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)

To assess the possibility that two conserved amino acids (glutamine 90 and asparagine 137) in O6-methylguanine-DNA methyltransferase (MGMT) are involved in protein-substrate contact and/or discrimination between favored and non-favored substrates, families of proteins mutant at these two sites were expressed in alkyltransferase-deficient bacteria and analyzed for stability, ability to repair O6-methylguanine (MG)-containing DNA, and ability to differentially repair a preferred (MG-containing DNA) versus a non-preferred (free base MG) substrate. All seven proteins mutant at glutamine 90 (except a proline mutant) were stable in bacteria and repaired MG-containing DNA (> 50% of wild-type levels). A representative glutamine 90 mutant protein was not, however, significantly different from the wild-type protein in the preferential repair of MG-containing DNA versus MG free base. Of eight proteins mutant at asparagine 137, only glutamine and serine mutants repaired MG-containing DNA to any degree (8.5% and 0.8% of wild-type respectively) and only the glutamine mutant protein was detectable in bacterial sonicates by Western blot analysis. Alanine and leucine mutant alkyltransferases, inactive and unstable as non-fusion proteins, could, however, be stably expressed in bacteria as glutathione S-transferase fusion proteins, although the proteins were still inactive in repair. These results suggest that while glutamine 90 has no direct role in MG-DNA methyltransferase-mediated repair or free base/lesioned DNA substrate specificity, asparagine 137 is important in both the stability and activity of the protein and may contribute to the formation or function of the active site of the protein.
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PMID:The role of two conserved amino acids, glutamine 90 and asparagine 137, in O6-methylguanine-DNA methyltransferase stability, activity and substrate specificity. 792 83

The human O6-methylguanine DNA methyltransferase (MGMT) repairs O6-methylguanine (O6-MG) in DNA at a much lower rate than the Escherichia coli Ada protein, and only MGMT repairs the altered base, O6-benzylguanine (O6-BG). The diversity in DNA repair properties between MGMT and Ada may be a result of divergent amino acid sequences outside their common proline-cysteine-histidine-arginine-valine (PCHRV) acceptor site. One notable sequence difference is an MGMT 28-amino acid carboxyl-terminal tail which is highly conserved among all mammalian alkyltransferases. The role of this tail sequence in substrate specificity was assessed by expressing full-length MGMT and Ada proteins, and mutant MGMT proteins lacking either 10 or 28 amino acids from the carboxyl terminus, as GST fusion proteins in alkyltransferase-deficient E. coli cells, and comparing rates of repair of O6-MG containing DNA and O6-BG by these fusion proteins at 4 degrees C and 37 degrees C. The MGMT carboxyl-terminal tail was not required for repair of O6-MG in DNA at 37 degrees C although the deletion of this tail sequence reversibly inhibited the ability of MGMT to repair O6-MG in DNA at 4 degrees C. Therefore, the absence of this region affects the ability of the protein to repair O6-MG in DNA at lower temperatures. Furthermore, removal of the tail sequence from MGMT decreased the rate of O6-BG repair 5-fold. We conclude that the 28-amino acid carboxyl-terminal MGMT tail, while not required for activity, modulates the rate of MGMT repair at reduced temperatures and plays a role in substrate specificity.
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PMID:The role of the carboxyl-terminal tail in human O6-methylguanine DNA methyltransferase substrate specificity and temperature sensitivity. 836 18

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 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

The expression of different genes potentially involved in DNA repair and in cell responses to chemotherapy was evaluated in 33 previously untreated ovarian cancer patients. In biopsies of the same patients the expression of repair genes O6-methylguanine DNA methyltransferase (MGMT), 3-methyladenine DNA glycosylase (MAG), ERCC1, MDR-1, DNA topoisomerase I, DNA topoisomerase IIalpha, and glutathione S-transferase-pi (GST-pi) was assessed by Northern blot analysis. No direct statistical correlation was found between the expression of these genes and the response to chemotherapy (mainly platinum-based with or without doxorubicin and cyclophosphamide). Univariate analysis showed a weak negative correlation (P = 0.037) between the expression of ERCC1 and mortality, whereas no statistically significant correlation was found for other parameters. The MDR-1 gene encoding for the P-glycoprotein P-170 was mostly undetectable in these patients (as assessed by Northern blotting), whereas relatively high levels of MAG and MGMT were found in the majority of patients. A statistically significant correlation was found between the expression of DNA topoisomerase I and the expression of either ERCC1 (P = 0.0026) or GST-pi (P = 0.0279).
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PMID:Expression of genes of potential importance in the response to chemotherapy and DNA repair in patients with ovarian cancer. 910 2

