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)

The vaccinia virus-encoded protein VP39 is a poly(A) polymerase subunit that stimulates the formation of long poly(A) tails as well as a cap-specific mRNA (nucleoside-2'-O-)-methyltransferase. We have carried out mutagenesis studies aimed at locating regions of VP39 which are important for these activities. The open reading frame encoding VP39 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. The affinity-purified protein had both mRNA modification activities, before and after removal of the glutathione S-transferase domain. Truncation, charge cluster-->Ala scanning, and Cys-->Ser substitution mutations of VP39 were made, and the proteins were synthesized, purified, and analyzed. Deletion of the RNA binding domain, experimentally localized within the carboxyl-terminal 112 amino acids, resulted in the loss of both mRNA modification activities. Eleven of the 21 charge cluster-->Ala mutated proteins had low to nondetectable methyltransferase activity. Four of those 11 also lacked adenylyl-transferase stimulatory function, whereas the remainder had amino acid substitutions that selectively affected methyltransferase activity. However, no mutated proteins lacking adenylyltransferase stimulatory function but possessing methyltransferase activity were isolated by the procedures used. Neither of the 2 cysteine residues in VP39 was necessary for either mRNA modification activity.
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PMID:Mutational analysis of a multifunctional protein, with mRNA 5' cap-specific (nucleoside-2'-O-)-methyltransferase and 3'-adenylyltransferase stimulatory activities, encoded by vaccinia virus. 805 Nov 70

The cloning and overexpression of the MspI DNA methyltransferase as a functional fusion with glutathione S-transferase is described. The fusion enzyme retains full biological activity and has been used to investigate the interaction of substrates and inhibitors with MspI DNA methyltransferase. The fusion enzyme has been purified to homogeneity in a single step on GSH-agarose and is free from contaminating exonuclease activity. The enzyme can be photolabelled with S-adenosyl-L-methionine and the level of incorporation of label is enhanced by the presence of a nonspecific DNA duplex. In the presence of a cognate oligodeoxynucleotide, no photolabelling was observed since methyl transfer occurs instead. The inclusion of a mechanism-based inhibitor of C-5 deoxycytidine DNA methylation (an oligodeoxynucleotide containing the base 2-pyrimidinone-1-beta-D-2'-deoxyribofuranoside in the position of the deoxycytidine to which methyl addition occurs), which is thought to form a covalent interaction with the reactive cysteine of such enzymes, led to an enhancement of S-adenosyl-L-methionine photolabelling which suggests that, in contrast with results obtained with EcoRII DNA methyltransferase [Som and Friedman (1991) J. Biol. Chem. 266, 2937-2945], methylcysteine is not the photolabelled product. The implications of the results obtained with this mechanism-based inhibitor are discussed with respect to other C-5-specific DNA methyltransferases. Gel-retardation assays in the presence of cognate oligodeoxynucleotides that contain the reactive pyrimidinone base in place of the deoxycytidine target base are described. These demonstrate that most probably a stable covalent bond is formed between the methyltransferase and this oligodeoxynucleotide. However, the alternative of extremely tight non-covalent binding cannot be rigorously excluded. Furthermore, the results from these experiments indicate that the reaction mechanism proceeds in a manner similar to that of HhaI DNA methyltransferase with sequence-specific DNA binding being followed by addition of S-adenosyl-L-methionine and concomitant isomerization of the ternary complex leading to methyl transfer. S-Adenosyl-L-homocysteine appears to inhibit the reaction pathway as a result of either competition with the methyl donor and potentiation of a high-affinity interaction between the enzyme and DNA in an abortive ternary complex or through an allosteric interaction.
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PMID:Determination of the order of substrate addition to MspI DNA methyltransferase using a novel mechanism-based inhibitor. 848 30

