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)

We have identified the major enzymatic activity responsible for the S-adenosyl-L-methionine-dependent methylation of arginine residues (EC 2.1.1.23) in proteins of the yeast Saccharomyces cerevisiae. The RMT1 (protein-arginine methyltransferase), formerly ODP1, gene product encodes a 348-residue polypeptide of 39.8 kDa that catalyzes both the NG-mono- and NG, NG-asymmetric dimethylation of arginine residues in a variety of endogenous yeast polypeptides. A yeast strain in which the chromosomal RMT1 gene was disrupted is viable, but the level of NG,NG-[3H]dimethylarginine residues detected in intact cells incubated with S-adenosyl-L-[methyl-3H]methionine is reduced to less than 15% of the levels found in the parent strain, while the NG-[3H]monomethylarginine content is reduced to less than 30%. We show that soluble extract from parent cell, but not from mutant rmt1 cells, catalyzes the in vitro methylation of endogenous polypeptides of 55, 41, 38, 34, and 30 kDa. The hypomethylated form of these five polypeptides, as well as that of several others, can be mono- and asymmetrically dimethylated by incubating the mutant rmt1 extract with a purified, bacterially produced, glutathione S-transferase-RMT1 fusion protein and S-adenosyl-L-[methyl-3H]methionine. This glutathione S-transferase-RMT1 fusion protein is also able to methylate a number of mammalian polypeptides including histones, recombinant heterogeneous ribonucleoprotein A1, cytochrome c, and myoglobin, but cannot methylate myelin basic protein. RMT1 appears to be a yeast homolog of a recently characterized mammalian protein-arginine methyltransferase whose activity may be modulated by mitotic stimulation of cells.
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PMID:The predominant protein-arginine methyltransferase from Saccharomyces cerevisiae. 864 69

Transforming growth factor-alpha (TGF-alpha) and hepatocyte growth factor (HGF) are strong hepatocyte mitogens and important regulators of liver regeneration. The TGF-alpha receptor EGFr appears primarily to mediate a proliferative signal, whereas mitogenic, motogenic, and morphogenic effects have been attributed to activation of the HGF receptor Met. We have studied the localization of Met and EGFr in normal and carcinogen-treated rat livers. Oval cells and preneoplastic lesions were induced by diethylnitrosamine initiation, followed by promotion with 2-acetylaminofluorene combined with a partial hepatectomy. Different liver cell populations and their receptor expression were characterized by two-color immunofluorescence and confocal laser scanning microscopy. Hepatocytes were detected by keratin K8 staining, and oval cells and bile ducts were recognized by keratin K19 expression. Enzyme-altered preneoplastic lesions ere identified by expression of placental glutathione S-transferase (GST-pi). Staining for these cellular markers was combined with immunodetection of EGFr and Met. Normal liver exhibited strong staining for EGFr in hepatocytes, whereas blood vessels, bile ducts, and some sinusoidal cells were Met-positive. In carcinogen-treated livers, oval cells showed Met but not EGFr immunostaining. GST-pi-positive foci displayed EGFr immunostaining at a similar intensity as surrounding hepatocytes, whereas Met was not detected. Our data indicate that putative liver cells (oval cells) have a growth receptor phenotype similar to that of bile ducts, whereas preneoplastic live lesions appear hepatocyte-like. These results indicate that the preferential proliferation of preneoplastic liver lesions compared to surrounding hepatocytes is not associated with an altered EGFr or Met phenotype.
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PMID:Differential distribution of Met and epidermal growth factor receptor in normal and carcinogen-treated rat liver. 864 82

