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Sample records for betaine-homocysteine methyltransferase expression

  1. Homocysteine homeostasis and betaine-homocysteine S-methyltransferase expression in the brain of hibernating bats.

    Directory of Open Access Journals (Sweden)

    Yijian Zhang

    Full Text Available Elevated homocysteine is an important risk factor that increases cerebrovascular and neurodegenerative disease morbidity. In mammals, B vitamin supplementation can reduce homocysteine levels. Whether, and how, hibernating mammals, that essentially stop ingesting B vitamins, maintain homocysteine metabolism and avoid cerebrovascular impacts and neurodegeneration remain unclear. Here, we compare homocysteine levels in the brains of torpid bats, active bats and rats to identify the molecules involved in homocysteine homeostasis. We found that homocysteine does not elevate in torpid brains, despite declining vitamin B levels. At low levels of vitamin B6 and B12, we found no change in total expression level of the two main enzymes involved in homocysteine metabolism (methionine synthase and cystathionine β-synthase, but a 1.85-fold increase in the expression of the coenzyme-independent betaine-homocysteine S-methyltransferase (BHMT. BHMT expression was observed in the amygdala of basal ganglia and the cerebral cortex where BHMT levels were clearly elevated during torpor. This is the first report of BHMT protein expression in the brain and suggests that BHMT modulates homocysteine in the brains of hibernating bats. BHMT may have a neuroprotective role in the brains of hibernating mammals and further research on this system could expand our biomedical understanding of certain cerebrovascular and neurodegenerative disease processes.

  2. Betaine-homocysteine methyltransferase: human liver genotype-phenotype correlation

    OpenAIRE

    Feng, Qiping; Kalari, Krishna; Fridley, Brooke L.; Jenkins, Gregory; Ji, Yuan; Abo, Ryan; Hebbring, Scott; Zhang, Jianping; Nye, Monica D.; Leeder, J Steven; Weinshilboum, Richard M.

    2010-01-01

    Betaine-homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT is highly expressed in the human liver. In the liver, BHMT catalyzes up to 50% of homocysteine metabolism. Understanding the relationship between BHMT genetic polymorphisms and function might increase our understanding of the role of this reaction in homocysteine remethylation and in S-adenosylmethionine-dependent methylation. To help achieve those goals, we measured levels of BHMT enzyme activity ...

  3. Corticoadrenal activity in rat regulates betaine-homocysteine S-methyltransferase expression with opposite effects in liver and kidney

    Indian Academy of Sciences (India)

    Osvaldo Fridman; Analía V Morales; Laura E Bortoni; Paula C Turk-Noceto; Elio A Prieto

    2012-03-01

    Betaine-homocysteine -methyltransferase (BHMT) is an enzyme that converts homocysteine (Hcy) to methionine using betaine as a methyl donor. Betaine also acts as osmolyte in kidney medulla, protecting cells from high extracellular osmolarity. Hepatic BHMT expression is regulated by salt intake. Hormones, particularly corticosteroids, also regulate BHMT expression in rat liver. We investigated to know whether the corticoadrenal activity plays a role in kidney BHMT expression. BHMT activity in rat kidneys is several orders of magnitude lower than in rat livers and only restricted to the renal cortex. This study confirms that corticosteroids stimulate BHMT activity in the liver and, for the first time in an animal model, also up-regulate the BHMT gene expression. Besides, unlike the liver, corticosteroids in rat kidney down-regulate BHMT expression and activity. Given that the classical effect of adrenocortical activity on the kidney is associated with sodium and water re-absorption by the distal tubule leading to volume expansion, by promoting lesser use of betaine as a methyl donor, corticosteroids would preserve betaine for its other role as osmoprotectant against changes in the extracellular osmotic conditions. We conclude that corticosteroids are, at least in part, responsible for the inhibition of BHMT expression and activity in rat kidneys.

  4. Betaine-homocysteine methyltransferase: human liver genotype-phenotype correlation

    Science.gov (United States)

    Feng, Qiping; Kalari, Krishna; Fridley, Brooke L.; Jenkins, Gregory; Ji, Yuan; Abo, Ryan; Hebbring, Scott; Zhang, Jianping; Nye, Monica D.; Leeder, J. Steven; Weinshilboum, Richard. M.

    2010-01-01

    Betaine-homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT is highly expressed in the human liver. In the liver, BHMT catalyzes up to 50% of homocysteine metabolism. Understanding the relationship between BHMT genetic polymorphisms and function might increase our understanding of the role of this reaction in homocysteine remethylation and in S-adenosylmethionine-dependent methylation. To help achieve those goals, we measured levels of BHMT enzyme activity and immunoreactive protein in 268 human hepatic surgical biopsy samples from adult subjects as well as 73 fetal hepatic tissue samples obtained at different gestational ages. BHMT protein levels were correlated significantly (p<0.001) with levels of enzyme activity in both fetal and adult tissue, but both were decreased in fetal tissue when compared with levels in the adult hepatic biopsies. To determine possible genotype-phenotype correlations, 12 tag SNPs for BHMT and the closely related BHMT2 gene were selected from SNPs observed during our own gene resequencing studies as well as from HapMap data were used to genotype DNA from the adult hepatic surgical biopsy samples, and genotype-phenotype association analysis was performed. Three SNPs (rs41272270, rs16876512, and rs6875201), located 28 kb upstream, in the 5′-UTR and in intron 1 of BHMT, respectively, were significantly correlated with both BHMT activity (p=3.41E-8, 2.55E-9 and 2.46E-10, respectively) and protein levels (p=5.78E-5, 1.08E-5 and 6.92E-6, respectively). We also imputed 230 additional SNPs across the BHMT and BHMT2 genes, identifying an additional imputed SNP, rs7700790, that was also highly associated with hepatic BHMT enzyme activity and protein. However, none of the 3 genotyped or one imputed SNPs displayed a “shift” during electrophoretic mobility shift assays. These observations may help us to understand individual variation in the regulation of BHMT in the human liver and its possible relationship

  5. Both the folate cycle and betaine-homocysteine methyltransferase contribute methyl groups for DNA methylation in mouse blastocysts.

    Science.gov (United States)

    Zhang, Baohua; Denomme, Michelle M; White, Carlee R; Leung, Kit-Yi; Lee, Martin B; Greene, Nicholas D E; Mann, Mellissa R W; Trasler, Jacquetta M; Baltz, Jay M

    2015-03-01

    The embryonic pattern of global DNA methylation is first established in the inner cell mass (ICM) of the mouse blastocyst. The methyl donor S-adenosylmethionine (SAM) is produced in most cells through the folate cycle, but only a few cell types generate SAM from betaine (N,N,N-trimethylglycine) via betaine-homocysteine methyltransferase (BHMT), which is expressed in the mouse ICM. Here, mean ICM cell numbers decreased from 18-19 in controls to 11-13 when the folate cycle was inhibited by the antifolate methotrexate and to 12-14 when BHMT expression was knocked down by antisense morpholinos. Inhibiting both pathways, however, much more severely affected ICM development (7-8 cells). Total SAM levels in mouse blastocysts decreased significantly only when both pathways were inhibited (from 3.1 to 1.6 pmol/100 blastocysts). DNA methylation, detected as 5-methylcytosine (5-MeC) immunofluorescence in isolated ICMs, was minimally affected by inhibition of either pathway alone but decreased by at least 45-55% when both BHMT and the folate cycle were inhibited simultaneously. Effects on cell numbers and 5-MeC levels in the ICM were completely rescued by methionine (immediate SAM precursor) or SAM. Both the folate cycle and betaine/BHMT appear to contribute to a methyl pool required for normal ICM development and establishing initial embryonic DNA methylation. PMID:25466894

  6. Structure-activity study of new inhibitors of human betaine-homocysteine S-methyltransferase

    Czech Academy of Sciences Publication Activity Database

    Vaněk, Václav; Buděšínský, Miloš; Kabeleová, Petra; Šanda, Miloslav; Kožíšek, Milan; Hančlová, Ivona; Mládková, Jana; Brynda, Jiří; Rosenberg, Ivan; Koutmos, M.; Garrow, T. A.; Jiráček, Jiří

    2009-01-01

    Roč. 52, č. 12 (2009), s. 3652-3665. ISSN 0022-2623 R&D Projects: GA MŠk 1M0508 Grant ostatní: GA MŠk(CZ) LC06077; NIH(US) R01TW0052501 Institutional research plan: CEZ:AV0Z40550506 Keywords : BHMT * betain * homocysteine * methionine * inhibitor Subject RIV: CE - Biochemistry Impact factor: 4.802, year: 2009

  7. Betaine:homocysteine methyltransferase--a new assay for the liver enzyme and its absence from human skin fibroblasts and peripheral blood lymphocytes.

    Science.gov (United States)

    Wang, J A; Dudman, N P; Lynch, J; Wilcken, D E

    1991-12-31

    Chronic elevation of plasma homocysteine is associated with increased atherogenesis and thrombosis, and can be lowered by betaine (N,N,N-trimethylglycine) treatment which is thought to stimulate activity of the enzyme betaine:homocysteine methyltransferase. We have developed a new assay for this enzyme, in which the products of the enzyme-catalysed reaction between betaine and homocysteine are oxidised by performic acid before being separated and quantified by amino acid analysis. This assay confirmed that human liver contains abundant betaine:homocysteine methyltransferase (33.4 nmol/h/mg protein at 37 degrees C, pH 7.4). Chicken and lamb livers also contain the enzyme, with respective activities of 50.4 and 6.2 nmol/h/mg protein. However, phytohaemagglutinin-stimulated human peripheral blood lymphocytes and cultured human skin fibroblasts contained no detectable betaine:homocysteine methyltransferase (less than 1.4 nmol/h/mg protein), even after cells were pre-cultured in media designed to stimulate production of the enzyme. The results emphasize the importance of the liver in mediating the lowering of elevated circulating homocysteine by betaine. PMID:1819467

  8. Double-Headed Sulfur-Linked Amino Acids As First Inhibitors for Betaine-Homocysteine S-Methyltransferase 2

    Czech Academy of Sciences Publication Activity Database

    Mládková, Jana; Vaněk, Václav; Buděšínský, Miloš; Elbert, Tomáš; Demianova, Zuzana; Garrow, T. A.; Jiráček, Jiří

    2012-01-01

    Roč. 55, č. 15 (2012), s. 6822-6831. ISSN 0022-2623 R&D Projects: GA ČR GA203/09/1919; GA ČR(CZ) GAP207/10/1277 Institutional support: RVO:61388963 Keywords : betaine * homocysteine * methionine * BHMT * inhibitor Subject RIV: CE - Biochemistry Impact factor: 5.614, year: 2012

  9. Alleviation of hepatic fat accumulation by betaine involves reduction of homocysteine via up-regulation of betaine-homocysteine methyltransferase (BHMT).

    Science.gov (United States)

    Ahn, Chul Won; Jun, Doo Sung; Na, Jong Deok; Choi, Yeo Jin; Kim, Young Chul

    2016-08-26

    We investigated the anti-lipogenic effect of betaine in rats fed methionine and choline-deficient diet (MCD). Intake of MCD for 3 wk resulted in a significant accumulation of hepatic lipids, which was prevented by betaine supplementation in drinking water (1%). Phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), sterol regulatory element-binding protein-1c (SREBP-1c), and liver kinase B1 (LKB1) was inhibited by MCD intake, and these changes were all inhibited by betaine feeding. Meanwhile, betaine supplementation reversed the reduction of methionine and S-adenosylmethionine (SAM), and the elevation of homocysteine levels in the liver, which could be attributable to the induction of betaine-homocysteine methyltransferase (BHMT) and methionine adenosyltransferase (MAT). Different cell lines were used to clarify the role of homocysteine on activation of the AMPK pathway. Homocysteine treatment decreased pAMPK, pACC, pSREBP-1c and pLKB1 in HepG2 cells. Metformin-induced activation of AMPK was also inhibited by homocysteine. Treatment with hydroxylamine, a cystathionine β-synthase inhibitor, resulted in a reduction of pAMPK, pACC and pSREBP-1c, accompanied by an elevation of intracellular homocysteine. Betaine treatment prevented the homocysteine-induced reduction of pAMPK, pACC, pSREBP-1c and pLKB1 in H4IIE cells, but not in HepG2 cells. Also the elevation of cellular homocysteine and inhibition of protein expression of BHMT were prevented by betaine only in H4IIE cells which express BHMT. The results suggest that the beneficial effect of betaine against hepatic lipid accumulation may be attributed, at least in part, to the depletion of homocysteine via up-regulation of BHMT in hepatocytes. PMID:27320863

  10. Specific potassium ion interactions facilitate homocysteine binding to betaine-homocysteine S-methyltransferase

    Czech Academy of Sciences Publication Activity Database

    Mládková, Jana; Hladílková, Jana; Diamond, C. E.; Tryon, K.; Yamada, K.; Garrow, T. A.; Jungwirth, Pavel; Koutmos, M.; Jiráček, Jiří

    2014-01-01

    Roč. 82, č. 10 (2014), s. 2552-2564. ISSN 0887-3585 R&D Projects: GA ČR(CZ) GAP207/10/1277; GA ČR GBP208/12/G016 Institutional support: RVO:61388963 Keywords : BHMT * homocysteine * potassium * crystal structure * molecular dynamics * simulations * enzyme kinetics Subject RIV: CE - Biochemistry Impact factor: 2.627, year: 2014

  11. The development of a new class of inhibitors for betaine-homocysteine S-methyltransferase

    Czech Academy of Sciences Publication Activity Database

    Pícha, Jan; Vaněk, Václav; Buděšínský, Miloš; Mládková, Jana; Garrow, T. A.; Jiráček, Jiří

    2013-01-01

    Roč. 65, July (2013), s. 256-275. ISSN 0223-5234 R&D Projects: GA ČR(CZ) GAP207/10/1277 Institutional support: RVO:61388963 Keywords : BHMT * inhibitor * homocysteine * phosphonate * phosphinate * amino acid derivative * bioisostere * S-alkylated homocysteine Subject RIV: CE - Biochemistry Impact factor: 3.432, year: 2013

  12. Effects of hyperhomocysteinemia and betaine-homocysteine S-methyltransferase inhibition on hepatocyte metabolites and the proteome

    Czech Academy of Sciences Publication Activity Database

    Selicharová, Irena; Kořínek, M.; Demianova, Zuzana; Chrudinová, Martina; Mládková, Jana; Jiráček, Jiří

    2013-01-01

    Roč. 1834, č. 8 (2013), s. 1596-1606. ISSN 1570-9639 R&D Projects: GA ČR(CZ) GAP207/10/1277 Institutional support: RVO:61388963 Keywords : apolipoprotein * fibrinogen * one-carbon metabolism * S-Adenosylmethionine * two-dimensional electrophoresis Subject RIV: CE - Biochemistry Impact factor: 3.191, year: 2013

  13. Dissecting the Catalytic Mechanism of Betaine - Homocysteine S-Methyltransferase by Use of Intrinsic Tryptophan Fluorescence and Site-Directed Mutagenesis

    Czech Academy of Sciences Publication Activity Database

    Castro, C.; Gratson, A. A.; Evans, J. C.; Jiráček, Jiří; Collinsová, Michaela; Ludwig, M. L.; Garrow, T. A.

    2004-01-01

    Roč. 43, č. 18 (2004), s. 5341-5351. ISSN 0006-2960 R&D Projects: GA AV ČR IAA4055302 Grant ostatní: NIH(US) GM16429; NIH(US) DK52501; Illinois Agricultural Experimental Station(US) ILLU-698-352 Institutional research plan: CEZ:AV0Z4055905 Keywords : BHMT * CBHcy * fluorescence Subject RIV: CE - Biochemistry Impact factor: 4.008, year: 2004

  14. Quantification of homocysteine-related metabolites and the role of betaine-homocysteine S-methyltransferase in HepG2 cells

    Czech Academy of Sciences Publication Activity Database

    Kořínek, M.; Šístek, V.; Mládková, Jana; Mikeš, P.; Jiráček, Jiří; Selicharová, Irena

    2013-01-01

    Roč. 27, č. 1 (2013), s. 111-121. ISSN 0269-3879 R&D Projects: GA ČR(CZ) GAP207/10/1277 Institutional support: RVO:61388963 Keywords : homocysteine * BHMT * LC-MS/MS * HepG2 * metabolites Subject RIV: CE - Biochemistry Impact factor: 1.662, year: 2013

  15. High level expression and purification of HhaI methyltransferase.

    OpenAIRE

    Wu, J. C.; Santi, D V

    1988-01-01

    A cloning system for the DNA-(cytosine-5)-methyltransferase MHhaI and high level expression of the enzyme are described. A parent plasmid was constructed from fragments of the MHhaI gene and synthetic oligonucleotides. The construct permits introduction of various restriction sites for cloning at precise positions near the initiation codon, and beyond the termination codon. The entire MHhaI coding sequence was introduced as a 1042 b.p. NdeI-XbaI fragment into the vector pAR3040 which contains...

