped structure surrounded by its globular outer domains that are connected to each other and the inner domain by 2-3-kDa mobile hinge regions. Two of the outer domains are approximately 10 kDa lipoyl domains, an NH2-terminal one, E2L1, and, after the first hinge region a second one, E2L2. The pyruvate dehydrogenase kinase binds tightly to the lipoyl domain region of the oligomeric E2 core and phosphorylates and inactivates the pyruvate dehydrogenase (E1) component. We wished to determine whether lipoyl domain constructs prepared by recombinant techniques from a cDNA for human E2 could bind the bovine E1 kinase and, that being the case, to pursue which lipoyl domain the kinase binds. We also wished to gain insights into how a molecule of kinase tightly bound to the E2 core can rapidly phosphorylate 20-30 molecules of the pyruvate dehydrogenase (E1) component which are also bound to an outer domain of the E2 core. We prepared recombinant constructs consisting of the entire lipoyl domain region or the individual lipoyl domains with or without the intervening hinge region. Constructs were made and used both as free lipoyl domains and fused to glutathione S-transferase (GST). Using GSH-Sepharose to selectively bind GST constructs, tightly bound kinase was shown to rapidly transfer in a highly preferential way from intact E2 core to GST constructs containing the E2L2 domain rather than to ones containing only the E2L1 domain. GST-E2L2-kinase complexes could be eluted from GSH-Sepharose with glutathione. Delipoylation of E2L2 by treatment with lipoamidase eliminated kinase binding supporting a direct role of the lipoyl prosthetic group in this association. Transfer to and selective binding of the kinase by E2L2 but not E2L1 was also demonstrated with free constructs using a sucrose gradient procedure to separate the large E2 core from the various lipoyl domain constructs. E2L2 but not E2L1 increased the activity of resolved kinase by up to 43%. We conclude that the kinase selectively binds to the inner lipoyl domain of E2 subunits and that this association involves its lipoyl prosthetic group. We further suggest that transfer of tightly bound kinase between E2L2 domains occurs by a direct interchange mechanism without formation of free kinase (model presented).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Binding of the pyruvate dehydrogenase kinase to recombinant constructs containing the inner lipoyl domain of the dihydrolipoyl acetyltransferase component. 782 13

Human T-cell lymphotropic virus type I (HTLV-I) transactivator Tax augments transcription from three (cyclic AMP response element (CRE)-containing 21-bp repeats in the viral long terminal repeat and several other cis regulatory elements, including the NF-kappa B binding sites and the serum response element. Tax does not bind DNA directly; rather, it acts via cellular sequence-specific DNA binding proteins to stimulate transcription. We have shown recently that Tax forms multiprotein complexes with the heterodimeric and homodimeric forms of a ubiquitous cellular transcription factor, CREB (CRE binding protein). In vitro selection for preferred Tax-CREB binding sites indicates that the Tax-CREB complex exhibits greatly increased DNA recognition specificity and assembles preferentially on CRE motifs, TGACGT/C, flanked by long runs of G (5') and/or C (3') residues, as found in the HTLV-I 21-bp repeats. The indirect tethering of Tax to the 21-bp repeats via CREB is crucial for Tax transactivation. We now report the domain organization of Tax by characterizing its mutants. Tax mutants with alterations in the NH2 terminus, including three deletion mutants, Tax(6-353), Tax(21-353), and Tax(89-353), and two amino acid substitution mutants, M1 (H3S) and M7 (C29A, P30S), all failed to interact with CREB in vitro. In contrast, a short COOH-terminal deletion, Tax(1-319), and a Tax mutant with amino acid substitutions near the COOH end, M47 (L319R, L320S), were able to interact with CREB and the 21-bp repeats to assemble ternary Tax-CREB-DNA complexes. As demonstrated earlier, M1, M7, and M47 all failed to transactivate the HTLV-I long terminal repeat. Our data indicate that the defects in M1 and M7 result from an inability to interact with CREB. In contrast, the COOH-terminal mutations in M47 most likely inactivated the transactivation domain of Tax. As anticipated, a Tax mutant, M22 (G137A, L138S) which activated transcription from the 21-bp repeats with reduced capacity and was defective in trans activating the NF-kappa B binding sites, continued to interact with CREB in vitro, albeit with a lower level of efficiency. Finally, a glutathione S-transferase (GST)-Tax fusion protein with the GST moiety fused to the NH2 terminus of Tax failed to interact with CREB. Removal of the GST domain from GST-Tax by thrombin restores Tax's ability to assemble a ternary Tax-CREB-21-bp-repeat complex.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Distinct regions in human T-cell lymphotropic virus type I tax mediate interactions with activator protein CREB and basal transcription factors. 785 24

The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) associate to form a large inner core with a protruding structure composed of three globular domains connected by mobile linker regions. This exterior region of E2 includes two lipoyl domains which engage not only in the intermediate reactions of the complex but also have integral roles in the kinase-phosphatase regulatory interconversion of the pyruvate dehydrogenase (E1) component. To facilitate understanding of these roles, lipoyl domain constructs of the E2 component of human PDC were expressed as glutathione S-transferase (GST)-linked fusion proteins from plasmid inserts prepared by polymerase chain reaction procedures. The NH2-terminal lipoyl domain, E2L1, and the interior lipoyl domain, E2L2, are connected by a 30-amino-acid hinge region, H1. Constructs designed and expressed were E2L1(1-98), E2L1.H1(1-128), E2L2(120-233), E2H1.L2(98-233), and E2L1.H1.L2(1-233), where numbers in parentheses give the amino acid sequence for the portions of the E2 component incorporated into a construct. The domains were expressed in Escherichia coli with and without lipoate supplementation. GST constructs were purified to homogeneity by affinity chromatography and selectively released by thrombin treatment. Sequencing of insert DNAs and NH2-terminal sequencing confirmed that domains were produced as designed. Measurement of masses by electrospray mass spectrometry indicated that constructs with lipoylated, nonlipoylated, and octanoylated forms were produced when expression was with E. coli grown without lipoate supplementation and that fully lipoylated forms were produced upon lipoate supplementation. The lipoylation status was confirmed, following delipoylation with Enterococcus faecalis lipoamidase, by the expected decrease in mass and by the observation in native gel electrophoresis of a shift to a slower mobility (possibly less compact) form. Constructs were used in E1-catalyzed reductive-acetylation reaction in proportion to their degree of lipoylation and were effective substrates in a NADH-dependent dihydrolipoyl dehydrogenase reduction reaction. Thus, we have produced lipoyl domain constructs that can be employed in sorting the specific roles of E2L1 and E2L2 in facilitating catalytic and regulatory processes.
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PMID:Recombinant expression and evaluation of the lipoyl domains of the dihydrolipoyl acetyltransferase component of the human pyruvate dehydrogenase complex. 786 52


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