M.EcoRV is an alpha-adenine DNA methyltransferase. According to structure predictions, the enzyme consists of a catalytic domain, which has a structure similar to all other DNA-methyltransferases, and a smaller DNA-recognition domain. We have investigated this enzyme by random mutagenesis, using error-prone PCR, followed by selection for catalytically inactive mutants. 20 single mutants were identified that are completely inactive in vivo as His6- and GST-fusion proteins. 13 of them could be overexpressed and purified. All of these mutants are also inactive in vitro. 5 of the mutations are located near the putative binding site for a flipped adenine residue (C192R, D193G, E212G, W231R, N239H). All of these variants bind to DNA, demonstrating the importance of this region of the protein in catalysis. Only the W231R mutant could be purified with high yields. It binds to DNA and AdoMet and, thus, behaves like a bona fide active site mutant. According to the structure prediction Trp231 corresponds to Val121 in M.HhaI, which forms a hydrophobic contact to the flipped target cytosine. 4 of the remaining purified variants are located within a small region of the putative DNA-recognition domain (F115S, F117L, S121P, C122Y). F117L, S121P and C122Y are unable to bind to DNA, suggesting a critical role of this region in DNA binding. Taken together, these results are in good agreement with the structural model of M.EcoRV.
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PMID:Functional mapping of the EcoRV DNA methyltransferase by random mutagenesis and screening for catalytically inactive mutants. 962 40

Cancer chemotherapy is the principal approach for urogenital cancers. However, the acquisition of resistance to anticancer agents is a critical factor that limits the successful treatment of malignancies. The multidrug resistant (MDR) phenotype has been widely recognized in cancer chemotherapy in urogenital tumors and the mechanisms underlying MDR have also been extensively studied. One of the principle mechanisms in MDR is caused by the overexpression of P-glycoprotein (P-gp), encoded by the multidrug resistance gene (MDR1). It functions as an ATP-dependent active efflux pump of chemotherapeutic agents in human cancer cells. Recently, other drug resistance proteins, including multidrug resistance-associated protein (MRP1) and cMOAT (or MRP2), were also identified from multidrug resistant cells. A functional analysis of MRP1 has shown that MRP1 may have the potential to act as a transporter of glutathione conjugates, which has been known as a central detoxification pathway in anticancer agents. Furthermore, several other resistance-related proteins (e.g. glutathione S-transferase, metallothionein, thioredoxin, topoisomerase I, II, O6-alkylguanine-DNA methyltransferase, etc.) have been found to be up- or down-regulated in resistant cells and these molecules are believed to contribute to the resistant phenotype as well. Based on the molecular characteristics identified in MDR, several experimental and clinical approaches have been studied to overcome MDR. One of these strategies is to reverse MDR by using such P-gp inhibitors as verapamil and cyclosporine A. In this review, we summarize the recent advances in MDR-related molecules and clinical trials to circumvent MDR in urogenital carcinomas.
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PMID:Mechanisms of drug resistance in chemotherapy for urogenital carcinoma. 1051 Aug 88

We have isolated a novel cDNA clone, named AZ2, from a cDNA library of mRNA prepared from C3H10T1/2 cells that had been transiently exposed to 5-azacytidine, a potent inhibitor of DNA methyltransferase. The elucidated nucleotide sequence revealed that the 5' region of the cDNA was rich in the CpG sequence. The AZ2 cDNA contained a 1215-nucleotide open reading frame, and the expected amino acid sequence had a molecular mass of 46090. The amount of the transcript increased on 5-azacytidine treatment of C3H10T1/2 cells, and the transcript was significantly expressed in mouse testis, brain, lung, kidney, heart and ovary. Specific antibodies raised against a fusion protein including glutathione S-transferase revealed a band of an approximately 48kDa translation product for testis, brain, lung, and cultured cells that ectopically expressed the AZ2 protein. The AZ2 protein was mainly localized in the cytoplasm. The amino-terminal part of the AZ2 protein was homologous to the previously reported TANK (Cheng and Baltimore, 1996. Genes Dev. 10, 963-973) and I-TRAF (Rothe, 1996. Proc. Natl. Acad. Sci. USA 93, 8241-8246), which participate in the signal transduction cascade from the tumor necrosis factor-receptor to the transcription factor, NFkappaB. Overexpression of AZ2 inhibited TNF alpha mediated NFkappaB activation. AZ2 could be a component of a regulator of the NFkappaB activation cascade.
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PMID:Isolation of the novel cDNA of a gene of which expression is induced by a demethylating stimulus. 1058 Jan 48