Xenobiotic metabolism can be influenced by various nutritional factors, including protein. In the present study, we have examined the effect of dietary protein (casein) levels on the ability of rat liver S9 to activate the promutagens aflatoxin B1 (AFB), 2-aminoanthracene (2AN) and benzo[a]pyrene (BAP) in strain TA98 using the spiral Salmonella mutagenicity assay. S9s were derived from individual male F344 rats fed for 6 weeks on semisynthetic diets containing 8%, 12% or 22% methionine-supplemented casein as the sole source of protein (diets were made isocaloric by adjusting the corn starch content). Rats were housed in large, raised-bed cages by groups of three per diet. S9 activation mixtures were prepared at 5 mg of S9 protein/ml of S9 mix. Slopes from the linear portions of the mutagenicity dose-response curves were analyzed by ANOVA comparisons. Assays used to elucidate the phase I activities of microsomal preparations were cytochrome P450 content, cytochrome-c reductase activity, flavin-containing monooxygenase activity, 7-ethoxyresorufin O-deethylation (EROD) activity, N-demethylation of benzphetamine and para-nitrophenol O-deethylation. Phase II activities in cytosolic preparations were assayed by estimation of glutathione (GSH) content and glutathione S-transferase activity through metabolism of 1-chloro-2,4-dinitrobenzene (CDNB). Increased levels of dietary casein increased liver wet weights and decreased the ability of the S9 to activate 2AN. Dietary casein levels did not influence the S9-mediated activation of BAP; and consistent but nonsignificant increases in activation of AFB were produced by S9 from animals fed the 22% casein diet. The phase I and phase II activities measured here were not altered significantly by dietary casein levels; thus, other, more specific enzymatic activities may account for the mutagenesis data. These results illustrate the complex interaction between dietary levels of casein and promutagen activation mechanisms, which prevents drawing broad generalizations regarding the influence of dietary casein levels on the capacity of hepatic S9s to activate promutagens.
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PMID:Effect of dietary casein levels on activation of promutagens in the spiral Salmonella mutagenicity assay. I. Studies with noninduced rat liver S9. 864 64

In the previous study (Mutation Res., this issue), we showed that increased levels of dietary casein as the sole protein source for male F344 rats decreased the ability of the uninduced liver S9s to activate 2-aminoanthracene (2AN) to a mutagen in strain TA98 using the spiral Salmonella mutagenicity assay. No effects of dietary casein levels were noted for the ability of uninduced liver S9s to activate the promutagens aflatoxin B1 (AFB) and benzo[a]pyrene (BAP). In the present study, we have extended this study to include liver S9s induced with either Aroclor 1254, phenobarbital or 3-methylcholanthrene (3MC). S9s were derived from individual male F344 rats fed for 6 weeks on semisynthetic diets containing 8%, 12% or 22% methionine-supplemented casein as the sole source of protein (diets were made isocaloric by adjusting the corn starch content). Rats were housed in large, raised-bed cages by groups of three/diet/inducing agent. S9 activation mixtures were prepared at 5 mg of S9 protein/ml of S9 mix. Slopes from the linear portions of the mutagenicity dose-response curves were analyzed by ANOVA comparisons. Assays used to elucidate the phase I activities of microsomal preparations were cytochrome P-450 content, cytochrome-c reductase activity, flavin-containing monooxygenase activity, 7-ethoxyresorufin O-deethylation (EROD) activity, N-demethylation of benzphetamine, and para-nitrophenol O-deethylation. Phase II activities were assayed by estimating glutathione (GSH) content and measuring the metabolism of 1-chloro-2,4-dinitrobenzene (CDNB) by glutathione S-transferase in cytosolic preparations. None of the phase I or phase II endpoints were significantly affected by dietary casein levels. In general, increasing levels of dietary casein resulted in increased body and liver wet weight and amount of S9 protein. Aroclor-induced S9s from rats fed the 22% or 12% casein diet were most effective at activating AFB, depending on the lot of Aroclor used for induction; these divergent results were replicated with two groups of rats for each lot of Aroclor. The observed differences between Aroclor lots are assumed to arise from variation in the mix of PCB isomers. The Aroclor-induced S9s did not exhibit any casein-related effects for the activation of BAP or 2AN. For 3MC-induced S9s, the 12% casein diets produced S9s with the highest ability to activate AFB and BAP when standardized for protein content. Phenobarbital-induced S9s did not demonstrate any dietary casein-related effects on the activation of the three model promutagens. These results illustrate the complex interaction between dietary levels of casein, enzyme inducing agent and promutagen.
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PMID:Effect of dietary casein levels on activation of promutagens in the spiral Salmonella mutagenicity assay. II. Studies with induced rat liver S9. 864 65