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

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

Grapevine virus B (GVB) is a tentative member of the genus Trichovirus. The 5'-terminal region of the RNA genome of GVB comprises 5437 nucleotides and has been sequenced by the dideoxynucleotide chain termination method. Evidence was obtained that the RNA is capped. Two putative open reading frames (ORFs) were identified. ORF 1 coded for a 194.7 kDa polypeptide with conserved motifs of replication-related proteins of positive-strand RNA viruses (i.e. methyltransferase, helicase and RNA-dependent RNA polymerase, in that order from the N to the C terminus). ORF 2 encoded a 20 kDa polypeptide that did not show any significant sequence homology with protein sequences from the databases. The biological function of this polypeptide was not determined. Although the 20 kDa product was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and an antiserum produced, it could not be identified in GVB-infected plant tissue extracts. The GVB genome had the same size as that of apple chlorotic leaf spot virus (ACLSV), the type species of the genus Trichovirus, but differed substantially in the number (five compared to three), size and order of genes. Differences existed also in the extent of sequence homology between polymerases, which did not cluster together in tentative phylogenetic trees. The results of this study show that definitive and tentative trichovirus species differ molecularly to an extent that may warrant a taxonomic revision of the genus.
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PMID:The nucleotide sequence and genomic organization of grapevine virus B. 888 2

The type I DNA methyltransferase M.EcoR124I consists of two methylation subunits (HsdM) and one DNA recognition subunit (HsdS). When expressed independently, HsdS is insoluble, but this subunit can be obtained in soluble form as a GST fusion protein. We show that the HsdS subunit, even as a fusion protein, is unable to form a discrete complex with its DNA recognition sequence. When HsdM is added to the HsdS fusion protein, discrete complexes are formed but these are unable to methylate DNA. The two complexes formed correspond to species with one or two copies of the HsdM subunit, indicating that blocking the N-terminus of HsdS affects one of the HsdM binding sites. However, removal of the GST moiety from such complexes results in tight and specific DNA binding and restores full methylation activity. The results clearly demonstrate the importance of the HsdM subunit for DNA binding, in addition to its catalytic role in the methyltransferase reaction.
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PMID:DNA binding and subunit interactions in the type I methyltransferase M.EcoR124I. 902 8

Resistance of tumor cells to chemotherapeutic drugs can not only be caused by treatment with antineoplastic agents but also by radiotherapy. The aim of this study was to analyze whether ionizing radiation can influence the mRNA expression of proteins which have been found to be involved in drug resistance of tumor cells. Human tumor cell lines (MCF-7, LXF and Sk-Mel) were treated with single doses of irradiation (5, 10 and 20 Gy). The expression of the resistance related proteins glutathione S-transferase-pi (GST-pi), topoisomerase II alpha (Topo II), thymidylate synthase (TS), O6-methylguanine-DNA-methyltransferase (MGMT), P-glycoprotein (Pgp), glutathione peroxidase (GPX) multidrug resistance-associated protein (MRP) and also of the heat-shock protein 70 (HSP 70) were determined at the mRNA level during the time interval from 1.5 to 72 h post-irradiation and compared with their corresponding controls. We also examined whether a relationship exists between these proteins and the proliferative activity (histone 3, Ki-67, statin) of the cells. We found that exposure of MCF-7, LXF and Sk-Mel cells to ionizing radiation increases the expression of the mRNA of GST-pi. Topo II, TS, HSP 70 and proliferation markers were also altered by exposure to ionizing radiation, but there was no common response of the three cell lines. No significant changes were observed in the expression of MGMT, Pgp, GPX and MRP after radiation treatment. Drug resistance tests revealed that irradiated MCF 7 cells were less sensitive to doxorubicin than non-irradiated control cells. Our results indicate that ionizing irradiation modifies the expression of some proteins involved in drug resistance and the response of MCF 7 cells to doxorubicin and may, therefore, play a role in clinical drug response.
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PMID:Messenger RNA expression of resistance factors in human tumor cell lines after single exposure to radiation. 941 87