  16. Gene expression in rats with Barrett's esophagus and esophageal adenocarcinoma induced by gastroduodenoesophageal reflux

    Institute of Scientific and Technical Information of China (English)

    Peng Cheng; Jun Gong; Tao Wang; Jie Chen; Gui-Sheng Liu; Ru Zhang

    2005-01-01

    AIM: To study the different gene expression profiles in rats with Barrett's esophagus (BE) and esophageal adenocarcinoma (EA) induced by gastro-duodenoesophageal reflux.METHODS: Esophagoduodenostomy was performed in 8-wk old Sprague-Dawley rats to induce gastro-duodenoesophageal reflux, and a group of rats that received sham operation served as control. Esophageal epithelial pathological tissues were dissected and frozen in liquid nitrogen immediately. The expression profiles of 4 096genes in EA and BE tissues were compared to normal esophagus epithelium in normal control (NC) by cDNA microarray.RESULTS: Four hundred and forty-eight genes in BE were more than three times different from those in NC, including 312 upregulated and 136 downregulated genes. Three hundred and seventy-seven genes in EA were more than three times different from those in NC, including 255upregulated and 142 downregulated genes. Compared to BE, there were 122 upregulated and 156 downregulated genes in EA. In the present study, the interested genes were those involved in carcinogenesis. Among them, the upregulated genes included cathepsin C, aminopeptidase M, arachidonic acid epoxygenase, tryptophan-2,3-dioxygenase, ubiquitin-conjugating enzyme, cyclic GMP-stimulated phosphodiesterase, tissue inhibitor of metalloproteinase-1, betaine-homocysteine methyltransferase, lysozyme, complement 4b binding protein,complement 9 protein, insulin-like growth factor binding protein, UDP-glucuronosyltransferase, tissue inhibitor of metalloproteinase-3, aldolase B, retinoid X receptor gamma, carboxylesterase and testicular cell adhesion molecule 1. The downregulated genes included glutathione synthetase, lecithin-cholesterol acyltransferase, p55CDC,heart fatty acid binding protein, cell adhesion regulator and endothelial cell selectin ligand.CONCLUSION: Esophageal epithelium exposed excessively to harmful ingredients of duodenal and gastric reflux may develop into BE and even EA gradually. The gene

  17. Vector design for expression of O6-methylguanine-DNA methyltransferase in hematopoietic cells

    OpenAIRE

    Schambach, Axel; Baum, Christopher

    2007-01-01

    Enhancing DNA repair activity of hematopoietic cells by stably integrating gene vectors that express O(6)-methylguanine-DNA-methyltransferase (MGMT) is of major interest for innovative approaches in tumor chemotherapy and for the control of hematopoietic chimerism in the treatment of multiple other acquired or inherited disorders. Crucial determinants of this selection principle are the stringency of treatment with O(6)-alkylating agents and the level of transgenic MGMT expression. Attempts t...

  18. Expression of O6-methylguanine DNA methyltransferase (MGMT) and its clinical significance in gastroenteropancreatic neuroendocrine neoplasm

    OpenAIRE

    Yang, Qiu-chen; Wang, Yu-Hong; Lin, Yuan; Xue, Ling; Chen, Yuan-Jia; Chen, Min-hu; Chen, Jie

    2014-01-01

    O6-methylguanine-DNA methyltransferase (MGMT) is a widespread DNA repair enzyme defending against mutation caused by guanine O6-alkylating agents. Until now, we know only little about the expression of MGMT in gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN). To study the expression of MGMT and its clinical significance in GEP-NEN, 174 specimens of GEP-NEN were examined, of which 152 specimens came from The First Affiliated Hospital, Sun Yat-sen University during October 1995 to Novem...

  19. Homocysteine Induces Collagen I Expression by Downregulating Histone Methyltransferase G9a

    OpenAIRE

    Lei, Wenjing; Long, Yanjun; LI, Shuang; Liu, Ze; Zhu, Fengxin; Hou, Fan Fan; Nie, Jing

    2015-01-01

    Hyperhomocysteinemia (HHcy) leads to several clinical manifestations including hepatic fibrosis. Excess deposition of extracellular matrix (ECM) components including collagen is the eponymous lesion of liver fibrosis. In this study, we demonstrated that elevated concentration of Hcy induced the expression of collagen type I in cultured human liver cells as well as in liver tissue of HHcy mice. Meanwhile, Hcy inhibited the expression of histone methyltransferase G9a. Mechanistically, silencing...

  20. Selenium induced selenocysteine methyltransferase gene expression and antioxidant enzyme activities in Astragalus chrysochlorus

    OpenAIRE

    Çakir, Özgür; Turgut-Kara, Neslihan; Ari, Şule

    2016-01-01

    Astragalus sp. are used in folk medicine because of their biological activities and are known for the ability to accumulate high levels of selenium (Se). The purpose of this study was to explore gene expression of selenocysteine methyltransferase (SMT), responsible for forming MeSeCys, and activities of ascorbate peroxidase (APX), peroxidase (POX), catalase (CAT) and glutathione reductase (GR) enzymes in callus tissues of Astragalus chrysochlorus growing in different Se-containing media. Quan...

  1. Histone H3K9 methyltransferase G9a represses PPARγ expression and adipogenesis

    OpenAIRE

    Wang, Lifeng; Xu, Shiliyang; Lee, Ji-Eun; Baldridge, Anne; Grullon, Sean; Peng, Weiqun; Ge, Kai

    2012-01-01

    PPARγ promotes adipogenesis while Wnt proteins inhibit adipogenesis. However, the mechanisms that control expression of these positive and negative master regulators of adipogenesis remain incompletely understood. By genome-wide histone methylation profiling in preadipocytes, we find that among gene loci encoding adipogenesis regulators, histone methyltransferase (HMT) G9a-mediated repressive epigenetic mark H3K9me2 is selectively enriched on the entire PPARγ locus. H3K9me2 and G9a levels dec...

  2. Transcriptome profiling of Set5 and Set1 methyltransferases: Tools for visualization of gene expression

    Directory of Open Access Journals (Sweden)

    Glòria Mas Martín

    2014-12-01

    Full Text Available Cells regulate transcription by coordinating the activities of multiple histone modifying complexes. We recently identified the yeast histone H4 methyltransferase Set5 and discovered functional overlap with the histone H3 methyltransferase Set1 in gene expression. Specifically, using next-generation RNA sequencing (RNA-Seq, we found that Set5 and Set1 function synergistically to regulate specific transcriptional programs at subtelomeres and transposable elements. Here we provide a comprehensive description of the methodology and analysis tools corresponding to the data deposited in NCBI's Gene Expression Omnibus (GEO under the accession number GSE52086. This data complements the experimental methods described in Mas Martín G et al. (2014 and provides the means to explore the cooperative functions of histone H3 and H4 methyltransferases in the regulation of transcription. Furthermore, a fully annotated R code is included to enable researchers to use the following computational tools: comparison of significant differential expression (SDE profiles; gene ontology enrichment of SDE; and enrichment of SDE relative to chromosomal features, such as centromeres, telomeres, and transposable elements. Overall, we present a bioinformatics platform that can be generally implemented for similar analyses with different datasets and in different organisms.

  3. MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF A JUVENILE HORMONE ACID METHYLTRANSFERASE EXPRESSED IN THE CORPORA ALLATA OF MOSQUITOES

    OpenAIRE

    Mayoral, Jaime G.; Nouzova, Marcela; Yoshiyama, Michiyo; Shinoda, Tetsuro; Hernandez-Martinez, Salvador; Dolghih, Elena; Turjanski, Adrian G; Roitberg, Adrian E.; Priestap, Horacio; Perez, Mario; Mackenzie, Lucy; Li, Yiping; Noriega, Fernando G.

    2008-01-01

    A juvenile hormone acid methyltransferase (JHAMT) was isolated as an abundant EST in a library of the corpora allata of the adult female mosquito Aedes aegypti. Its full-length cDNA encodes a 278-aa protein that has 43 % amino acid identity with BmJHAMT, a juvenile hormone acid methyltransferase previously cloned from Bombyx mori. Heterologous expression produced a recombinant protein that metabolizes farnesoic acid (FA) into methyl farnesoate, as well as juvenile hormone acid into juvenile h...

  4. Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2012-05-15

    DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

  5. Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

    International Nuclear Information System (INIS)

    DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

  6. Identification and expression profiling of DNA methyltransferases during development and stress conditions in Solanaceae.

    Science.gov (United States)

    Kumar, Rahul; Chauhan, Pankaj Kumar; Khurana, Ashima

    2016-09-01

    DNA methyltransferase (DMTase) enzymes contribute to plant development and stress responses by de novo establishment and subsequent maintenance of DNA methylation during replication. However, the molecular mechanism underlying this activity remains obscure, especially in crop species. Using DMTase homolog complement in six Solanaceae species, we demonstrated here that their number remained conserved in Solanum lineage, whereas it was expanded in both pepper and Nicotiana benthamiana. Non-synonymous vs synonymous (Ka/Ks) substitution ratio revealed that most of the Solanaceous DMTase homologs undergo purifying selection. The genomic sequences of tomato DMT homologs in its wild relative, Solanum pennellii, remained highly conserved in their exons and methyltransferase domains. Structure analysis further revealed highly similar folding of DMTase homologs and conservation in the residues participating in protein-protein interaction in Solanum lineage, whereas a considerable diversification was observed of pepper homologs. Transcript profiling of DMTases highlighted both similar and distinct expression patterns of tomato homologs in other species during fruit development and stress responses. Overall, our analysis provides a strong basis for in-depth exploration of both conserved as well as distinct functions of tomato DMTase homologs in other economically important Solanaceae species. PMID:27380018

  7. Expression of O6-methylguanine DNA methyltransferase (MGMT) and its clinical significance in gastroenteropancreatic neuroendocrine neoplasm

    Science.gov (United States)

    Yang, Qiu-Chen; Wang, Yu-Hong; Lin, Yuan; Xue, Ling; Chen, Yuan-Jia; Chen, Min-Hu; Chen, Jie

    2014-01-01

    O6-methylguanine-DNA methyltransferase (MGMT) is a widespread DNA repair enzyme defending against mutation caused by guanine O6-alkylating agents. Until now, we know only little about the expression of MGMT in gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN). To study the expression of MGMT and its clinical significance in GEP-NEN, 174 specimens of GEP-NEN were examined, of which 152 specimens came from The First Affiliated Hospital, Sun Yat-sen University during October 1995 to November 2013, 22 specimens came from Peking Union Medical College Hospital during September 2004 to April 2010. MGMT protein was detected with EnVision immunohistochemical staining method. Clinicopathological factors were also collected and analyzed. We observed that the overall expression rate of MGMT was 83.9%. Over expression of MGMT protein was not associated with sex, age, functional status, primary tumor location, grading, classification, TNM stage and metastasis (P > 0.05). Kaplan-Meier analysis revealed that there was no significant difference in survival between MGMT-positive and MGMT-negative tumors of GEP-NEN patients (χ2 = 0.887, P = 0.346). In multivariate analyses carried out by Cox proportional hazards regression model, MGMT expression was also not an independent predictors of survival. These results demonstrated that MGMT protein was highly expressed in GEP-NEN. MGMT deficiency rate was similar in pancreatic NEN and in gastrointestinal NEN. MGMT expression was not correlated with prognosis of GEP-NEN. PMID:25120800

  8. Expression of a functional jasmonic acid carboxyl methyltransferase is negatively correlated with strawberry fruit development.

    Science.gov (United States)

    Preuß, Anja; Augustin, Christiane; Figueroa, Carlos R; Hoffmann, Thomas; Valpuesta, Victoriano; Sevilla, José F; Schwab, Wilfried

    2014-09-15

    The volatile metabolite methyl jasmonate (MeJA) plays an important role in intra- and interplant communication and is involved in diverse biological processes. In this study, we report the cloning and functional characterization of a S-adenosyl-l-methionine:jasmonic acid carboxyl methyltransferase (JMT) from Fragaria vesca and Fragaria×ananassa. Biochemical assays and comprehensive transcript analyses showed that JMT has been erroneously annotated as gene fusion with a carboxyl methyltransferase (CMT) (gene15184) in the first published genome sequence of F. vesca. Recombinant FvJMT catalyzed the formation of MeJA with KM value of 22.3μM while FvCMT and the fusion protein were almost inactive. Activity of JMT with benzoic acid and salicylic acid as substrates was less than 1.5% of that with JA. Leucine at position 245, an amino acid missing in other JMT sequences is essential for activity of FvJMT. In accordance with MeJA levels, JMT transcript levels decreased steadily during strawberry fruit ripening, as did the expression levels of JA biosynthesis and regulatory genes. It appears that CMT has originated by a recent duplication of JMT and lost its enzymatic activity toward JA. In the newest version of the strawberry genome sequence (June 2014) CMT and JMT are annotated as separate genes in accordance with differential temporal and spatial expression patterns of both genes in Fragaria sp. In conclusion, MeJA, the inactive derivative of JA, is probably involved in early steps of fruit development by modulating the levels of the active plant hormone JA. PMID:25046752

  9. O6-Methylguanine DNA methyltransferase protein expression in tumor cells predicts outcome of temozolomide therapy in glioblastoma patients

    OpenAIRE

    Spiegl-Kreinecker, Sabine; Pirker, Christine; Filipits, Martin; Lötsch, Daniela; Buchroithner, Johanna; Pichler, Josef; Silye, Rene; Weis, Serge; Micksche, Michael; Fischer, Johannes; Berger, Walter

    2009-01-01

    O6-Methylguanine DNA methyltransferase (MGMT) is implicated as a major predictive factor for treatment response to alkylating agents including temozolomide (TMZ) of glioblastoma multiforme (GBM) patients. However, whether the MGMT status in GBM patients should be detected at the level of promoter methylation or protein expression is still a matter of debate. Here, we compared promoter methylation (by methylation-specific polymerase chain reaction) and protein expression (by Western blot) in t...

  10. Expression of nicotinamide N-methyltransferase in hepatocellular carcinoma is associated with poor prognosis

    Directory of Open Access Journals (Sweden)

    Do In-Gu

    2009-02-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC is the most common tumor in the adult liver, with high relapse and mortality rates despite diverse treatment modalities. In this study, nicotinamide N-methyltransferase (NNMT, a key enzyme in drug metabolism, was investigated as a potential prognostic factor. Methods Frozen tumors and non-cancerous surrounding tissues from 120 patients with primary HCC were studied. Expressions of NNMT and internal control genes were measured by real-time reverse-transcription PCR (RT-PCR. The relationship of NNMT mRNA level with clinicopathologic parameters and clinical outcome was evaluated. Results NNMT mRNA level is markedly reduced in HCCs compared to non-cancerous surrounding tissues (P Conclusion The results of this study indicated that NNMT gene expression is associated with tumor stage and DFS time in HCC cases. Because of the broad substrate specificity of NNMT, which could alter the efficacy and adverse effects of chemotherapy, NNMT merits further investigation regarding its role as a prognostic factor with a larger cohort of HCC patients.

  11. Hyperhomocysteinemia causes premature hearing loss in C57BL/6J mice

    OpenAIRE

    Martínez-Vega, Raquel; Varela, Gregorio; Partearroyo, Teresa; Pajares, María A.; Varela-Nieto, Isabel

    2012-01-01

    [Introduction]: Alterations in plasma homocysteine (pHcy) levels and betaine homocysteine methyltransferase (BHMT) expression have been reported in genetic mouse models of human deafness. BHMT is one of the enzymes responsible for Hcy remethylation leading to methionine synthesis, its changes being one of the causes of increased pHcy levels and indirectly altering production of glutathione (2). Nutritional factors modulate Hcy metabolism, and hence we have studied the impact of a dietary-indu...

  12. Interplay among coactivator-associated arginine methyltransferase 1, CBP, and CIITA in IFN-γ-inducible MHC-II gene expression

    OpenAIRE

    Zika, Eleni; Fauquier, Lucas; Vandel, Laurence; Ting, Jenny P -Y

    2005-01-01

    Class II major histocompatibility (MHC-II) genes are prototype targets of IFN-γ. IFN-γ activates the expression of the non-DNA-binding master regulator of MHC-II, class II transactivator (CIITA), which is crucial for enhanceosome formation and gene activation. This report shows the importance of the histone methyltransferase, coactivator-associated arginine methyltransferase (CARM1/PRMT4), during IFN-γ-induced MHC-II gene activation. It also demonstrates the coordinated regulation of CIITA, C...

  13. O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation

    OpenAIRE

    Simone Kreth; Niklas Thon; Sabina Eigenbrod; Juergen Lutz; Carola Ledderose; Rupert Egensperger; Tonn, Joerg C.; Kretzschmar, Hans A.; Ludwig C Hinske; Kreth, Friedrich W.

    2011-01-01

    Background: We analyzed prospectively whether MGMT (O(6)-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylatio...

  14. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    International Nuclear Information System (INIS)

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  15. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Jinsong, E-mail: pangjs542@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Dong, Mingyue; Li, Ning; Zhao, Yanli [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Liu, Bao, E-mail: baoliu@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China)

    2013-03-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  16. Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

    Energy Technology Data Exchange (ETDEWEB)

    Sontag, Ryan L.; Weber, Thomas J.

    2012-05-04

    In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediated toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.

  17. Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase.