The TIS21 immediate-early gene and leukemia-associated BTG1 gene encode proteins with similar sequences. Two-hybrid analysis identified a protein that interacts with TIS21 and BTG1. Sequence motifs associated with S-adenosyl-L-methionine binding suggested this protein might have methyltransferase activity. A glutathione S-transferase (GST) fusion of the putative methyltransferase modifies arginine residues, in appropriate protein substrates, to form NG-monomethyl and NG,NG-dimethylarginine (asymmetric). We term the protein- arginine N-methyltransferase (EC 2.1.1.23) gene "PRMT1, " for protein-arginine methyltransferase 1. GST-TIS21 and GST-BTG1 fusion proteins qualitatively and quantitatively modulate endogenous PRMT1 activity, using control and hypomethylated RAT1 cell extracts as methyl-accepting substrates. PRMT1 message appears ubiquitous, and is constitutive in mitogen-stimulated cells. Modulation of PRMT1 activity by transiently expressed regulatory subunits may be an additional mode of signal transduction following ligand stimulation.
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PMID:The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a protein-arginine N-methyltransferase. 866 46

A gene encoding a new heat shock protein that may function as a molecular chaperone for the retinoblastoma protein (Rb) was characterized. The cDNA fragment was isolated by using the yeast two-hybrid system and Rb as bait. The open reading frame of the longest cDNA codes for a protein with substantial sequence homology to members of the hsp90 family. Antibodies prepared against fusions between glutathione S-transferase and portions of this new heat shock protein specifically recognized a 75-kDa cellular protein, hereafter designated hsp75, which is expressed ubiquitously and located in the cytoplasm. A unique LxCxE motif in hsp75, but not in other hsp90 family members, appears to be important for binding to the simian virus 40 T-antigen-binding domain of hypophosphorylated Rb, since a single mutation changing the cysteine to methionine abolishes the binding. In mammalian cells, Rb formed complexes with hsp75 under two special physiological conditions: (i) during M phase, when the envelope that separates the nuclear and cytoplasmic compartments broke down, and (ii) after heat shock, when hsp75 moved from its normal cytoplasmic location into the nucleus. In vitro, hsp75 had a biochemical activity to refold denatured Rb into its native conformation. Taken together, these results suggest that Rb may be a physiological substrate for the hsp75 chaperone molecule. The discovery of a heat shock protein that chaperones Rb identifies a mechanism, in addition to phosphorylation, by which Rb is regulated in response to progression of the cell cycle and to external stimuli.
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PMID:A new member of the hsp90 family of molecular chaperones interacts with the retinoblastoma protein during mitosis and after heat shock. 875 26

The alpha-, pi-, and mu-class glutathione S-transferases utilize a hydrogen bond between a conserved tyrosine and glutathione (GSH) to stabilize the nucleophilic thiolate anion, as Tyr-OH...-SG. This hydrogen bond is critical for efficient detoxication catalysis. The detailed structure of this hydrogen bond, however, is controlled by active site features which are not conserved across class boundaries. The alpha-class GST A1-1 has a cluster of aromatic residues on one side of the ring of the catalytic tyrosine, Tyr-9. Also, a hydrophobic Met-16 side chain is packed against the edge of the ring of Tyr-9. Molecular modeling and ab initio calculations suggested that substitution of Phe-220 with tyrosine could generate an aromatic on-face hydrogen bond (pi...HO-Ar) between the ring of Tyr-9 and the hydroxyl group of Tyr-220, and this would lower the pKa of enzyme-bound GSH. Therefore, Phe-220 was replaced by Tyr in the rat A1-1 isozyme. Also, Met-16 was replaced by Thr in order to investigate the effect of a hydrogen bond donor at the Tyr-9 ring edge. UV spectroscopic titration of GST.GSH and steady-state kinetic analysis indicate that substitution of Tyr at Phe-220 results in a decrease of the pKa of the cofactor, whereas substitution of Met-16 with Thr results in an increase of this pKa. Also, the pKa of Tyr-9 in the absence of substrates was determined directly by fluorescence titration. Substitutions F220Y and M16T resulted in a decrease of 0.5 pKa unit and an increase of 0.6 pKa unit, respectively. Together, these results indicate that a weak hydrogen bond between the engineered Tyr-220 side chain and the aromatic ring face of the catalytic Tyr-9 decreases the pKa of GSH and Tyr-9, and this alters the pH dependence of the enzymatic reaction.
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PMID:Rational modulation of the catalytic activity of A1-1 glutathione S-transferase: evidence for incorporation of an on-face (pi...HO-Ar) hydrogen bond at tyrosine-9. 881 Aug 97