Protein arginine methyltransferase was recently identified to be associated with some proteins in signal transduction pathways. N-Arginine methylation in RNA binding proteins with arginine- and glycine-rich RGG motifs is known to be the major protein methylation in cells. Considering that arginine methylation might be involved in certain human disorders, we used human lymphoblastoid cells that can be easily prepared from lymphocytes as a model system to study the methylation. Lymphoblastoid cells grown in the presence of 20 microM indirect methyltransferase inhibitor adenosine dialdehyde (AdOx) for 72 h appeared to accumulate high levels of hypomethylated proteins for the endogenous protein methyltransferase or recombinant glutathion S-transferase-fused yeast arginine methyltransferase (RMT1). Analysis of methyl-accepting polypeptides in AdOx-treated lymphoblastoid cells by SDS-PAGE and fluorography showed that many polypeptides between 29,000 and 90,000 Da were methylated by the endogenous methyltransferase. A few polypeptides could be methylated to a higher extent upon the addition of yeast GST-RMT1 fusion protein. A peptide (GGRGRGGGF) could compete for the majority of the methyl-accepting protein substrates in the AdOx-treated lymphoblastoid cell extracts, whether or not exogenous yeast RMT1 was included in the reaction. When the arginine residues in the peptide were replaced by lysine, no competition was observed. The results indicated that the protein methyl acceptors in lymphoblastoid cells share similar RGG motifs and that arginine residues should be the site of methylation.
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PMID:Protein N-arginine methylation in adenosine dialdehyde-treated lymphoblastoid cells. 950 Aug 43

Carcinogen-resistant inbred DRH rats developed from the Donryu strain showed a remarkably low incidence of liver tumors when they were fed diets containing hepatocarcinogens such as 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). In this work, we examined various characteristics of male DRH and Donryu rats during 3'-Me-DAB administration for 8 weeks. 32P-Postlabeling analysis showed that essentially similar levels of DNA-adducts were generated by the metabolites of 3'-Me-DAB in the livers of these two strains of rats at several time points. However, both GADD45 (growth arrest and DNA damage-inducible) and O6-methylguanine methyltransferase (putatively DNA damage-inducible) mRNA levels were increased significantly in Donryu rat livers, but were increased to a lesser extent in DRH rats. [3H]Thymidine incorporation into hepatic DNA began to increase around 10 to 20 days after the start of 3'-Me-DAB administration in Donryu rats probably due to DNA repair, while no significant change occurred in DRH rats under the same conditions. Furthermore, inductions of heme oxygenase (due to degradation of heme-proteins) and hepatocyte growth factor (HGF; cell death and regeneration of hepatocytes) mRNAs were greater in Donryu rat livers than those of DRH, suggesting that the former were more sensitive to cytotoxic effects of 3'-Me-DAB than the latter. Another remarkable difference observed between these two strains was the significant induction of cytochrome P-450 2E1 mRNA in Donryu rat livers; this may contribute to the generation of reactive oxygen intermediates. Finally, increases of glutathione S-transferase (P-form) and gamma-glutamyltranspeptidase mRNAs as marker enzymes of preneoplastic changes of hepatocytes were clearly seen only in Donryu rat livers at 6 to 8 weeks after the start of 3'-Me-DAB administration. These results indicate that the different susceptibility to hepatocarcinogenesis between these two strains of rats may arise from events other than the DNA adduct formation.
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PMID:Different responses other than the formation of DNA-adducts between the livers of carcinogen-resistant rats (DRH) and carcinogen-sensitive rats (Donryu) to 3'-methyl-4-dimethylaminoazobenzene administration. 976 15

Recent evidence suggests that tumor cells may release DNA into the circulation, which is enriched in the serum and plasma, allowing detection of ras and p53 mutations and microsatellite alterations in the serum DNA of cancer patients. We examined whether aberrant DNA methylation might also be found in the serum of patients with non-small cell lung cancer. We tested 22 patients with non-small cell lung cancer using methylation-specific PCR, searching for promoter hypermethylation of the tumor suppressor gene p16, the putative metastasis suppressor gene death-associated protein kinase, the detoxification gene glutathione S-transferase P1, and the DNA repair gene O6-methylguanine-DNA-methyltransferase. Aberrant methylation of at least one of these genes was detected in 15 of 22 (68%) NSCLC tumors but not in any paired normal lung tissue. In these primary tumors with methylation, 11 of 15 (73%) samples also had abnormal methylated DNA in the matched serum samples. Moreover, none of the sera from patients with tumors not demonstrating methylation was positive. Abnormal promoter methylation in serum DNA was found in all tumor stages. Although these results need to be confirmed in larger studies and in other tumor types, detection of aberrant promoter hypermethylation of cancer-related genes in serum may be useful for cancer diagnosis or the detection of recurrence.
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PMID:Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. 989 87


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