    Science.gov (United States)

    Byeon, Yeong; Back, Kyoungwhan

    2016-08-01

    Melatonin is a well-known bioactive molecule produced in animals and plants and a well-studied natural compound. Two enzymatic steps are required for the biosynthesis of melatonin from serotonin. First, serotonin N-acetyltransferase (SNAT) catalyzes serotonin to N-acetylserotonin (NAS) followed by the action of N-acetylserotonin O-methyltransferase (ASMT), resulting in the synthesis of O-methylated NAS, also known as melatonin. Attempts to document melatonin production in Escherichia coli have been unsuccessful to date due to either low enzyme activity or inactive ASMT expression. Here, we employed caffeic acid O-methyltransferase (COMT) instead of ASMT, as COMT is a multifunctional enzyme that has ASMT activity as well. Among several combinations of dual expression cassettes, recombinant E. coli that expressed sheep SNAT with rice COMT produced a high quantity of melatonin, which was measured in a culture medium (1.46 mg/L in response to 1 mM serotonin). This level was several orders of magnitude higher than that produced in transgenic rice and tomato overexpressing sheep SNAT and ASMT, respectively. This heterologous expression system can be widely employed to screen various putative SNAT or ASMT genes from animals and plants as well as to overproduce melatonin in various useful microorganisms. PMID:27005412

  18. Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1

    International Nuclear Information System (INIS)

    Isolated modules of mouse coactivator-associated arginine methyltransferase 1 encompassing the protein arginine N-methyltransferase catalytic domain have been overexpressed, purified and crystallized. X-ray diffraction data have been collected and have enabled determination of the structures by multiple isomorphous replacement using anomalous scattering. Coactivator-associated arginine methyltransferase 1 (CARM1) plays a crucial role in gene expression as a coactivator of several nuclear hormone receptors and also of non-nuclear receptor systems. Its recruitment by the transcriptional machinery induces protein methylation, leading to chromatin remodelling and gene activation. CARM128–507 and two structural states of CARM1140–480 were expressed, purified and crystallized. Crystals of CARM128–507 belong to space group P6222, with unit-cell parameters a = b = 136.0, c = 125.3 Å; they diffract to beyond 2.5 Å resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM128–507 was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1140–480 belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 Å; they diffract to beyond 2.7 Å resolution and contain two monomers in the asymmetric unit. Crystals of CARM1140–480 in complex with S-adenosyl-l-homocysteine belong to space P21212, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 Å; they diffract to beyond 2.6 Å resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1140–480 were solved by molecular-replacement techniques from the structure of CARM128–507

  19. Expression of the O6-methylguanine-DNA methyltransferase gene MGMT in MER+ and MER- human tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Fornace, A.J. Jr.; Papathanasiou, M.A.; Hollander, M.C.; Yarosh, D.B. (National Cancer Institute, Bethesda, MD (USA))

    1990-12-15

    DNA probes prepared from human O6-methylguanine-DNA methyltransferase complementary DNA were hybridized to mRNA isolated from human liver and fifteen human tumor cell lines proficient (Mer+) or deficient (Mer-) in transferase activity. Liver and Mer+ cells contained levels of transferase-specific mRNA that correlated with their transferase activity levels, whereas Mer- cells contained undetectable amounts of transferase mRNA. The mRNA levels were not induced in human cells by treatments that induce other DNA damage-inducible genes. These results demonstrate that in human cells the transferase gene is constitutively expressed, that its expression is related to activity levels, and that in Mer- tumor cells the expression of the transferase gene is probably blocked at the level of mRNA production.

  20. Immunohistochemical Assessment of O6-Methylguanine-DNA Methyltransferase (MGMT) and Its Relationship with p53 Expression in Endometrial Cancers

    Science.gov (United States)

    Lee, Kyung Eun

    2013-01-01

    O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein, the loss of MGMT expression was commonly known due to hypermethylation of CpG islands in its promoter region. Overexpression of p53 protein may be associated with downregulated MGMT expression in brain tumors. The aims of this study were to investigate the role of MGMT expression loss and its correlation with p53 overexpression in endometrial cancers. MGMT and p53 expression was examined in formalin-fixed, paraffin-embedded tissues from 36 endometrial cancer cases using immnunohistochemical staining. The loss of MGMT expression was detected in 11 (30.6%) out of the 36 endometrial cancers and p53 immunoreactivity was detected in 23 (63.9%) out of the 36 endometrial cancers. Ten (90.9%) of the 11 cases with negative MGMT immunoreactivity showed positive p53 expression, so the loss of MGMT expression was significantly associated with the p53 overexpression (P=0.03). These findings suggest that the loss of MGMT expression may be one of factors capable of p53 overexpression in endometrial cancer. Further studies are needed to define the relation between MGMT and p53 for examining the mechanisms of tissue-specific MGMT expression. PMID:25337565

  1. The decrease in histone methyltransferase EZH2 in response to fluid shear stress alters endothelial gene expression and promotes quiescence.

    Science.gov (United States)

    Maleszewska, Monika; Vanchin, Byambasuren; Harmsen, Martin C; Krenning, Guido

    2016-01-01

    High uniform fluid shear stress (FSS) is atheroprotective and preserves the endothelial phenotype and function through activation of downstream mediators such as MAPK7 (Erk5). Endothelial cells respond to FSS thanks to mechanotransduction. However, how the resulting signaling is integrated and resolved at the epigenetic level remains elusive. We hypothesized that Polycomb methyltransferase EZH2 is involved in the effects of FSS in human endothelial cells. We showed that FSS decreases the expression of the Polycomb methyltransferase EZH2. Despite simultaneous activation of MAPK7, MAPK7 pathway does not directly influence the transcription of EZH2. Interestingly though, the knockdown of EZH2 activates the protective MAPK7 signaling in endothelial cells, even in the absence of FSS. To understand the influence of the FSS-decreased expression of EZH2 on endothelial transcriptome, we performed RNA-seq and differential gene expression analysis. We identified candidate groups of genes dependent on both EZH2 and FSS. Among those, Gene Ontology overrepresentation analysis revealed highly significant enrichment of the cell cycle-related genes, suggesting changes in proliferation. Indeed, the depletion of EZH2 strongly inhibited endothelial proliferation, indicating cell cycle arrest. The concomitant decrease in CCNA expression suggests the transition of endothelial cells into a quiescent phenotype. Further bioinformatical analysis suggested TXNIP as a possible mediator between EZH2 and cell cycle-related gene network. Our data show that EZH2 is a FSS-responsive gene. Decreased EZH2 levels enhance the activation of the atheroprotective MAPK7 signaling. Decrease in EZH2 under FSS mediates the decrease in the expression of the network of cell cycle-related genes, which allows the cells to enter quiescence. EZH2 is therefore important for the protective effects of FSS in endothelium. PMID:26416763

  2. O-methylguanine-DNA methyltransferase (MGMT mRNA expression predicts outcome in malignant glioma independent of MGMT promoter methylation.

    Directory of Open Access Journals (Sweden)

    Simone Kreth

    Full Text Available BACKGROUND: We analyzed prospectively whether MGMT (O(6-methylguanine-DNA methyltransferase mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. METHODOLOGY/PRINCIPAL FINDINGS: ADULT PATIENTS WITH A HISTOLOGICALLY PROVEN MALIGNANT ASTROCYTOMA (GLIOBLASTOMA: N = 53, anaplastic astrocytoma: N = 10 were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA database (N = 229 glioblastomas. Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001; the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001; however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6 did significantly worse than those with low transcriptional activity (p<0.01. Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6 did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001. Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT m

  3. Verminderte Expression von O6-Methylguanin-DNA-Methyltransferase bei Gliomen durch Promotormethylierung

    OpenAIRE

    Nickolay, Carla

    2009-01-01

    Pro Jahr erkranken 10 von 100 000 Menschen an einem malignen Hirntumor. Die Prognose ist schlecht. Die Therapie schließt Chirurgie, Bestrahlung und Chemotherapie ein. Das DNA-Reparaturenzym O6-Methylguanin-DNA-Methyltransferase, MGMT, erkennt DNA-Schäden, die durch alkylierende Substanzen wie Temozolomid entstanden sind. Übermäßige Methylierung der Promotorregion von MGMT führt zum Funktionsverlust des Enzyms. Dieser kann zu einer erhöhten Sensibilität für Alkylanzien führen. In der Therapie ...

  4. Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

    International Nuclear Information System (INIS)

    A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A)+ RNA

  5. Expression, Purification and Activity Assay of the Recombinant Protein of Catechol-O-Methyltransferase from Chinese White Shrimp (Fenneropenaeus chinensis

    Directory of Open Access Journals (Sweden)

    Dian-Xiang Li

    2010-01-01

    Full Text Available Problem statement: We have previously cloned a gene of Chinese white shrimp Catechol O-Methyltransferase (designated Fc-COMT and characterized the gene expression pattern. In this study, expression and purification as well as activity assay of the recombinant Fc-COMT was further conducted. Approach: Using pET-30a (+ as a prokaryotic expression vector, the recombinant Fc- COMT was expressed in the supernatant of Escherichia coli lysate and easily purified by His-Bind resin chromatography. SDS-PAGE analysis showed that the molecular mass of recombinant Fc-COMT was approximately 30,000 Da, in good agreement with the software-predicted molecular weight. The enzymatic activity of recombinant Fc-COMT was tested using Dihydroxybenzoic Acid (DHBAc as a substrate. Results: The methyl products of DHBAc, Vanillic Acid (VA and Isovanillic Acid (IVA, were detected in the enzymatic reaction mixture with recombinant Fc-COMT by High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS. Conclusion: The recombinant Fc-COMT has catalytic activity of transferring methyl group from S-Adenosyl-L-Methionine (SAM to the 3’ hydroxyl or 4’ hydroxyl group of benzyl ring of DHBAc.

  6. Clinical significance of epigenetic silencing and re-expression of O6-methylguanine-DNA methyltransferase using epigenetic agents in laryngeal carcinoma

    OpenAIRE

    Yang, Jing; ZHU, XIN-BING; He, Li-xia; GU, ZHAO-WEI; JIN, MING-ZHU; JI, WEN-YUE

    2014-01-01

    The aim of the present study was to investigate the association between O6-methylguanine-DNA methyltransferase (MGMT) gene expression levels, and DNA methylation status and histone modifications in laryngeal squamous cell carcinoma (LSCC). Chromatin immunoprecipitation, methylation-specific polymerase chain reaction (PCR), and reverse transcription-quantitative PCR were performed to analyze histone modifications, DNA methylation status and mRNA expression levels in the promoter region of the ...

  7. The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development

    Science.gov (United States)

    Ortega-Molina, Ana; Boss, Isaac W.; Canela, Andres; Pan, Heng; Jiang, Yanwen; Zhao, Chunying; Jiang, Man; Hu, Deqing; Agirre, Xabier; Niesvizky, Itamar; Lee, Ji-Eun; Chen, Hua-Tang; Ennishi, Daisuke; Scott, David W.; Mottok, Anja; Hother, Christoffer; Liu, Shichong; Cao, Xing-Jun; Tam, Wayne; Shaknovich, Rita; Garcia, Benjamin A.; Gascoyne, Randy D.; Ge, Kai; Shilatifard, Ali; Elemento, Olivier; Nussenzweig, Andre; Melnick, Ari M.; Wendel, Hans-Guido

    2015-01-01

    The lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL). However, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center (GC) involution, impedes B cell differentiation and class switch recombination (CSR). Integrative genomic analyses indicate that KMT2D affects H3K4 methylation and expression of a specific set of genes including those in the CD40, JAK-STAT, Toll-like receptor, and B cell receptor pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3, and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell activating pathways. PMID:26366710

  8. The expression of spinal methyl-CpG-binding protein 2, DNA methyltransferases and histone deacetylases is modulated in persistent pain states

    Directory of Open Access Journals (Sweden)

    Tochiki Keri K

    2012-02-01

    Full Text Available Abstract Background DNA CpG methylation is carried out by DNA methyltransferases and induces chromatin remodeling and gene silencing through a transcription repressor complex comprising the methyl-CpG-binding protein 2 (MeCP2 and a subset of histone deacetylases. Recently, we have found that MeCP2 activity had a crucial role in the pattern of gene expression seen in the superficial dorsal horn rapidly after injection of Complete Freund's Adjuvant (CFA in the rat ankle joint. The aim of the present study was to analyse the changes in expression of MeCP2, DNA methyltransferases and a subset of histone deacetylases in the superficial dorsal horn during the maintenance phase of persistent pain states. In this process, the cell specific expression of MeCP2 was also investigated. Results Using immunohistochemistry, we found that neurones, oligodendrocytes and astrocytes expressed MeCP2. Microglia, oligodendrocyte precursor cells and Schwann cells never showed any positive stain for MeCP2. Quantitative analyses showed that MeCP2 expression was increased in the superficial dorsal horn 7 days following CFA injection in the ankle joint but decreased 7 days following spared nerve injury. Overall, the expression of DNA methyltransferases and a subset of histone deacetylases followed the same pattern of expression. However, there were no significant changes in the expression of the MeCP2 targets that we had previously shown are regulated in the early time points following CFA injection in the ankle joint. Finally, the expression of MeCP2 was also down regulated in damaged dorsal root ganglion neurones following spared nerve injury. Conclusion Our results strongly suggest that changes in chromatin compaction, regulated by the binding of MeCP2 complexes to methylated DNA, are involved in the modulation of gene expression in the superficial dorsal horn and dorsal root ganglia during the maintenance of persistent pain states.

  9. Expression of O6-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue

    OpenAIRE

    Ishiguro, Kimiko; Shyam, Krishnamurthy; Penketh, Philip G.; Baumann, Raymond P.; Sartorelli, Alan C.; Rutherford, Thomas J.; Ratner, Elena S.

    2013-01-01

    The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions, depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive ...

  10. In planta production of the highly potent resveratrol analogue pterostilbene via stilbene synthase and O-methyltransferase co-expression

    Energy Technology Data Exchange (ETDEWEB)

    Rimando A. M.; Liu C.; Pan, Z.; Polashock, J. J.; Dayan, F. E., Mizuno, C. S.; Snook, M. E.; Baerson, S. R.

    2012-04-01

    Resveratrol and related stilbenes are thought to play important roles in defence responses in several plant species and have also generated considerable interest as nutraceuticals owing to their diverse health-promoting properties. Pterostilbene, a 3,5-dimethylether derivative of resveratrol, possesses properties similar to its parent compound and, additionally, exhibits significantly higher fungicidal activity in vitro and superior pharmacokinetic properties in vivo. Recombinant enzyme studies carried out using a previously characterized O-methyltransferase sequence from Sorghum bicolor (SbOMT3) demonstrated its ability to catalyse the A ring-specific 3,5-bis-O-methylation of resveratrol, yielding pterostilbene. A binary vector was constructed for the constitutive co-expression of SbOMT3 with a stilbene synthase sequence from peanut (AhSTS3) and used for the generation of stably transformed tobacco and Arabidopsis plants, resulting in the accumulation of pterostilbene in both species. A reduced floral pigmentation phenotype observed in multiple tobacco transformants was further investigated by reversed-phase HPLC analysis, revealing substantial decreases in both dihydroquercetin-derived flavonoids and phenylpropanoid-conjugated polyamines in pterostilbene-producing SbOMT3/AhSTS3 events. These results demonstrate the potential utility of this strategy for the generation of pterostilbene-producing crops and also underscore the need for the development of additional approaches for minimizing concomitant reductions in key phenylpropanoid-derived metabolites.

  11. DNA methyltransferase expressions in Japanese rice fish (Oryzias latipes) embryogenesis is developmentally regulated and modulated by ethanol and 5-azacytidine.

    Science.gov (United States)

    Dasmahapatra, Asok K; Khan, Ikhlas A

    2015-01-01

    We aimed to investigate the impact of the epigenome in inducting fetal alcohol spectrum disorder (FASD) phenotypes in Japanese rice fish embryogenesis. One of the significant events in epigenome is DNA methylation which is catalyzed by DNA methyltransferase (DNMT) enzymes. We analyzed DNMT enzyme mRNA expressions in Japanese rice fish development starting from fertilized eggs to hatching and also in embryos exposed for first 48h of development either to ethanol (300mM) or to 5-azacytidine (5-azaC; 2mM), an inhibitor of DNMT enzyme activity. As observed in FASD phenotypes, 5-azaC exposure was able to induce microcephaly and craniofacial cartilage deformities in Japanese rice fish. Moreover, we have observed that expression of DNMTs (dnmt1, dnmt3aa, and dnmt3bb.1) are developmentally regulated; high mRNA copies were found in early stages (1-2day-post-fertilization, dpf), followed by gradual reduction until hatched. In ethanol-treated embryos, compared to controls, dnmt1 mRNA is in reduced level in 2dpf and in enhanced level in 6dpf embryos. While dnmt3aa and 3bb.1 remained unaltered. In contrast, embryos exposed to 5-azaC have an enhanced level of dnmt1 and dnmt3bb.1 mRNAs both in 2 and 6dpf embryos while dnmt3aa is enhanced only in 6dpf embryos. Moreover, endocannabinoid receptor 1a (cnr1a) mRNA which was found to be reduced by ethanol remained unaltered and cnr1b and cnr2 mRNAs, which were remained unaltered by ethanol, were increased significantly by 5-azaC in 6dpf embryos. This study indicates that the craniofacial defects observed in FASD phenotypes are the results of dysregulations in DNMT expressions. PMID:26183885

  12. Interplay among coactivator-associated arginine methyltransferase 1, CBP, and CIITA in IFN-gamma-inducible MHC-II gene expression.