VP39 is a bifunctional vaccinia virus protein that acts as both a cap- dependent 2'-O-Methyltransferase and a poly(A) polymerase processivity factor. An analysis of C-terminal truncation mutants of a GST-VP39 fusion protein indicated the presence of a protease-sensitive C-terminal "tail" 36-43 amino acids in length that is non-essential for VP39 function. Fourteen new VP39 pointmutants, containing either single or multiple-clustered amino acid substitutions, were expressed in Escherichia coli. Of the eight that retained either one or both of the activities of VP39, seven were specifically methyltransferase-defective. None was specifically defective in adenylyltransferase stimulation. The nature of the methyltransferase defects in 10 of the methyltransferase-specific defectives, identified both herein and in a previous study (Schnierle BS, Gershon PD, Moss B, 1994, J biol Chem 269:20700-20706), was investigated using two novel substrate-binding assays. Three of the mutants (and possible a fourth), whose lesions were juxtaposed and centrally located within VP39, exhibited anomalous S-adenosyl-(L)-methionine (AdoMet) binding behavior, identifying residues important for AdoMet binding and possible also for catalysis. A surface plasmon resonance-based assay measured the interaction of VP39 with uncapped and 5'-cap 0-terminated oligo(A). A cap 0- dependent association-rate enhancement was observed for wild-type VP39 and 4 of the 10 mutant proteins. Two others were identified as defective in cap binding, and a third as partially defective. The lesions within the latter three mutants were closely apposed, and located toward the N-terminus of VP39. We have thus identified regions of VP39 important for interaction with its two substrates for cap-dependent methyltransferase activity: AdoMet and cap 0.
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PMID:Methyltransferase-specific domains within VP-39, a bifunctional protein that participates in the modification of both mRNA ends. 884

Human CD7 is a 40 kDa protein expressed on thymocytes, early T, B, NK and myeloid lineage cells in bone marrow, and on mature T and NK cells. Previous studies suggested human CD7 may be involved in T and NK cell activation and/or adhesion, and that CD7-mediated cell activation may be transduced via the lipid kinase phosphatidylinositol 3-kinase (Pi3-kinase), a heterodimeric cytosolic protein consisting of an 85 kDa adaptor subunit that is coupled to a 110 kDa catalytic subunit. It has recently been shown that a sequence motif present in the cytoplasmic tall of both human and mouse CD7 bound with high affinity to recombinant SH2 domains present in the p85 subunit of Pi3-kinase. In this work, we used co-precipitation with anti-CD7 mAb 3A1 and recombinant p85 SH2-GST fusion proteins and peptide competition analysis to demonstrate that the cytoplasmic tail of CD7 interacts with a functional Pi3-kinase via the pTyr-X-X-Met motif. Furthermore, we show that cross-linking of CD7 markedly increased the amount of Pi3-kinase activity associated with CD7. The interaction of CD7 with the Pi3-kinase signal transduction pathway provides a mechanism for the previously observed functional responses attributed to CD7-mediated T and NK cell activation.
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PMID:Functional association of CD7 with phosphatidylinositol 3-kinase: interaction via a YEDM motif. 891 88

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein causes cellular transformation by deregulating important cellular processes such as DNA repair, transcription, signal transduction, proliferation, and growth. Although it is clear that normal cell cycle control is deregulated during HTLV-1-induced cellular transformation, the effects of Tax on cell cycle control are not well understood. Flow cytometric analyses of human T cells indicate that cell cycle arrest in late G1, at or before the G1/S restriction point, by p16INK4a is relieved by Tax. Furthermore, Tax-dependent stimulation of 5-bromo-2'-deoxyuridine incorporation and transcriptional activation is inhibited by p16INK4a. This result suggests that p16INK4a is able to block Tax-dependent stimulation of DNA synthesis and cell cycle progression into S phase. In vitro binding assays with recombinant glutathione S-transferase fusion proteins and [35S]methionine-labeled proteins indicate that Tax binds specifically with p16INK4a but not with either p21cip1 or p27kip1. Furthermore, sequential immunoprecipitation assays with specific antisera and [35S]methionine-labeled cell lysates subsequent to coexpression with Tax and p16INK4a indicate that the two proteins form complexes in vivo. Immunocomplex kinase assays with cyclin-dependent kinase 4 antiserum indicate that Tax blocks the inhibition of cdk4 kinase activity by p16INK4a. This study identifies p16INK4a as a novel cellular target for Tax and suggests that the inactivation of p16INK4a function is a mechanism of cell cycle deregulation by Tax.
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PMID:Human T-cell leukemia virus type 1 Tax releases cell cycle arrest induced by p16INK4a. 903 27


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