Glutathione S-transferases, enzymes that defend cells against damage mediated by oxidant and electrophilic carcinogens, may be critical determinants of cancer pathogenesis. We report here that the pathogenesis of hepatocellular carcinoma (HCC), one of the most common cancers in the world, frequently involves an accumulation of somatic <CpG island> DNA methylation changes at GSTP1, the gene encoding the pi-class glutathione S-transferase. For our study, Hep3B HCC cells and a cohort of 20 HCC tissue specimens were subjected to analysis for GSTP1 expression and for somatic GSTP1 alterations. GSTP1 <CpG island> DNA hypermethylation in HCC DNA was assessed by Southern blot analysis, via a polymerase chain reaction (PCR) assay, and by using a genomic sequencing approach. Hep3B HCC cells failed to express GSTP1 mRNA or GSTP1 polypeptides. Similarly, HCC cells in 19 of 20 HCC cases were devoid of GSTP1 polypeptides. By Southern blot analysis, DNA from Hep3B HCC cells displayed abnormal GSTP1 <CpG island> hypermethylation. Treatment of Hep3B HCC cells in vitro with the DNA methyltransferase inhibitor 5-aza-deoxycytidine both reversed GSTP1 <CpG island> DNA hypermethylation and restored GSTP1 expression. Using a PCR assay, somatic GSTP1 <CpG island> DNA hypermethylation was also detected in HCC DNA from 17 of 20 HCC cases. Genomic sequencing analyses, undertaken to map 5-methyldeoxycytidine nucleotides located at the GSTP1 transcriptional regulatory region, frequently detected somatic DNA hypermethylation near the gene promoter in HCC DNA. The data indicate that GSTP1 <CpG island> DNA hypermethylation changes appear frequently in human HCC. In addition, the data raise the possibility that somatic GSTP1 inactivation, via <CpG island> hypermethylation, may contribute to the pathogenesis of HCC.
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PMID:GSTP1 CpG island DNA hypermethylation in hepatocellular carcinomas. 1071 33

DRH strain rats were established by inbreeding a closed colony of Donryu rats continuously fed the chemical hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene for over 10 years. They are highly resistant to chemical induction of liver cancer and preneoplastic lesions. We studied the genetic basis of DRH resistance to preneoplastic lesions by analyzing 108 (F344 x DRH)F2 male rats fed 3'-methyl-4-dimethylaminoazobenzene for 7 weeks. Five parameters of preneoplastic liver lesions were selected for quantitative analysis: (a) number of glutathione S-transferase placental form-positive foci per unit area of liver section; (b) percentage area occupied by the foci; (c) average size of foci; (d) glutathione S-transferase placental form mRNA level; and (e) gamma-glutamyltranspeptidase mRNA level. Furthermore, O6-methylguanine DNA methyltransferase and mannose 6-phosphatase/insulin-like growth factor 2 receptor mRNA levels were quantified. Composite interval mapping analysis showed that there were two remarkably significant clusters of quantitative trait loci affecting preneoplastic liver lesions on chromosomes 1 and 4. These clusters were designated collectively as Drh1 and Drh2, respectively. The functions of the recessive DRH allele of Drh1 and the semidominant DRH allele of Drh2 were to suppress the phenotypes of precancerous lesions. Each cluster showed two to three subpeaks in linkage likelihood plots, suggesting the presence of several closely linked quantitative trait loci affecting preneoplastic lesions. Possible candidate genes at each locus will be discussed. Expression of O6-methylguanine DNA methyltransferase and mannose 6-phosphatase/insulin-like growth factor 2 receptor did not affect DRH resistance to hepatocarcinogenesis, although they were polymorphic between DRH and F344 rats.
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PMID:Genetic resistance to chemical carcinogen-induced preneoplastic hepatic lesions in DRH strain rats. 1085 Apr 31


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