    Science.gov (United States)

    Zika, Eleni; Fauquier, Lucas; Vandel, Laurence; Ting, Jenny P-Y

    2005-11-01

    Class II major histocompatibility (MHC-II) genes are prototype targets of IFN-gamma. IFN-gamma activates the expression of the non-DNA-binding master regulator of MHC-II, class II transactivator (CIITA), which is crucial for enhanceosome formation and gene activation. This report shows the importance of the histone methyltransferase, coactivator-associated arginine methyltransferase (CARM1/PRMT4), during IFN-gamma-induced MHC-II gene activation. It also demonstrates the coordinated regulation of CIITA, CARM1, and the acetyltransferase cyclic-AMP response element binding (CREB)-binding protein (CBP) during this process. CARM1 synergizes with CIITA in activating MHC-II transcription and synergy is abrogated when an arginine methyltransferase-defective CARM1 mutant is used. Protein-arginine methyltransferase 1 has much less effect on MHC-II transcription. Specific RNA interference reduced CARM1 expression as well as MHC-II expression. The recruitment of CARM1 to the promoter requires endogenous CIITA and results in methylation of histone H3-R17; hence, CIITA is an upstream regulator of histone methylation. Previous work has shown that CARM1 can methylate CBP at three arginine residues. Using wild-type CBP and a mutant of CBP lacking the CARM1-targeted arginine residues (R3A), we show that arginine methylation of CBP is required for IFN-gamma induction of MHC-II. A kinetic analysis shows that CIITA, CARM1, and H3-R17 methylation all precede CBP loading on the MHC-II promoter during IFN-gamma treatment. These results suggest functional and temporal relationships among CIITA, CARM1, and CBP for IFN-gamma induction of MHC-II. PMID:16254053

  13. TGF-β regulates DNA methyltransferase expression in prostate cancer, correlates with aggressive capabilities, and predicts disease recurrence.

    Directory of Open Access Journals (Sweden)

    Qiang Zhang

    Full Text Available BACKGROUND: DNA methyltransferase (DNMT is one of the major factors mediating the methylation of cancer related genes such as TGF-β receptors (TβRs. This in turn may result in a loss of sensitivity to physiologic levels of TGF-β in aggressive prostate cancer (CaP. The specific mechanisms of DNMT's role in CaP remain undetermined. In this study, we describe the mechanism of TGF-β-mediated DNMT in CaP and its association with clinical outcomes following radical prostatectomy. METHODOLOGY/PRINCIPAL FINDINGS: We used human CaP cell lines with varying degrees of invasive capability to describe how TGF-β mediates the expression of DNMT in CaP, and its effects on methylation status of TGF-β receptors and the invasive capability of CaP in vitro and in vivo. Furthermore, we determined the association between DNMT expression and clinical outcome after radical prostatectomy. We found that more aggressive CaP cells had significantly higher TGF-β levels, increased expression of DNMT, but reduced TβRs when compared to benign prostate cells and less aggressive prostate cancer cells. Blockade of TGF-β signaling or ERK activation (p-ERK was associated with a dramatic decrease in the expression of DNMT, which results in a coincident increase in the expression of TβRs. Blockade of either TGF-β signaling or DNMT dramatically decreased the invasive capabilities of CaP. Inhibition of TGF-β in an TRAMP-C2 CaP model in C57BL/6 mice using 1D11 was associated with downregulation of DNMTs and p-ERK and impairment in tumor growth. Finally, independent of Gleason grade, increased DNMT1 expression was associated with biochemical recurrence following surgical treatment for prostate cancer. CONCLUSIONS AND SIGNIFICANCE: Our findings demonstrate that CaP derived TGF-β may induce the expression of DNMTs in CaP which is associated with methylation of its receptors and the aggressive potential of CaP. In addition, DNMTs is an independent predictor for disease

  14. Continuous and low-energy 125I seed irradiation changes DNA methyltransferases expression patterns and inhibits pancreatic cancer tumor growth

    Directory of Open Access Journals (Sweden)

    Gong Yan-fang

    2011-04-01

    Full Text Available Abstract Background Iodine 125 (125I seed irradiation is an effective treatment for unresectable pancreatic cancers. However, the radiobiological mechanisms underlying brachytherapy remain unclear. Therefore, we investigated the influence of continuous and low-energy 125I irradiation on apoptosis, expression of DNA methyltransferases (DNMTs and cell growth in pancreatic cancers. Materials and methods For in vitro 125I seed irradiation, SW-1990 cells were divided into three groups: control (0 Gy, 2 Gy, and 4 Gy. To create an animal model of pancreatic cancer, the SW 1990 cells were surgically implanted into the mouse pancreas. At 10 d post-implantation, the 30 mice with pancreatic cancer underwent 125I seed implantation and were separated into three groups: 0 Gy, 2 Gy, and 4 Gy group. At 48 or 72 h after irradiation, apoptosis was detected by flow cytometry; changes in DNMTs mRNA and protein expression were assessed by real-time PCR and western blotting analysis, respectively. At 28 d after 125I seed implantation, in vivo apoptosis was evaluated with TUNEL staining, while DNMTs protein expression was detected with immunohistochemical staining. The tumor volume was measured 0 and 28 d after 125I seed implantation. Results 125I seed irradiation induced significant apoptosis, especially at 4 Gy. DNMT1 and DNMT3b mRNA and protein expression were substantially higher in the 2 Gy group than in the control group. Conversely, the 4 Gy cell group exhibited significantly decreased DNMT3b mRNA and protein expression relative to the control group. There were substantially more TUNEL positive in the 125I seed implantation treatment group than in the control group, especially at 4 Gy. The 4 Gy seed implantation group showed weaker staining for DNMT1 and DNMT3b protein relative to the control group. Consequently, 125I seed implantation inhibited cancer growth and reduced cancer volume. Conclusion 125I seed implantation kills pancreatic cancer cells, especially

  15. The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases

    Institute of Scientific and Technical Information of China (English)

    Chi-NeuTsai

    2005-01-01

    The latent membrane protein (LMP1) of Epstein-Barr virus (EBV) is expressed in EBV-associated nasopharyngeal carcinoma, which isnotoriously metastatic. Although it Is established that LMP1 represses E-cadherin expression and enhances the invasive ability of carcinoma cells, the mechanism underlying this repression remains to be elucidated. In this study, we demonstrate that LMP1 induces the expression and activity of the DNA methyltransferases 1, 3a, and 3b, using real-time reverse transcription-PCR and enzyme activity assay. This results in hypermethylation of the E-cadherin promoter and down-regulation of E-cadherin gene expression, as revealed by methylation-specific PCR, real-time reverse transcription-PeR and Western blotting data. The DNA methyltransferase inhibitor, 5'-Aza-2'dC, restores E-cadherin promoter activity and protein expression in LMPl-expressing cells, which in turn blocks cell migration ability, as demonstrated by the Transwell cell migration assay. Our findings suggest that LMP1 down-regulates E-cadherin gene expression and induces cell migration activity by using cellular DNA methylation machinery.

  16. Recombinant expression of rat glycine N-methyltransferase and evidence for contribution of N-terminal acetylation to co-operative binding of S-adenosylmethionine.

    Science.gov (United States)

    Ogawa, H; Gomi, T; Takata, Y; Date, T; Fujioka, M

    1997-10-15

    An expression vector was constructed that produced rat glycine N-methyltransferase in Escherichia coli. Recombinant glycine N-methyltransferase was purified to homogeneity by DEAE-cellulose and gel-filtration chromatography, with a yield of more than 80 mg of pure enzyme from a 1 litre culture. HPLC of tryptic peptides and analysis of isolated peptides showed that the recombinant enzyme was structurally identical with the liver enzyme except for the absence of N-terminal blocking. The alpha-amino group of rat glycine N-methyltransferase is blocked by acetylation [Ogawa, Konishi, Takata, Nakashima and Fujioka (1987) Eur. J. Biochem. 168, 141-151]. In contrast with the liver enzyme, which shows sigmoidal kinetics toward S-adenosylmethionine at all pH values tested [Ogawa and Fujioka (1982) J. Biol. Chem. 257, 3447-3452], the recombinant enzyme exhibited hyperbolic kinetics at low pH and sigmoidal rate behaviour at high pH. The Hill coefficient increased with increasing pH and a pKa of 8.11 was obtained in this transition. The values of Vmax and Km for glycine were not different between the two enzymes. These results suggest that elimination of the positive charge at the N-terminal end either by acetylation or deprotonation is required for co-operative behaviour. PMID:9359408

  17. Cloning, expression, purification and initial crystallographic studies of UbiG: a methyltransferase involved in ubiquinone biosynthesis in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Costa, M.A.F.; Magalhaes, R.D.; Nagem, R.A.P. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil); Ferreira-Junior, J.R.; Barros, M.H. [Universidade de Sao Paulo (USP), SP (Brazil)

    2012-07-01

    Full text: Ubiquinone is a molecule that functions as an electron carrier in the respiratory chain in living organisms. Some clinical phenotypes, including, encephalomyopathy, has been associated with ubiquinone deficiency, raising the interest in the biosynthetic pathway of this molecule. This pathway was proposed mainly from the results of the genetic analysis of mutants of E. coli. UbiG is a methyltransferase involved in ubiquinone biosynthesis in E. coli. In this work we have cloned, expressed, purified and made initial crystallographic assessments of UbiG for later determination of its three-dimensional structure. The gene encoding UbiG was amplified from E. coli genomic DNA by polymerase chain reaction. The 753 bases pairs amplicon was inserted into the expression plasmid pMCSG7 by ligation independent cloning system and transformed into BL21(DE3) E. coli strain. The expression of UbiG, verified by SDS polyacrylamide gel, showed a protein of approximately 29kDa after IPTG induction. The recombinant UbiG, in the soluble fraction of the cellular lysate, was purified by affinity chromatography and the molecular weight of recombinant UbiG of approximately 29 kDa was confirmed by mass spectrometry. After removal of His-tag by TEV protease, another affinity chromatography was performed and UbiG, without His-tag, was observed in flow-through fraction. In Size-Exclusion Chromatography (SEC), the recombinant UbiG showed a unique peak with correct molecular weight of a monomer. Analysis of CD indicated that recombinant UbiG has 31,80% of alpha helix at 20 deg C and DLS showed that 70.9% of the sample is still monomeric in solution even five days after purification. Initial crystallization studies were performed with Crystal Screen 1 and Crystal Screen 2 from Hampton Research. Needle-shaped microcrystals of UbiG were obtained using a precipitant solution consisting of 0,1M lithium sulfate, 0,1M Tris pH 7,5 and 30% w/v polyethylene glycol 4,000. (author)

  18. Cloning, expression, purification and initial crystallographic studies of UbiG: a methyltransferase involved in ubiquinone biosynthesis in Escherichia coli

    International Nuclear Information System (INIS)

    Full text: Ubiquinone is a molecule that functions as an electron carrier in the respiratory chain in living organisms. Some clinical phenotypes, including, encephalomyopathy, has been associated with ubiquinone deficiency, raising the interest in the biosynthetic pathway of this molecule. This pathway was proposed mainly from the results of the genetic analysis of mutants of E. coli. UbiG is a methyltransferase involved in ubiquinone biosynthesis in E. coli. In this work we have cloned, expressed, purified and made initial crystallographic assessments of UbiG for later determination of its three-dimensional structure. The gene encoding UbiG was amplified from E. coli genomic DNA by polymerase chain reaction. The 753 bases pairs amplicon was inserted into the expression plasmid pMCSG7 by ligation independent cloning system and transformed into BL21(DE3) E. coli strain. The expression of UbiG, verified by SDS polyacrylamide gel, showed a protein of approximately 29kDa after IPTG induction. The recombinant UbiG, in the soluble fraction of the cellular lysate, was purified by affinity chromatography and the molecular weight of recombinant UbiG of approximately 29 kDa was confirmed by mass spectrometry. After removal of His-tag by TEV protease, another affinity chromatography was performed and UbiG, without His-tag, was observed in flow-through fraction. In Size-Exclusion Chromatography (SEC), the recombinant UbiG showed a unique peak with correct molecular weight of a monomer. Analysis of CD indicated that recombinant UbiG has 31,80% of alpha helix at 20 deg C and DLS showed that 70.9% of the sample is still monomeric in solution even five days after purification. Initial crystallization studies were performed with Crystal Screen 1 and Crystal Screen 2 from Hampton Research. Needle-shaped microcrystals of UbiG were obtained using a precipitant solution consisting of 0,1M lithium sulfate, 0,1M Tris pH 7,5 and 30% w/v polyethylene glycol 4,000. (author)

  19. EXPRESSION OF THE O6-METHYLGUANINE-DNA METHYLTRANSFERASE GENE IN EIGHT HUMAN TUMOR CELL LINES

    Institute of Scientific and Technical Information of China (English)

    陈建敏; 章扬培; 吴英

    1994-01-01

    O6-methylguanine-DNA methltransferase(MGMT) gene expression in 6 Mer+(HeLa S3,SMMC-7721,SGC-7901,B-239,AGZY83-a,and Cc801)and 2Mer-(SHG-44,AND HeLa MR) haman tumor cell lines was examined.Southern blot analysis revealed no deletion,amplification,or rearrangement of the MGMT gene in these cell lines.However,-1.0kb transcripts were detected in the 6 Mer+ cell lines but not in the 2 Mer- cell lines by Northern blot analysis.Furthermore,a rough correlation between MGMT activity and mRNA level in these cell lines was observed.These results suggest that transcriptional regulation of the MGMT gene is the molecular basis of the absence of MGMT activity in Mer- cell lines.

  20. Impaired Homocysteine Transmethylation and Protein-Methyltransferase Activity Reduce Expression of Selenoprotein P: Implications for Obesity and Metabolic Syndrome

    Science.gov (United States)

    Obesity causes Metabolic Syndrome and Type-II Diabetes, disrupting hepatic function, methionine (Met)/homocysteine (Hcy) transmethylation and methyltransferase (PRMT) activities. Selenoprotein P (SEPP1), exported from the liver, is the predominate form of plasma selenium (Se) and the physiological S...

  1. Molecular cloning, characterization and expression of the caffeic acid O-methyltransferase (COMT) ortholog from kenaf (Hibiscus cannabinus)

    Science.gov (United States)

    We cloned the full-length of the gene putatively encoding caffeic acid O-methyltransferase (COMT) from kenaf (Hibiscus cannabinus L.) using degenerate primers and the RACE (rapid amplification of cDNA ends) method. Kenaf is an herbaceous and rapidly growing dicotyledonous plant with great potential ...

  2. Direct methylation of FXR by Set7/9, a lysine methyltransferase, regulates the expression of FXR target genes

    OpenAIRE

    Balasubramaniyan, Natarajan; Ananthanarayanan, Meena; Suchy, Frederick J.

    2012-01-01

    The farnesoid X receptor (FXR) is a ligand (bile acid)-dependent nuclear receptor that regulates target genes involved in every aspect of bile acid homeostasis. Upon binding of ligand, FXR recruits an array of coactivators and associated proteins, some of which have intrinsic enzymatic activity that modify histones or even components of the transcriptional complex. In this study, we show chromatin occupancy by the Set7/9 methyltransferase at the FXR response element (FXRE) and direct methylat...

  3. Histone methyltransferases in cancer

    DEFF Research Database (Denmark)

    Albert, Mareike; Helin, Kristian

    2009-01-01

    Cancer is perceived as a heterogeneous group of diseases that is characterized by aberrant patterns of gene expression. In the last decade, an increasing amount of data has pointed to a key role for epigenetic alterations in human cancer. In this review, we focus on a subclass of epigenetic...... regulators, namely histone methyltransferases (HMTs). Several HMTs have been linked to different types of cancer; however, in most cases we only have limited knowledge regarding the molecular mechanisms by which the HMTs contribute to disease development. We summarize the current knowledge regarding some of...

  4. Cloning, expression, purification, crystallization and preliminary X-ray analysis of NodS N-methyltransferase from Bradyrhizobium japonicum WM9

    International Nuclear Information System (INIS)

    The NodS N-methyltransferase, an enzyme participating in the biosynthesis of the bacterial nodulation (Nod) factor necessary to establish symbiotic nitrogen fixation with a legume plant host, has been crystallized in the apo form as well as in complex with SAH. SAH is a byproduct of SAM degradation during the SAM-dependent methylation reaction. The Nod factor (NF) is a rhizobial signal molecule that is involved in recognition of a legume host and the formation of root and stem nodules. Some unique enzymes are involved in the biosynthesis of NF, which is a variously but specifically substituted lipochitooligosaccharide. One of these enzymes is NodS, an N-methyltransferase that methylates end-deacetylated chitooligosaccharide substrates. In the methylation reaction, NodS uses S-adenosyl-l-methionine (SAM) as a methyl donor. To date, no structural information is available about NodS from any rhizobium. X-ray crystallographic studies of the NodS protein from Bradyrhizobium japonicum WM9, which infects the legumes lupin and serradella, have been undertaken. The nodS gene was cloned and the recombinant protein was expressed in Escherichia coli cells using natural amino acids and as an SeMet derivative. NodS without ligands was crystallized in the presence of PEG 3350 and MgCl2. The protein was also crystallized in complex with S-adenosyl-l-homocysteine (SAH) in the presence of PEG 8000 and MgCl2. SAH is produced from SAM as a byproduct of the methylation reaction. The crystals of apo NodS are tetragonal and diffracted X-rays to 2.42 Å resolution. The NodS–SAH complex crystallizes in an orthorhombic space group and the crystals diffracted X-rays to 1.85 Å resolution

  5. Methylation-related chromatin structure is associated with exclusion of transcription factors from and suppressed expression of the O-6-methylguanine DNA methyltransferase gene in human glioma cell lines.

    OpenAIRE

    Costello, J F; Futscher, B.W.; Kroes, R A; Pieper, R O

    1994-01-01

    There is considerable interest in identifying factors responsible for expression of the O-6-methylguanine DNA methyltransferase (MGMT) gene, as MGMT is a major determinant in the response of glioma cells to the chemotherapeutic agent 1,3 bis(2-chloroethyl)-1-nitrosourea. Recently we have shown that MGMT expression is correlated in a direct, graded fashion with methylation in the body of the MGMT gene and in an inverse, graded fashion with promoter methylation in human glioma cell lines. To de...

  6. Benzo[a]pyrene diol epoxide suppresses retinoic acid receptor-β2 expression by recruiting DNA (cytosine-5--methyltransferase 3A

    Directory of Open Access Journals (Sweden)

    Xu Xiao-Chun

    2010-04-01

    Full Text Available Abstract Tobacco smoke is an important risk factor for various human cancers, including esophageal cancer. How benzo [a]pyrene diol epoxide (BPDE, a carcinogen present in tobacco smoke as well as in environmental pollution, induces esophageal carcinogenesis has yet to be defined. In this study, we investigated the molecular mechanism responsible for BPDE-suppressed expression of retinoic acid receptor-beta2 (RAR-β2 in esophageal cancer cells. We treated esophageal cancer cells with BPDE before performing methylation-specific polymerase chain reaction (MSP to find that BPDE induced methylation of the RAR-β2 gene promoter. We then performed chromatin immunoprecipitation (ChIP assays to find that BPDE recruited genes of the methylation machinery into the RAR-β2 gene promoter. We found that BPDE recruited DNA (cytosine-5--methyltransferase 3 alpha (DNMT3A, but not beta (DNMT3B, in a time-dependent manner to methylate the RAR-β2 gene promoter, which we confirmed by reverse transcription-polymerase chain reaction (RT-PCR analysis of the reduced RAR-β2 expression in these BPDE-treated esophageal cancer cell lines. However, BPDE did not significantly change DNMT3A expression, but it slightly reduced DNMT3B expression. DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-Aza and DNMT3A small hairpin RNA (shRNA vector antagonized the effects of BPDE on RAR-β2 expressions. Transient transfection of the DNMT3A shRNA vector also antagonized BPDE's effects on expression of RAR-β2, c-Jun, phosphorylated extracellular signal-regulated protein kinases 1/2 (ERK1/2, and cyclooxygenase-2 (COX-2, suggesting a possible therapeutic effect. The results of this study form the link between the esophageal cancer risk factor BPDE and the reduced RAR-β2 expression.

  7. Arginine Methyltransferases Are Regulated by Epstein-Barr Virus in B Cells and Are Differentially Expressed in Hodgkin’s Lymphoma

    Directory of Open Access Journals (Sweden)

    Sarah Leonard

    2012-09-01

    Full Text Available Although there is increasing evidence that aberrant expression of those enzymes which control protein arginine methylation contribute to carcinogenesis, their de-regulation by oncogenic viruses in primary cells has yet to be reported. We first show that the protein arginine methyltransferases, CARM1, PRMT1 and PRMT5 are strongly expressed in Hodgkin Reed-Sternberg (HRS cells, and up-regulated in Hodgkin's lymphoma (HL cell lines. Given that Epstein-Barr virus (EBV can be detected in approximately 50% of primary HL, we next examined how EBV infection of germinal centre (GC B cells, the presumptive precursors of HRS cells, modulated the expression of these proteins. EBV infection of GC B cells was followed by the up-regulation of CARM1, PRMT1 and PRMT5, and by the down-regulation of the arginine deiminase, PADI4. Latent membrane protein 1 (LMP1, the major EBV transforming gene was shown to induce PRMT1 in GC B cells and in a stably transfected B cell line. The recent development of compounds which inhibit PRMT-mediated reactions provides a compelling case for continuing to dissect the contribution of virus induced changes in these proteins to lymphomagenesis.

  8. Developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters DNA methyltransferase (dnmt) expression in zebrafish (Danio rerio)

    Energy Technology Data Exchange (ETDEWEB)

    Aluru, Neelakanteswar, E-mail: naluru@whoi.edu [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Kuo, Elaine [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Stanford University, 450 Serra Mall, Stanford, CA 94305 (United States); Helfrich, Lily W. [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Northwestern University, 633 Clark St, Evanston, IL 60208 (United States); Karchner, Sibel I. [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Linney, Elwood A. [Department of Molecular Genetics and Microbiology, Duke University Medical Center, Box 3020, Durham, NC 27710 (United States); Pais, June E. [New England Biolabs, 240 County Road, Ipswich, MA 01938 (United States); Franks, Diana G. [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States)

    2015-04-15

    DNA methylation is one of the most important epigenetic modifications involved in the regulation of gene expression. The DNA methylation reaction is catalyzed by DNA methyltransferases (DNMTs). Recent studies have demonstrated that toxicants can affect normal development by altering DNA methylation patterns, but the mechanisms of action are poorly understood. Hence, we tested the hypothesis that developmental exposure to TCDD affects dnmt gene expression patterns. Zebrafish embryos were exposed to 5 nM TCDD for 1 h from 4 to 5 h post-fertilization (hpf) and sampled at 12, 24, 48, 72, and 96 hpf to determine dnmt gene expression and DNA methylation patterns. We performed a detailed analysis of zebrafish dnmt gene expression during development and in adult tissues. Our results demonstrate that dnmt3b genes are highly expressed in early stages of development, and dnmt3a genes are more abundant in later stages. TCDD exposure upregulated dnmt1 and dnmt3b2 expression, whereas dnmt3a1, 3b1, and 3b4 are downregulated following exposure. We did not observe any TCDD-induced differences in global methylation or hydroxymethylation levels, but the promoter methylation of aryl hydrocarbon receptor (AHR) target genes was altered. In TCDD-exposed embryos, AHR repressor a (ahrra) and c-fos promoters were differentially methylated. To characterize the TCDD effects on DNMTs, we cloned the dnmt promoters with xenobiotic response elements and conducted AHR transactivation assays using a luciferase reporter system. Our results suggest that ahr2 can regulate dnmt3a1, dnmt3a2, and dnmt3b2 expression. Overall, we demonstrate that developmental exposure to TCDD alters dnmt expression and DNA methylation patterns. - Highlights: • TCDD altered the dnmt expression in a gene and developmental time-specific manner. • TCDD hypermethylated ahrra and hypomethylated c-fos proximal promoter regions. • Functional analysis suggests that ahr2 can regulate dnmt3a1, 3a2, and 3b2 expression. • Dnmt

  9. Developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters DNA methyltransferase (dnmt) expression in zebrafish (Danio rerio)

    International Nuclear Information System (INIS)

    DNA methylation is one of the most important epigenetic modifications involved in the regulation of gene expression. The DNA methylation reaction is catalyzed by DNA methyltransferases (DNMTs). Recent studies have demonstrated that toxicants can affect normal development by altering DNA methylation patterns, but the mechanisms of action are poorly understood. Hence, we tested the hypothesis that developmental exposure to TCDD affects dnmt gene expression patterns. Zebrafish embryos were exposed to 5 nM TCDD for 1 h from 4 to 5 h post-fertilization (hpf) and sampled at 12, 24, 48, 72, and 96 hpf to determine dnmt gene expression and DNA methylation patterns. We performed a detailed analysis of zebrafish dnmt gene expression during development and in adult tissues. Our results demonstrate that dnmt3b genes are highly expressed in early stages of development, and dnmt3a genes are more abundant in later stages. TCDD exposure upregulated dnmt1 and dnmt3b2 expression, whereas dnmt3a1, 3b1, and 3b4 are downregulated following exposure. We did not observe any TCDD-induced differences in global methylation or hydroxymethylation levels, but the promoter methylation of aryl hydrocarbon receptor (AHR) target genes was altered. In TCDD-exposed embryos, AHR repressor a (ahrra) and c-fos promoters were differentially methylated. To characterize the TCDD effects on DNMTs, we cloned the dnmt promoters with xenobiotic response elements and conducted AHR transactivation assays using a luciferase reporter system. Our results suggest that ahr2 can regulate dnmt3a1, dnmt3a2, and dnmt3b2 expression. Overall, we demonstrate that developmental exposure to TCDD alters dnmt expression and DNA methylation patterns. - Highlights: • TCDD altered the dnmt expression in a gene and developmental time-specific manner. • TCDD hypermethylated ahrra and hypomethylated c-fos proximal promoter regions. • Functional analysis suggests that ahr2 can regulate dnmt3a1, 3a2, and 3b2 expression. • Dnmt

  10. Molecular cloning and characterization of juvenile hormone acid methyltransferase in the honey bee, Apis mellifera, and its differential expression during caste differentiation.

    Science.gov (United States)

    Li, Wenfeng; Huang, Zachary Y; Liu, Fang; Li, Zhiguo; Yan, Limin; Zhang, Shaowu; Chen, Shenglu; Zhong, Boxiong; Su, Songkun

    2013-01-01

    Juvenile hormone acid methyltransferase (JHAMT) is an enzyme involved in one of the final steps of juvenile hormone biosynthesis in insects. It transfers a methyl group from S-adenosyl-L-methionine (SAM) to the carboxyl group of either farnesoic acid (FA) or JH acid (JHA). Several genes coding for JHAMT have been cloned and characterized from insects from different orders, and they have been shown to play critical roles in metamorphosis and reproduction. However, the significance of JHAMT in Hymenopteran insects is unknown. We used RACE amplification method to clone JHAMT cDNA from the honey bee, Apis mellifera (AmJHAMT). The full length cDNA of AmJHAMT that we cloned is 1253bp long and encodes a 278-aa protein that shares 32-36% identity with known JHAMTs. A SAM-binding motif, conserved in the SAM-dependent methyltransferase (SAM-MT) superfamily, is present in AmJHAMT. Its secondary structure also contains a typical SAM-MT fold. Most of the active sites bound with SAM and substrates (JHA or FA) are conserved in AmJHAMT as in other JHAMT orthologs. Phylogenetic analysis clustered AmJHAMT with the other orthologs from Hymenoptera to form a major clade in the phylogenetic tree. Purified recombinant AmJHAMT protein expressed in E. coli was used to produce polyclonal antibodies and to verify the identity of AmJHAMT by immunoblotting and mass spectrometry. Quantitative RT-PCR and immunoblotting analyses revealed that queen larvae contained significantly higher levels of AmJHAMT mRNA and protein than worker larvae during the periods of caste development. The temporal profiles of both AmJHAMT mRNA and protein in queens and workers showed a similar pattern as the JH biosynthesis. These results suggest that the gene that we cloned codes for a functional JHAMT that catalyzes the final reactions of JH biosynthesis in honey bees. In addition, AmJHAMT may play an important role in honey bee caste differentiation. PMID:23874662

  11. Molecular cloning and characterization of juvenile hormone acid methyltransferase in the honey bee, Apis mellifera, and its differential expression during caste differentiation.

    Directory of Open Access Journals (Sweden)

    Wenfeng Li

    Full Text Available Juvenile hormone acid methyltransferase (JHAMT is an enzyme involved in one of the final steps of juvenile hormone biosynthesis in insects. It transfers a methyl group from S-adenosyl-L-methionine (SAM to the carboxyl group of either farnesoic acid (FA or JH acid (JHA. Several genes coding for JHAMT have been cloned and characterized from insects from different orders, and they have been shown to play critical roles in metamorphosis and reproduction. However, the significance of JHAMT in Hymenopteran insects is unknown. We used RACE amplification method to clone JHAMT cDNA from the honey bee, Apis mellifera (AmJHAMT. The full length cDNA of AmJHAMT that we cloned is 1253bp long and encodes a 278-aa protein that shares 32-36% identity with known JHAMTs. A SAM-binding motif, conserved in the SAM-dependent methyltransferase (SAM-MT superfamily, is present in AmJHAMT. Its secondary structure also contains a typical SAM-MT fold. Most of the active sites bound with SAM and substrates (JHA or FA are conserved in AmJHAMT as in other JHAMT orthologs. Phylogenetic analysis clustered AmJHAMT with the other orthologs from Hymenoptera to form a major clade in the phylogenetic tree. Purified recombinant AmJHAMT protein expressed in E. coli was used to produce polyclonal antibodies and to verify the identity of AmJHAMT by immunoblotting and mass spectrometry. Quantitative RT-PCR and immunoblotting analyses revealed that queen larvae contained significantly higher levels of AmJHAMT mRNA and protein than worker larvae during the periods of caste development. The temporal profiles of both AmJHAMT mRNA and protein in queens and workers showed a similar pattern as the JH biosynthesis. These results suggest that the gene that we cloned codes for a functional JHAMT that catalyzes the final reactions of JH biosynthesis in honey bees. In addition, AmJHAMT may play an important role in honey bee caste differentiation.

  12. Production of Two Novel Methoxy-Isoflavones from Biotransformation of 8-Hydroxydaidzein by Recombinant Escherichia coli Expressing O-Methyltransferase SpOMT2884 from Streptomyces peucetius

    Directory of Open Access Journals (Sweden)

    Chien-Min Chiang

    2015-11-01

    Full Text Available Biotransformation of 8-hydroxydaidzein by recombinant Escherichia coli expressing O-methyltransferase (OMT SpOMT2884 from Streptomyces peucetius was investigated. Two metabolites were isolated and identified as 7,4′-dihydroxy-8-methoxy-isoflavone (1 and 8,4′-dihydroxy-7-methoxy-isoflavone (2, based on mass, 1H-nuclear magnetic resonance (NMR and 13C-NMR spectrophotometric analysis. The maximum production yields of compound (1 and (2 in a 5-L fermenter were 9.3 mg/L and 6.0 mg/L, respectively. The two methoxy-isoflavones showed dose-dependent inhibitory effects on melanogenesis in cultured B16 melanoma cells under non-toxic conditions. Among the effects, compound (1 decreased melanogenesis to 63.5% of the control at 25 μM. This is the first report on the 8-O-methylation activity of OMT toward isoflavones. In addition, the present study also first identified compound (1 with potent melanogenesis inhibitory activity.

  13. Reduced Euchromatin histone methyltransferase 1 causes developmental delay, hypotonia, and cranial abnormalities associated with increased bone gene expression in Kleefstra syndrome mice.

    Science.gov (United States)

    Balemans, Monique C M; Ansar, Muhammad; Oudakker, Astrid R; van Caam, Arjan P M; Bakker, Brenda; Vitters, Elly L; van der Kraan, Peter M; de Bruijn, Diederik R H; Janssen, Sanne M; Kuipers, Arthur J; Huibers, Manon M H; Maliepaard, Eliza M; Walboomers, X Frank; Benevento, Marco; Nadif Kasri, Nael; Kleefstra, Tjitske; Zhou, Huiqing; Van der Zee, Catharina E E M; van Bokhoven, Hans

    2014-02-15

    Haploinsufficiency of Euchromatin histone methyltransferase 1 (EHMT1), a chromatin modifying enzyme, is the cause of Kleefstra syndrome (KS). KS is an intellectual disability (ID) syndrome, with general developmental delay, hypotonia, and craniofacial dysmorphisms as additional core features. Recent studies have been focused on the role of EHMT1 in learning and memory, linked to the ID phenotype of KS patients. In this study we used the Ehmt1(+/-) mouse model, and investigated whether the core features of KS were mimicked in these mice. When comparing Ehmt1(+/-) mice to wildtype littermates we observed delayed postnatal growth, eye opening, ear opening, and upper incisor eruption, indicating a delayed postnatal development. Furthermore, tests for muscular strength and motor coordination showed features of hypotonia in young Ehmt1(+/-) mice. Lastly, we found that Ehmt1(+/-) mice showed brachycephalic crania, a shorter or bent nose, and hypertelorism, reminiscent of the craniofacial dysmorphisms seen in KS. In addition, gene expression analysis revealed a significant upregulation of the mRNA levels of Runx2 and several other bone tissue related genes in P28 Ehmt1(+/-) mice. Runx2 immunostaining also appeared to be increased. The mRNA upregulation was associated with decreased histone H3 lysine 9 dimethylation (H3K9me2) levels, the epigenetic mark deposited by Ehmt1, in the promoter region of these genes. Together, Ehmt1(+/-) mice indeed recapitulate KS core features and can be used as an animal model for Kleefstra syndrome. The increased expression of bone developmental genes in the Ehmt1(+/-) mice likely contributes to their cranial dysmorphisms and might be explained by diminished Ehmt1-induced H3K9 dimethylation. PMID:24362066

  14. DNA methyltransferase 1, 3a, and 3b expression in hepatitis C associated human hepatocellular carcinoma and their clinicopathological association.

    Science.gov (United States)

    Siddiqui, Nadir Naveed; Ul Haq, Ahtesham; Siddiqui, Owais Ali; Khan, Rizma

    2016-08-01

    Identification of biomarker will obligate a substantial influence on various cancer management and treatment. We hypothesize that genetic/proteomic and epigenetic studies should be uncovering modifications which may be independently or jointly affect the expression of the genes that are involved in the progression of liver cancer (LC). For this purpose, we examined the effect of expressional changes of DNMTs on HCV infected LC of different genotypes. We found that both mRNA and protein expression levels of DNMT1, 3a, and 3b were upregulated in genotype 1b and 3a HCV infected patients as compared to control. However, DNMT3b mRNA levels did not change in genotypes 2a, 3, and 4, but were upregulated at the protein level by genotype 1b, 2a, and 3a. Furthermore, no significant changes were observed for DNMTs investigated in sample expressing the genotypes 5 and 6. Our findings suggest that HCV at least in part by altering DNMTs expression may play a significant role in HCC progression. PMID:26850594

  15. Synthesis of Lysine Methyltransferase Inhibitors

    Science.gov (United States)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  16. The association of betaine, homocysteine and related metabolites with cognitive function in Dutch elderly people

    NARCIS (Netherlands)

    Eussen, S.J.P.M.; Ueland, P.M.; Clarke, R.; Blom, H.J.; Hoefnagels, W.H.L.; Staveren, van W.A.; Groot, de C.P.G.M.

    2007-01-01

    The importance of the one-carbon metabolites, choline and homocysteine, to brain function is well known. However, the associations between the one-carbon metabolites choline, betaine, methionine and dimethylglycine with cognition in elderly are unclear. We therefore examined the associations of thes

  17. The association of betaine, homocysteine and related metabolites with cognitive function in Dutch elderly people

    OpenAIRE

    Eussen, S.J.P.M.; Ueland, P. M.; Clarke, R; Blom, H.J.; Hoefnagels, W.H.L.; Staveren, van, R.; Groot, de, W.T.

    2007-01-01

    The importance of the one-carbon metabolites, choline and homocysteine, to brain function is well known. However, the associations between the one-carbon metabolites choline, betaine, methionine and dimethylglycine with cognition in elderly are unclear. We therefore examined the associations of these metabolites with cognition in a double-blind, placebo-controlled trial. Individuals (n 195) were randomized to receive daily oral capsules with either 1000 ¿g cobalamin (vitamin B12), or 1000 ¿g ...

  18. The association of betaine, homocysteine and related metabolites with cognitive function in Dutch elderly people.

    NARCIS (Netherlands)

    Eussen, S.; Ueland, P.M.; Clarke, R.; Blom, H.J.; Hoefnagels, W.H.L.; Staveren, W.A. van; Groot, L.C. de

    2007-01-01

    The importance of the one-carbon metabolites, choline and homocysteine, to brain function is well known. However, the associations between the one-carbon metabolites choline, betaine, methionine and dimethylglycine with cognition in elderly are unclear. We therefore examined the associations of thes

  19. Resistance to ketolide antibiotics by coordinated expression of rRNA methyltransferases in a bacterial producer of natural ketolides

    DEFF Research Database (Denmark)

    Almutairi, Mashal M; Park, Sung Ryeol; Rose, Simon;

    2015-01-01

    Ketolides are promising new antimicrobials effective against a broad range of Gram-positive pathogens, in part because of the low propensity of these drugs to trigger the expression of resistance genes. A natural ketolide pikromycin and a related compound methymycin are produced by Streptomyces...... activation by ketolide antibiotics. The resistance genes and the induction mechanism remain fully functional when transferred to heterologous bacterial hosts. The anticipated wide use of ketolide antibiotics could promote horizontal transfer of these highly efficient resistance genes to pathogens. Taken...... together, these findings emphasized the need for surveillance of pikR1/pikR2-based bacterial resistance and the preemptive development of drugs that can remain effective against the ketolide-specific resistance mechanism....

  20. Loss of LSD1 (lysine-specific demethylase 1) suppresses growth and alters gene expression of human colon cancer cells in a p53- and DNMT1(DNA methyltransferase 1)-independent manner

    OpenAIRE

    Jin, Lihua; Hanigan, Christin L.; Wu, Yu; Wang, Wei; Park, Ben Ho; Woster, Patrick M.; Casero, Robert A.

    2012-01-01

    Epigenetic silencing of gene expression is important in cancer. Aberrant DNA CpG island hypermethylation and histone modifications are involved in the aberrant silencing of tumour-suppressor genes. LSD1 (lysine-specific demethylase 1) is a H3K4 (histone H3 Lys4) demethylase associated with gene repression and is overexpressed in multiple cancer types. LSD1 has also been implicated in targeting p53 and DNMT1 (DNA methyltransferase 1), with data suggesting that the demethylating activity of LSD...

  1. Faithful inheritance of cytosine methylation patterns in repeated sequences of the allotetraploid tobacco correlates with the expression of DNA methyltransferase gene families from both parental genomes

    Czech Academy of Sciences Publication Activity Database

    Fulneček, Jaroslav; Matyášek, Roman; Kovařík, Aleš

    2009-01-01

    Roč. 281, č. 4 (2009), s. 407-420. ISSN 1617-4615 R&D Projects: GA ČR(CZ) GA204/06/1432; GA ČR(CZ) GA521/07/0116 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : cytosine methylation * DNA (cytosine-5) methyltransferase * allopolyploidy Subject RIV: BO - Biophysics Impact factor: 2.579, year: 2009

  2. Betaine as a determinant of postmethionine load total plasma homocysteine before and after B-vitamin supplementation.

    NARCIS (Netherlands)

    Holm, P.I.; Bleie, O.; Ueland, P.M.; Lien, E.A.; Refsum, H.; Nordrehaug, J.E.; Nygard, O.

    2004-01-01

    OBJECTIVE: Betaine is a substrate in the betaine-homocysteine methyltransferase reaction, converting homocysteine to methionine. There are only sparse data on plasma betaine as a determinant of the plasma total homocysteine (tHcy) concentration. METHODS AND RESULTS: Ninety patients undergoing corona

  3. Genomic and proteomic analyses reveal multiple homologs of genes encoding enzymes of the methanol:coenzyme M methyltransferase system that are differentially expressed in methanol- and acetate-grown Methanosarcina thermophila.

    Science.gov (United States)

    Ding, Yan-Huai R; Zhang, Shi-Ping; Tomb, Jean-Francois; Ferry, James G

    2002-09-24

    Each of the genomic sequences of Methanosarcina acetivorans, Methanosarcina mazei, and Methanosarcina thermophila revealed two homologs of mtaA, three homologs of mtaB, and three homologs of mtaC encoding enzymes specific for methanogenesis from methanol. Two-dimensional gel electrophoretic analyses of polypeptides from M. thermophila established that methanol induces the expression of mtaA-1, mtaB-1, mtaB-2, mtaB-3, mtaC-1, mtaC-2, and mtaC-3 whereas mtaB-3 and mtaC-3 are constitutively expressed in acetate-grown cells. The gene product of one of three mttC homologs, encoding trimethylamine-specific methyltransferase I, was detected in methanol- but not acetate-grown M. thermophila. A postulated role for the multiple homologs is discussed. PMID:12393212

  4. Isolation and Characterization of Suv39h2, a Second Histone H3 Methyltransferase Gene That Displays Testis-Specific Expression

    OpenAIRE

    O'Carroll, Dónal; Scherthan, Harry; Peters, Antoine H. F. M.; Opravil, Susanne; Haynes, Andrew R.; Laible, Götz; Rea, Stephen; Schmid, Manfred; Lebersorger, Angelika; Jerratsch, Martin; Sattler, Lydia; Mattei, Marie G.; Denny, Paul; Brown, Stephen D. M.; Schweizer, Dieter

    2000-01-01

    Higher-order chromatin has been implicated in epigenetic gene control and in the functional organization of chromosomes. We have recently discovered mouse (Suv39h1) and human (SUV39H1) histone H3 lysine 9-selective methyltransferases (Suv39h HMTases) and shown that they modulate chromatin dynamics in somatic cells. We describe here the isolation, chromosomal assignment, and characterization of a second murine gene, Suv39h2. Like Suv39h1, Suv39h2 encodes an H3 HMTase that shares 59% identity w...

  5. Alternative splicing regulates the expression of G9A and SUV39H2 methyltransferases, and dramatically changes SUV39H2 functions

    OpenAIRE

    Mauger, Oriane; Klinck, Roscoe; Chabot, Benoit; Muchardt, Christian; Allemand, Eric; Batsche, Eric

    2015-01-01

    International audience Alternative splicing is the main source of proteome diversity. Here, we have investigated how alternative splicing affects the function of two human histone methyltransferases (HMTase): G9A and SUV39H2. We show that exon 10 in G9A and exon 3 in SUV39H2 are alternatively included in a variety of tissues and cell lines, as well as in a different species. The production of these variants is likely tightly regulated because both constitutive and alternative splicing fact...

  6. RFX1 regulates CD70 and CD11a expression in lupus T cells by recruiting the histone methyltransferase SUV39H1

    OpenAIRE

    Zhao, Ming; Wu, Xiaoyan; Zhang, Qing; Luo, Shuangyan; Liang, Gongping; Su, Yuwen; Tan, Yixin; Lu, Qianjin

    2010-01-01

    Introduction Regulatory factor X-box 1 (RFX1) can interact with DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1), and RFX1 down-regulation contributes to DNA hypomethylation and histone H3 hyperacetylation at the cluster of differentiation (CD) 11a and CD70 promoters in CD4+ T cells of patients with systemic lupus erythematosus (SLE). This leads to CD11a and CD70 overexpression, thereby triggering autoimmune responses. In order to provide more insight into the epigenetic mech...

  7. Melatonin synthesis: Acetylserotonin O-methyltransferase (ASMT) is strongly expressed in a subpopulation of pinealocytes in the male rat pineal gland

    DEFF Research Database (Denmark)

    Rath, Martin Fredensborg; Coon, Steven L.; Amaral, Fernanda G.;

    2016-01-01

    The rat pineal gland has been extensively used in studies of melatonin synthesis. However, the cellular localization of melatonin synthesis in this species has not been investigated. Here we focus on the localization of melatonin synthesis using immunohistochemical methods to detect the last enzyme...... in melatonin synthesis, acetylserotonin O-methyltransferase (ASMT), and in situ hybridization techniques to study transcripts encoding ASMT and two other enzymes in melatonin synthesis, tryptophan hydroxylase 1 (TPH1) and aralkylamine N-acetyltransferase (AANAT). In sections of the rat pineal gland......, marked cell-to-cell differences were found in ASMT immunostaining intensity and in the abundance of Tph1, Aanat and Asmt transcripts. ASMT immunoreactivity was localized to the cytoplasm in pinealocytes in the parenchyma of the superficial pineal gland, and immunopositive pinealocytes were also detected...

  8. Melatonin Synthesis: Acetylserotonin O-Methyltransferase (ASMT) Is Strongly Expressed in a Subpopulation of Pinealocytes in the Male Rat Pineal Gland.

    Science.gov (United States)

    Rath, Martin F; Coon, Steven L; Amaral, Fernanda G; Weller, Joan L; Møller, Morten; Klein, David C

    2016-05-01

    The rat pineal gland has been extensively used in studies of melatonin synthesis. However, the cellular localization of melatonin synthesis in this species has not been investigated. Here we focus on the localization of melatonin synthesis using immunohistochemical methods to detect the last enzyme in melatonin synthesis, acetylserotonin O-methyltransferase (ASMT), and in situ hybridization techniques to study transcripts encoding ASMT and two other enzymes in melatonin synthesis, tryptophan hydroxylase (TPH)-1 and aralkylamine N-acetyltransferase. In sections of the rat pineal gland, marked cell-to-cell differences were found in ASMT immunostaining intensity and in the abundance of Tph1, Aanat, and Asmt transcripts. ASMT immunoreactivity was localized to the cytoplasm in pinealocytes in the parenchyma of the superficial pineal gland, and immunopositive pinealocytes were also detected in the pineal stalk and in the deep pineal gland. ASMT was found to inconsistently colocalize with S-antigen, a widely used pinealocyte marker; this colocalization was seen in cells throughout the pineal complex and also in displaced pinealocyte-like cells of the medial habenular nucleus. Inconsistent colocalization between ASMT and TPH protein was also detected in the pineal gland. ASMT protein was not detected in extraepithalamic parts of the central nervous system or in peripheral tissues. The findings in this report are of special interest because they provide reason to suspect that melatonin synthesis varies significantly among individual pinealocytes. PMID:26950199

  9. Expression of cell wall related genes in basal and ear internodes of silking brown-midrib-3, caffeic acid O-methyltransferase (COMT down-regulated, and normal maize plants

    Directory of Open Access Journals (Sweden)

    Martinant Jean-Pierre

    2008-06-01

    Full Text Available Abstract Background Silage maize is a major forage and energy resource for cattle feeding, and several studies have shown that lignin content and structure are the determining factors in forage maize feeding value. In maize, four natural brown-midrib mutants have modified lignin content, lignin structure and cell wall digestibility. The greatest lignin reduction and the highest cell wall digestibility were observed in the brown-midrib-3 (bm3 mutant, which is disrupted in the caffeic acid O-methyltransferase (COMT gene. Results Expression of cell wall related genes was investigated in basal and ear internodes of normal, COMT antisens (AS225, and bm3 maize plants of the INRA F2 line. A cell wall macro-array was developed with 651 gene specific tags of genes specifically involved in cell wall biogenesis. When comparing basal (older lignifying and ear (younger lignifying internodes of the normal line, all genes known to be involved in constitutive monolignol biosynthesis had a higher expression in younger ear internodes. The expression of the COMT gene was heavily reduced, especially in the younger lignifying tissues of the ear internode. Despite the fact that AS225 transgene expression was driven only in sclerenchyma tissues, COMT expression was also heavily reduced in AS225 ear and basal internodes. COMT disruption or down-regulation led to differential expressions of a few lignin pathway genes, which were all over-expressed, except for a phenylalanine ammonia-lyase gene. More unexpectedly, several transcription factor genes, cell signaling genes, transport and detoxification genes, genes involved in cell wall carbohydrate metabolism and genes encoding cell wall proteins, were differentially expressed, and mostly over-expressed, in COMT-deficient plants. Conclusion Differential gene expressions in COMT-deficient plants highlighted a probable disturbance in cell wall assembly. In addition, the gene expressions suggested modified chronology of the

  10. Proteomics reveal a concerted upregulation of methionine metabolic pathway enzymes, and downregulation of carbonic anhydrase-III, in betaine supplemented ethanol-fed rats

    OpenAIRE

    Kharbanda, Kusum K; Vigneswara, Vasanthy; McVicker, Benita L.; Newlaczyl, Anna U.; Bailey, Kevin; Tuma, Dean; David E Ray; Carter, Wayne G.

    2009-01-01

    We employed a proteomic profiling strategy to examine the effects of ethanol and betaine diet supplementation on major liver protein level changes. Male Wistar rats were fed control, ethanol or betaine supplemented diets for 4 weeks. Livers were removed and liver cytosolic proteins resolved by one-dimensional and two-dimensional separation techniques. Significant upregulation of betaine homocysteine methyltransferase-1, methionine adenosyl transferase-1, and glycine N-methyltransferase were t...

  11. O6-Methylguanine-DNA methyltransferase protein expression by immunohistochemistry in brain and non-brain systemic tumours: systematic review and meta-analysis of correlation with methylation-specific polymerase chain reaction

    International Nuclear Information System (INIS)

    The DNA repair protein O6-Methylguanine-DNA methyltransferase (MGMT) confers resistance to alkylating agents. Several methods have been applied to its analysis, with methylation-specific polymerase chain reaction (MSP) the most commonly used for promoter methylation study, while immunohistochemistry (IHC) has become the most frequently used for the detection of MGMT protein expression. Agreement on the best and most reliable technique for evaluating MGMT status remains unsettled. The aim of this study was to perform a systematic review and meta-analysis of the correlation between IHC and MSP. A computer-aided search of MEDLINE (1950-October 2009), EBSCO (1966-October 2009) and EMBASE (1974-October 2009) was performed for relevant publications. Studies meeting inclusion criteria were those comparing MGMT protein expression by IHC with MGMT promoter methylation by MSP in the same cohort of patients. Methodological quality was assessed by using the QUADAS and STARD instruments. Previously published guidelines were followed for meta-analysis performance. Of 254 studies identified as eligible for full-text review, 52 (20.5%) met the inclusion criteria. The review showed that results of MGMT protein expression by IHC are not in close agreement with those obtained with MSP. Moreover, type of tumour (primary brain tumour vs others) was an independent covariate of accuracy estimates in the meta-regression analysis beyond the cut-off value. Protein expression assessed by IHC alone fails to reflect the promoter methylation status of MGMT. Thus, in attempts at clinical diagnosis the two methods seem to select different groups of patients and should not be used interchangeably

  12. The Construction of a Mammalian Transfection Vector For Expression of Cytosine-5 Specific DNA Methyltransferase Gene M.Msp1 In Cultured Cells

    OpenAIRE

    ÖZDEMİR, Öztürk

    1998-01-01

    The expression vectors are designed for expression and purification of normal or recombi-nant gene of interest. There is a wide variety of stable and transferable selectable mammalian expres-sion vectors.The vector pCDM8 and its derivatives, pcDNAI/Ampicilline are widely used for cloning and analyzing of genes in higher eukaryotic cells. The vectors pRC/CMV, pcDNA3 and pRC/RSV are designed for high-level expression of recombinant genes in mammalian cells. In the present study, we have be...

  13. Differential gene expression in the liver of the African lungfish, Protopterus annectens, after 6 months of aestivation in air or 1 day of arousal from 6 months of aestivation.

    Directory of Open Access Journals (Sweden)

    Kum C Hiong

    Full Text Available The African lungfish, Protopterus annectens, can undergo aestivation during drought. Aestivation has three phases: induction, maintenance and arousal. The objective of this study was to examine the differential gene expression in the liver of P. annectens after 6 months (the maintenance phase of aestivation as compared with the freshwater control, or after 1 day of arousal from 6 months aestivation as compared with 6 months of aestivation using suppression subtractive hybridization. During the maintenance phase of aestivation, the mRNA expression of argininosuccinate synthetase 1 and carbamoyl phosphate synthetase III were up-regulated, indicating an increase in the ornithine-urea cycle capacity to detoxify ammonia to urea. There was also an increase in the expression of betaine homocysteine-S-transferase 1 which could reduce and prevent the accumulation of hepatic homocysteine. On the other hand, the down-regulation of superoxide dismutase 1 expression could signify a decrease in ROS production during the maintenance phase of aestivation. In addition, the maintenance phase was marked by decreases in expressions of genes related to blood coagulation, complement fixation and iron and copper metabolism, which could be strategies used to prevent thrombosis and to conserve energy. Unlike the maintenance phase of aestivation, there were increases in expressions of genes related to nitrogen, carbohydrate and lipid metabolism and fatty acid transport after 1 day of arousal from 6 months aestivation. There were also up-regulation in expressions of genes that were involved in the electron transport system and ATP synthesis, indicating a greater demand for metabolic energy during arousal. Overall, our results signify the importance of sustaining a low rate of waste production and conservation of energy store during the maintenance phase, and the dependence on internal energy store for repair and structural modification during the arousal phase, of

  14. The prognostic impact of O 6- methylguanine DNA methyltransferase and epidermal growth factor receptor expressions on primary gliosarcoma: A clinicopathologic and immunohistochemical study of seven cases at a single institution

    Directory of Open Access Journals (Sweden)

    Jui-Wei Lin

    2011-01-01

    Full Text Available Context: Gliosarcoma is an uncommon variant of glioblastoma characterized by a biphasic tissue pattern of glial and mesenchymal differentiation. O 6- methylguanine DNA methyltransferase (MGMT is a DNA repair protein that removes mutagenic and cytotoxic adducts from O 6 -guanine in DNA. Lack of MGMT protein expression immunohistochemically is related to drug responses in patients of malignant glioma treated with alkylating agents. Epidermal growth factor receptor (EGFR is the most frequently amplified gene in glioblastoma and associated with tumor invasiveness, angiogenesis, poor survival, and resistance to radiation therapy. Aims: To elucidate the relationship between the statuses of the MGMT as well as EGFR proteins and the prognosis. The study was undertaken on samples received at the Department of Pathology from 2003 to 2009. Materials and Methods: Clinicopathologic and immunohistochemical study of seven cases was performed. Results: This series included three men and four women with a mean age of 49.3 years at first surgery. The median progression-free survival (PFS was 22.2 months and 8.6 months for primary tumors with 0 to 1+ and 2+ to 3+ MGMT staining, respectively; the median overall survival (OS was 27.5 months and 14.2 months for primary tumors with 0 to 1+ and 2+ to 3+ MGMT staining, respectively. The median PFS was 17.2 months and 11.2 months for primary tumors with 0 to 1+ and 2+ to 3+ EGFR staining, respectively; the median OS was 20.4 months and 17.7 months for primary tumors with 0 to 1+ and 2+ to 3+ EGFR staining, respectively. Conclusions: The series showed that MGMT and EGFR protein expressions were both unfavorable prognostic factors for patients with gliosarcoma.

  15. Epigenetic Regulation of Surfactant Protein A Gene (SP-A) Expression in Fetal Lung Reveals a Critical Role for Suv39h Methyltransferases during Development and Hypoxia ▿

    OpenAIRE

    Benlhabib, Houda; Mendelson, Carole R.

    2011-01-01

    SP-A gene expression is developmentally regulated in fetal lung. Cyclic AMP (cAMP) induction of SP-A expression in human fetal lung type II cells is O2 dependent and is mediated by increased binding of TTF-1/Nkx2.1 and NF-κB to a critical response element (TBE). This is associated with increased acetylation and decreased methylation of H3K9 at the TBE. Using chromatin immunoprecipitation analysis of fetal lung between 15.5 and 19.0 days of gestation, we observed that the developmental inducti...

  16. Cloning, expression, crystallization and preliminary X-ray analysis of the XMT and DXMT N-methyltransferases from Coffea canephora (robusta)

    International Nuclear Information System (INIS)

    The genes encoding XMT and DXMT, the enzymes from Coffea canephora (robusta) that catalyse the three independent N-methyl transfer reactions in the caffeine-biosynthesis pathway, have been cloned and the proteins have been expressed in Escherichia coli. Both proteins have been crystallized in the presence of the demethylated cofactor S-adenosyl-l-cysteine (SAH) and substrate (xanthosine for XMT and theobromine for DXMT). Caffeine is a secondary metabolite produced by a variety of plants including Coffea canephora (robusta) and there is growing evidence that caffeine is part of a chemical defence strategy protecting young leaves and seeds from potential predators. The genes encoding XMT and DXMT, the enzymes from Coffea canephora (robusta) that catalyse the three independent N-methyl transfer reactions in the caffeine-biosynthesis pathway, have been cloned and the proteins have been expressed in Escherichia coli. Both proteins have been crystallized in the presence of the demethylated cofactor S-adenosyl-l-cysteine (SAH) and substrate (xanthosine for XMT and theobromine for DXMT). The crystals are orthorhombic, with space group P212121 for XMT and C2221 for DXMT. X-ray diffraction to 2.8 Å for XMT and to 2.5 Å for DXMT have been collected on beamline ID23-1 at the ESRF

  17. Functional Role of G9a Histone Methyltransferase in Cancer

    OpenAIRE

    Casciello, Francesco; Windloch, Karolina; Gannon, Frank; Lee, Jason S.

    2015-01-01

    Post-translational modifications of DNA and histones are epigenetic mechanisms, which affect the chromatin structure, ultimately leading to gene expression changes. A number of different epigenetic enzymes are actively involved in the addition or the removal of various covalent modifications, which include acetylation, methylation, phosphorylation, ubiquitination, and sumoylation. Deregulation of these processes is a hallmark of cancer. For instance, G9a, a histone methyltransferase responsib...

  18. Molecular identification of carnosine N-methyltransferase as chicken histamine N-methyltransferase-like protein (hnmt-like.

    Directory of Open Access Journals (Sweden)

    Jakub Drozak

    Full Text Available Anserine (beta-alanyl-N(Pi-methyl-L-histidine, a naturally occurring derivative of carnosine (beta-alanyl-L-histidine, is an abundant constituent of skeletal muscles and brain of many vertebrates. Although it has long been proposed to serve as a proton buffer, radicals scavenger and transglycating agent, its physiological function remains obscure. The formation of anserine is catalyzed by carnosine N-methyltransferase which exhibits unknown molecular identity. In the present investigation, we have purified carnosine N-methyltransferase from chicken pectoral muscle about 640-fold until three major polypeptides of about 23, 26 and 37 kDa coeluting with the enzyme were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in an identification of histamine N-methyltransferase-like (HNMT-like protein as the only meaningful candidate. Analysis of GenBank database records indicated that the hnmt-like gene might be a paralogue of histamine N-methyltransferase gene, while comparison of their protein sequences suggested that HNMT-like protein might have acquired a new activity. Chicken HNMT-like protein was expressed in COS-7 cells, purified to homogeneity, and shown to catalyze the formation of anserine as confirmed by both chromatographic and mass spectrometry analysis. Both specificity and kinetic studies carried out on the native and recombinant enzyme were in agreement with published data. Particularly, several compounds structurally related to carnosine, including histamine and L-histidine, were tested as potential substrates for the enzyme, and carnosine was the only methyl group acceptor. The identification of the gene encoding carnosine N-methyltransferase might be beneficial for estimation of the biological functions of anserine.

  19. Association between BHMT gene rs3733890 polymorphism and cancer risk: evidence from a meta-analysis

    OpenAIRE

    Zhang, Li; Xu, Yue; Yan, Cunye; Hao, Zongyao; Zhou, Jun; Song, Fan; TAI, SHENG; Yang, Cheng; Liang, Chaozhao

    2016-01-01

    Yue Xu,1,* Cunye Yan,2,* Zongyao Hao,1 Jun Zhou,1 Song Fan,1 Sheng Tai,1 Cheng Yang,1 Li Zhang,1 Chaozhao Liang1 1Department of Urology, The First Affiliated Hospital of Anhui Medical University and Institute of Urology, 2First School of Clinical Medicine, Anhui Medical University, Hefei, Anhui, People’s Republic of China *These authors contributed equally to this work Background and objective: The gene betaine-homocysteine methyltransferase (BHMT) has drawn much attenti...

  20. Inhibition of G9a Histone Methyltransferase Converts Bone Marrow Mesenchymal Stem Cells to Cardiac Competent Progenitors

    OpenAIRE

    Jinpu Yang; Keerat Kaur; Li Lin Ong; Eisenberg, Carol A; Leonard M. Eisenberg

    2015-01-01

    The G9a histone methyltransferase inhibitor BIX01294 was examined for its ability to expand the cardiac capacity of bone marrow cells. Inhibition of G9a histone methyltransferase by gene specific knockdown or BIX01294 treatment was sufficient to induce expression of precardiac markers Mesp1 and brachyury in bone marrow cells. BIX01294 treatment also allowed bone marrow mesenchymal stem cells (MSCs) to express the cardiac transcription factors Nkx2.5, GATA4, and myocardin when subsequently exp...

  1. Genetic examination of SETD7 and SUV39H1/H2 methyltransferases and the risk of diabetes complications in patients with type 1 diabetes

    DEFF Research Database (Denmark)

    Syreeni, Anna; El-Osta, Assam; Forsblom, Carol;

    2011-01-01

    episodes of hyperglycemia. Epigenetic modifications mediated by histone methyltransferases are associated with gene-activating events that promote enhanced expression of key proinflammatory molecules implicated in vascular injury. In this study, we investigated genetic polymorphisms of the SETD7, SUV39H1......, and SUV39H2 methyltransferases as predictors of risk for micro- and macrovascular complications in type 1 diabetes....

  2. DNA Methyltransferases Inhibitors from Natural Sources.

    Science.gov (United States)

    Zwergel, Clemens; Valente, Sergio; Mai, Antonello

    2016-01-01

    DNA methyltransferases (DNMTs) catalyze the methylation at cytosine-C5 mainly in a CpG dinucleotide context. Although DNA methylation is essential for fundamental processes like embryonic development or differentiation, aberrant expression and/or activities of DNMTs are involved in several pathologies, from neurodegeneration to cancer. DNMTs inhibition can arrest tumor growth, cells invasiveness and induce differentiation, whereas their increased expression is shown in numerous cancer types. Moreover, hypermethylated promoters of tumor suppressor genes lead to their silencing. Hence, the use of specific inhibitors of DNMT might reactivate those genes and stop or even reverse the aberrant cell processes. To date, the only approved DNMTs inhibitors for therapy belong to the nucleoside-based family of drugs, but they display relevant side effects as well as high chemical instability. Thus, there is a keen interest actually exists to develop novel, potent and safe inhibitors possessing a nonnucleoside structure. Increasing literature evidence is highlighting that natural sources could help the researchers to achieve this goal. Indeed, several polyphenols, flavonoids, antraquinones, and others are described able to inhibit DNMTs activity and/or expression, thus decreasing the methylation/silencing of different genes involved in tumorigenesis. These events can lead to re-expression of such genes and to cell death in diverse cancer cell lines. Epigallocatechin-3-gallate (1) and laccaic acid A (11) resulted the most effective DNMT1 inhibitors with submicromolar IC50 values, acting as competitive inhibitors. Compound 1 and 11 both displayed gene demethylation and re-activation in several cancers. However, all of the natural compounds described in this review showed important results, from gene reactivation to cell growth inhibition. Moreover, some of them displayed interesting activity even in rodent cancer models and very recently entered clinical trials. PMID:26303417

  3. Enhanced Levels of microRNA-125b in Vascular Smooth Muscle Cells of Diabetic db/db Mice Lead to Increased Inflammatory Gene Expression by Targeting the Histone Methyltransferase Suv39h1

    OpenAIRE

    Villeneuve, Louisa M.; KATO, MITSUO; Reddy, Marpadga A.; Wang, Mei; Lanting, Linda; Natarajan, Rama

    2010-01-01

    OBJECTIVE Diabetes remains a major risk factor for vascular complications that seem to persist even after achieving glycemic control, possibly due to “metabolic memory.” Using cultured vascular smooth muscle cells (MVSMC) from type 2 diabetic db/db mice, we recently showed that decreased promoter occupancy of the chromatin histone H3 lysine-9 methyltransferase Suv39h1 and the associated repressive epigenetic mark histone H3 lysine-9 trimethylation (H3K9me3) play key roles in sustained inflamm...

  4. A SABATH Methyltransferase from the moss Physcomitrella patens catalyzes

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Nan [ORNL; Ferrer, Jean-Luc [Universite Joseph Fourier, France; Moon, Hong S [Department of Plant Sciences, University of Tennessee; Kapteyn, Jeremy [Institute of Biological Chemistry, Washington State University; Zhuang, Xiaofeng [Department of Plant Sciences, University of Tennessee; Hasebe, Mitsuyasu [Laboratory of Evolutionary Biology, National Institute for Biology, 38 Nishigounaka; Stewart, Neal C. [Department of Plant Sciences, University of Tennessee; Gang, David R. [Institute of Biological Chemistry, Washington State University; Chen, Feng [University of Tennessee, Knoxville (UTK)

    2012-01-01

    Known SABATH methyltransferases, all of which were identified from seed plants, catalyze methylation of either the carboxyl group of a variety of low molecular weight metabolites or the nitrogen moiety of precursors of caffeine. In this study, the SABATH family from the bryophyte Physcomitrella patens was identified and characterized. Four SABATH-like sequences (PpSABATH1, PpSABATH2, PpSABATH3, and PpSABATH4) were identified from the P. patens genome. Only PpSABATH1 and PpSABATH2 showed expression in the leafy gametophyte of P. patens. Full-length cDNAs of PpSABATH1 and PpSABATH2 were cloned and expressed in soluble form in Escherichia coli. Recombinant PpSABATH1 and PpSABATH2 were tested for methyltransferase activity with a total of 75 compounds. While showing no activity with carboxylic acids or nitrogen-containing compounds, PpSABATH1 displayed methyltransferase activity with a number of thiols. PpSABATH2 did not show activity with any of the compounds tested. Among the thiols analyzed, PpSABATH1 showed the highest level of activity with thiobenzoic acid with an apparent Km value of 95.5 lM, which is comparable to those of known SABATHs. Using thiobenzoic acid as substrate, GC MS analysis indicated that the methylation catalyzed by PpSABATH1 is on the sulfur atom. The mechanism for S-methylation of thiols catalyzed by PpSABATH1 was partially revealed by homology-based structural modeling. The expression of PpSABATH1 was induced by the treatment of thiobenzoic acid. Further transgenic studies showed that tobacco plants overexpressing PpSABATH1 exhibited enhanced tolerance to thiobenzoic acid, suggesting that PpSABATH1 have a role in the detoxification of xenobiotic thiols.

  5. Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability

    OpenAIRE

    Lee, Jae-Young; Lee, Se-Hwan; Heo, Sun-Hee; Kim, Kwang-Soo; Kim, Changhoon; Kim, Dae-Kwan; Ko, Jeong-Jae; Park, Kyung-Soon

    2015-01-01

    G9a is a lysine methyltransferase (KMTase) for histone H3 lysine 9 that plays critical roles in a number of biological processes. Emerging evidence suggests that aberrant expression of G9a contributes to tumor metastasis and maintenance of a malignant phenotype in cancer by inducing epigenetic silencing of tumor suppressor genes. Here, we show that G9a regulates Sox2 protein stability in breast cancer cells. When G9a lysine methyltransferase activity was chemically inhibited in the ER(+) brea...

  6. Effect of alternative temozolomide schedules on glioblastoma O 6-methylguanine-DNA methyltransferase activity and survival

    OpenAIRE

    Robinson, C G; Palomo, J M; Rahmathulla, G; McGraw, M; Donze, J; L. Liu; Vogelbaum, M A

    2010-01-01

    Background: O 6-methylguanine-DNA methyltransferase (MGMT) expression in glioblastoma correlates with temozolomide resistance. Dose-intense temozolomide schedules deplete MGMT activity in peripheral blood mononuclear cells; however, no published data exist evaluating the effect of temozolomide schedules on intracranial tumour MGMT activity. Methods: Human glioblastoma cells (GBM43) with an unmethylated MGMT promoter were implanted intracranially in immunodeficient rodents. Three weeks later, ...

  7. Caffeine synthase and related methyltransferases in plants.

    Science.gov (United States)

    Misako, Kato; Kouichi, Mizuno

    2004-05-01

    Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in high concentrations in tea and coffee and it is also found in a number of beverages such as coca cola. It is necessary to elucidate the caffeine biosynthetic pathway and to clone the genes related to the production of caffeine not only to determine the metabolism of the purine alkaloid but also to control the content of caffeine in tea and coffee. The available data support the operation of a xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway as the major route to caffeine. Since the caffeine biosynthetic pathway contains three S-adenosyl-L-methionine (SAM) dependent methylation steps, N-methyltransferases play important roles. This review focuses on the enzymes and genes involved in the methylation of purine ring. Caffeine synthase, the SAM-dependent methyltransferase involved in the last two steps of caffeine biosynthesis, was originally purified from young tea leaves (Camellia sinensis). The isolated cDNA, termed TCS1, consists of 1,483 base pairs and encodes a protein of 369 amino acids. Subsequently, the homologous genes that encode caffeine biosynthetic enzymes from coffee (Coffea arabica) were isolated. The recombinant proteins are classified into the three types on the basis of their substrate specificity i.e. 7-methylxanthosine synthase, theobromine synthase and caffeine synthase. The predicted amino acid sequences of caffeine biosynthetic enzymes derived from C. arabica exhibit more than 80% homology with those of the clones and but show only 40% homology with TCS1 derived from C. sinensis. In addition, they share 40% homology with the amino acid sequences of salicylic carboxyl methyltransferase, benzoic acid carboxyl methyltransferase and jasmonic acid carboxyl methyltransferase which belong to a family of motif B' methyltransferases which are novel plant methyltransferases with motif B' instead of motif B as the conserved region. PMID:14977590

  8. Directed evolution of improved zinc finger methyltransferases.

    Directory of Open Access Journals (Sweden)

    Brian Chaikind

    Full Text Available The ability to target DNA methylation toward a single, user-designated CpG site in vivo may have wide applicability for basic biological and biomedical research. A tool for targeting methylation toward single sites could be used to study the effects of individual methylation events on transcription, protein recruitment to DNA, and the dynamics of such epigenetic alterations. Although various tools for directing methylation to promoters exist, none offers the ability to localize methylation solely to a single CpG site. In our ongoing research to create such a tool, we have pursued a strategy employing artificially bifurcated DNA methyltransferases; each methyltransferase fragment is fused to zinc finger proteins with affinity for sequences flanking a targeted CpG site for methylation. We sought to improve the targeting of these enzymes by reducing the methyltransferase activity at non-targeted sites while maintaining high levels of activity at a targeted site. Here we demonstrate an in vitro directed evolution selection strategy to improve methyltransferase specificity and use it to optimize an engineered zinc finger methyltransferase derived from M.SssI. The unusual restriction enzyme McrBC is a key component of this strategy and is used to select against methyltransferases that methylate multiple sites on a plasmid. This strategy allowed us to quickly identify mutants with high levels of methylation at the target site (up to ∼80% and nearly unobservable levels of methylation at a off-target sites (<1%, as assessed in E. coli. We also demonstrate that replacing the zinc finger domains with new zinc fingers redirects the methylation to a new target CpG site flanked by the corresponding zinc finger binding sequences.

  9. Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

    International Nuclear Information System (INIS)

    DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

  10. Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yuan [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); Wu, Keqiang [Institute of Plant Biology, National Taiwan University, Taipei 106, Taiwan (China); Dhaubhadel, Sangeeta [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); An, Lizhe, E-mail: lizhean@lzu.edu.cn [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Tian, Lining, E-mail: tianl@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada)

    2010-05-28

    DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

  11. Structural Biology of Human H3K9 Methyltransferases

    Energy Technology Data Exchange (ETDEWEB)

    Wu, H.; Min, J; Lunin, V; Antoshenko, T; Dombrovsk, L; Zeng, H; Allali-Hassani, A; Campagna-Slater, V; Vedadi, M; et. al.

    2010-01-01

    SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. Post-translational modifications of histone proteins regulate chromatin compaction, mediate epigenetic regulation of transcription, and control cellular differentiation in health and disease. Methylation of histone tails is one of the fundamental events of epigenetic signaling. Tri-methylation of lysine 9 of histone 3 (H3K9) mediates chromatin recruitment of HP1, heterochromatin condensation and gene silencing. Similarly, methylation of H3K27 and H4K20 are associated with a repressed state of chromatin, whereas expressed genes are methylated at H3K4, H3K36 and H3K79. Histone methyltransferases are divided into protein arginine methyltransferases (PRMTs) and histone lysine methyltransferases (HKMTs). HKMTs catalyze the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) to a substrate lysine and, with the exception of DOT1L, are all organized around a canonical SET domain. The structures of a number of HKMTs have been reported, including ternary complexes of human orthologs with co-factor and substrate peptides (SETD7-H3K4, SETD8-H4K20 and MLL1-H3K4), as well

  12. Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity

    OpenAIRE

    Kraus, Daniel; Yang, Qin; Kong, Dong; Banks, Alexander S.; Zhang, Lin; Rodgers, Joseph T; Pirinen, Eija; Pulinilkunnil, Thomas C.; Gong, Fengying; Wang, Ya-chin; Cen, Yana; Sauve, Anthony A.; Asara, John M.; Peroni, Odile D.; Monia, Brett P.

    2014-01-01

    In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes1. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity2. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respect...

  13. A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids.

    Science.gov (United States)

    Levac, Dylan; Cázares, Paulo; Yu, Fang; De Luca, Vincenzo

    2016-04-01

    Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases. PMID:26848097

  14. Hepatic Methionine Homeostasis Is Conserved in C57BL/6N Mice on High-Fat Diet Despite Major Changes in Hepatic One-Carbon Metabolism

    Science.gov (United States)

    Dahlhoff, Christoph; Desmarchelier, Charles; Sailer, Manuela; Fürst, Rainer W.; Haag, Alexander; Ulbrich, Susanne E.; Hummel, Björn; Obeid, Rima; Geisel, Jürgen; Bader, Bernhard L.; Daniel, Hannelore

    2013-01-01

    Obesity is an underlying risk factor in the development of cardiovascular disease, dyslipidemia and non-alcoholic fatty liver disease (NAFLD). Increased hepatic lipid accumulation is a hallmark in the progression of NAFLD and impairments in liver phosphatidylcholine (PC) metabolism may be central to the pathogenesis. Hepatic PC biosynthesis, which is linked to the one-carbon (C1) metabolism by phosphatidylethanolamine N-methyltransferase, is known to be important for hepatic lipid export by VLDL particles. Here, we assessed the influence of a high-fat (HF) diet and NAFLD status in mice on hepatic methyl-group expenditure and C1-metabolism by analyzing changes in gene expression, protein levels, metabolite concentrations, and nuclear epigenetic processes. In livers from HF diet induced obese mice a significant downregulation of cystathionine β-synthase (CBS) and an increased betaine-homocysteine methyltransferase (BHMT) expression were observed. Experiments in vitro, using hepatoma cells stimulated with peroxisome proliferator activated receptor alpha (PPARα) agonist WY14,643, revealed a significantly reduced Cbs mRNA expression. Moreover, metabolite measurements identified decreased hepatic cystathionine and L-α-amino-n-butyrate concentrations as part of the transsulfuration pathway and reduced hepatic betaine concentrations, but no metabolite changes in the methionine cycle in HF diet fed mice compared to controls. Furthermore, we detected diminished hepatic gene expression of de novo DNA methyltransferase 3b but no effects on hepatic global genomic DNA methylation or hepatic DNA methylation in the Cbs promoter region upon HF diet. Our data suggest that HF diet induces a PPARα-mediated downregulation of key enzymes in the hepatic transsulfuration pathway and upregulates BHMT expression in mice to accommodate to enhanced dietary fat processing while preserving the essential amino acid methionine. PMID:23472083

  15. Structural basis for S-adenosylmethionine binding and methyltransferase activity by mitochondrial transcription factor B1.

    Science.gov (United States)

    Guja, Kip E; Venkataraman, Krithika; Yakubovskaya, Elena; Shi, Hui; Mejia, Edison; Hambardjieva, Elena; Karzai, A Wali; Garcia-Diaz, Miguel

    2013-09-01

    Eukaryotic transcription factor B (TFB) proteins are homologous to KsgA/Dim1 ribosomal RNA (rRNA) methyltransferases. The mammalian TFB1, mitochondrial (TFB1M) factor is an essential protein necessary for mitochondrial gene expression. TFB1M mediates an rRNA modification in the small ribosomal subunit and thus plays a role analogous to KsgA/Dim1 proteins. This modification has been linked to mitochondrial dysfunctions leading to maternally inherited deafness, aminoglycoside sensitivity and diabetes. Here, we present the first structural characterization of the mammalian TFB1 factor. We have solved two X-ray crystallographic structures of TFB1M with (2.1 Å) and without (2.0 Å) its cofactor S-adenosyl-L-methionine. These structures reveal that TFB1M shares a conserved methyltransferase core with other KsgA/Dim1 methyltransferases and shed light on the structural basis of S-adenosyl-L-methionine binding and methyltransferase activity. Together with mutagenesis studies, these data suggest a model for substrate binding and provide insight into the mechanism of methyl transfer, clarifying the role of this factor in an essential process for mitochondrial function. PMID:23804760

  16. Catechol-O-Methyltransferase Polymorphism and Endometriosis

    OpenAIRE

    Wieser, Fritz; Wenzl, Rene; Tempfer, Clemens; Worda, Christoph; Huber, Johannes; Schneeberger, Christian

    2002-01-01

    Purpose: Catechol-O-methyltransferase (COMT) inactivates the estradiol metabolites, 2-hydroxy and 4-hydroxy catechols, which have been implicated in the pathogenesis of endometriosis. A COMT valine to methionine polymorphism (G-to-A) in exon 4 of the COMT gene is polymorphic in the human population, with 25% of Caucasians being homozygous for the low-activity allele (COMT-L) of the enzyme. In a case-control study we investigated whether this COMT polymorphism is associated with endometriosis.

  17. Structural Chemistry of Human RNA Methyltransferases.

    Science.gov (United States)

    Schapira, Matthieu

    2016-03-18

    RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors. PMID:26566070

  18. Structural basis for S-adenosylmethionine binding and methyltransferase activity by mitochondrial transcription factor B1

    OpenAIRE

    Guja, Kip E.; Venkataraman, Krithika; Yakubovskaya, Elena; Hui SHI; Mejia, Edison; Hambardjieva, Elena; Karzai, A. Wali; Garcia-Diaz, Miguel

    2013-01-01

    Eukaryotic transcription factor B (TFB) proteins are homologous to KsgA/Dim1 ribosomal RNA (rRNA) methyltransferases. The mammalian TFB1, mitochondrial (TFB1M) factor is an essential protein necessary for mitochondrial gene expression. TFB1M mediates an rRNA modification in the small ribosomal subunit and thus plays a role analogous to KsgA/Dim1 proteins. This modification has been linked to mitochondrial dysfunctions leading to maternally inherited deafness, aminoglycoside sensitivity and di...

  19. Deficiency of Glycine N-Methyltransferase Aggravates Atherosclerosis in Apolipoprotein E–Null Mice

    OpenAIRE

    Chen, Chien-Yu; Ching, Li-Chieh; Liao, Yi-Jen; Yu, Yuan-Bin; Tsou, Chia-Yuan; Shyue, Song-Kun; Chen, Yi-Ming Arthur; Lee, Tzong-Shyuan

    2012-01-01

    The mechanism underlying the dysregulation of cholesterol metabolism and inflammation in atherogenesis is not understood fully. Glycine N-methyltransferase (GNMT) has been implicated in hepatic lipid metabolism and the pathogenesis of liver diseases. However, little is known about the significance of GNMT in atherosclerosis. We showed the predominant expression of GNMT in foamy macrophages of mouse atherosclerotic aortas. Genetic deletion of GNMT exacerbated the hyperlipidemia, inflammation a...

  20. The histone methyltransferase and putative oncoprotein MMSET is overexpressed in a large variety of human tumors

    DEFF Research Database (Denmark)

    Hudlebusch, Heidi Rye; Santoni-Rugiu, Eric; Simon, Ronald;

    2011-01-01

    Multiple myeloma SET (Suppressor of variegation, Enhancer of zeste, and Trithorax) domain (MMSET) is a histone lysine methyltransferase deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation and poor prognosis. With the aim of understanding, if MMSET can be involve...... in other types of cancer we investigated the expression of MMSET protein in different types of human tumors....

  1. Genetic Contribution of Catechol-O-methyltransferase Polymorphism in Patients with Migraine without Aura

    OpenAIRE

    Park, Jeong Wook; Lee, Kwang Soo; Kim, Joong Seok; Kim, Yeong In; Shin, Hae Eun

    2007-01-01

    Background Recent genetic association studies have investigated the possible genetic role of the dopaminergic system in migraine. Catechol-O-methyltransferase (COMT) is an enzyme that plays a crucial role in the metabolism of dopamine and its genetic polymorphism is associated with three- to fourfold variation of enzymatic activity. Objectives The objective of this study was to elucidate the role of the COMT polymorphism in the genetic susceptibility to migraine and its phenotypic expression ...

  2. miR-221/222 Target the DNA Methyltransferase MGMT in Glioma Cells

    OpenAIRE

    Cristina Quintavalle; Davide Mangani; Giuseppina Roscigno; Giulia Romano; Angel Diaz-Lagares; Margherita Iaboni; Elvira Donnarumma; Danilo Fiore; Pasqualino De Marinis; Ylermi Soini; Manel Esteller; Gerolama Condorelli

    2013-01-01

    Glioblastoma multiforme (GBM) is one of the most deadly types of cancer. To date, the best clinical approach for treatment is based on administration of temozolomide (TMZ) in combination with radiotherapy. Much evidence suggests that the intracellular level of the alkylating enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) impacts response to TMZ in GBM patients. MGMT expression is regulated by the methylation of its promoter. However, evidence indicates that this is not the only regula...

  3. CpG underrepresentation and the bacterial CpG-specific DNA methyltransferase M.MpeI

    OpenAIRE

    Wojciechowski, Marek; Czapinska, Honorata; Bochtler, Matthias

    2013-01-01

    Cytosine methylation promotes deamination. In eukaryotes, CpG methylation is thought to account for CpG underrepresentation. Whether scarcity of CpGs in prokaryotic genomes is diagnostic for methylation is not clear. Here, we report that Mycoplasms tend to be CpG depleted and to harbor a family of constitutively expressed or phase variable CpG-specific DNA methyltransferases. The very CpG poor Mycoplasma penetrans and its constitutively active CpG-specific methyltransferase M.MpeI were chosen...

  4. Comparative Distribution and Retention of Arsenic in Arsenic (+3 Oxidation State) Methyltransferase Knockout and Wild Type Mice

    Science.gov (United States)

    The mouse arsenic (+3 oxidation state) methyltransferase (As3mt) gene encodes a ~ 43 kDa protein that catalyzes conversion of inorganic arsenic into methylated products. Heterologous expression of AS3MT or its silencing by RNA interference controls arsenic methylation phenotypes...

  5. Substrate recognition and modification by the nosiheptide resistance methyltransferase.

    Directory of Open Access Journals (Sweden)

    Sitao Yin

    Full Text Available The proliferation of antibiotic resistant pathogens is an increasing threat to the general public. Resistance may be conferred by a number of mechanisms including covalent or mutational modification of the antibiotic binding site, covalent modification of the drug, or the over-expression of efflux pumps. The nosiheptide resistance methyltransferase (NHR confers resistance to the thiazole antibiotic nosiheptide in the nosiheptide producer organism Streptomyces actuosus through 2'O-methylation of 23S rRNA at the nucleotide A1067. Although the crystal structures of NHR and the closely related thiostrepton-resistance methyltransferase (TSR in complex with the cofactor S-Adenosyl-L-methionine (SAM are available, the principles behind NHR substrate recognition and catalysis remain unclear.We have analyzed the binding interactions between NHR and model 58 and 29 nucleotide substrate RNAs by gel electrophoresis mobility shift assays (EMSA and fluorescence anisotropy. We show that the enzyme binds to RNA as a dimer. By constructing a hetero-dimer complex composed of one wild-type subunit and one inactive mutant NHR-R135A subunit, we show that only one functional subunit of the NHR homodimer is required for its enzymatic activity. Mutational analysis suggests that the interactions between neighbouring bases (G1068 and U1066 and A1067 have an important role in methyltransfer activity, such that the substitution of a deoxy sugar spacer (5' to the target nucleotide achieved near wild-type levels of methylation. A series of atomic substitutions at specific positions on the substrate adenine show that local base-base interactions between neighbouring bases are important for methylation.Taken together these data suggest that local base-base interactions play an important role in aligning the substrate 2' hydroxyl group of A1067 for methyl group transfer. Methylation of nucleic acids is playing an increasingly important role in fundamental biological processes

  6. New erythromycin derivatives from Saccharopolyspora erythraea using sugar O-methyltransferases from the spinosyn biosynthetic gene cluster.

    Science.gov (United States)

    Gaisser, S; Lill, R; Wirtz, G; Grolle, F; Staunton, J; Leadlay, P F

    2001-09-01

    Using a previously developed expression system based on the erythromycin-producing strain of Saccharopolyspora erythraea, O-methyltransferases from the spinosyn biosynthetic gene cluster of Saccharopolyspora spinosa have been shown to modify a rhamnosyl sugar attached to a 14-membered polyketide macrolactone. The spnI, spnK and spnH methyltransferase genes were expressed individually in the S. erythraea mutant SGT2, which is blocked both in endogenous macrolide biosynthesis and in ery glycosyltransferases eryBV and eryCIII. Exogenous 3-O-rhamnosyl-erythronolide B was efficiently converted into 3-O-(2'-O-methylrhamnosyl)-erythronolide B by the S. erythraea SGT2 (spnI) strain only. When 3-O-(2'-O-methylrhamnosyl)-erythronolide B was, in turn, fed to a culture of S. erythraea SGT2 (spnK), 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was identified in the culture supernatant, whereas S. erythraea SGT2 (spnH) was without effect. These results confirm the identity of the 2'- and 3'-O-methyltransferases, and the specific sequence in which they act, and they demonstrate that these methyltransferases may be used to methylate rhamnose units in other polyketide natural products with the same specificity as in the spinosyn pathway. In contrast, 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was found not to be a substrate for the 4'-O-methyltransferase SpnH. Although rhamnosylerythromycins did not serve directly as substrates for the spinosyn methyltransferases, methylrhamnosyl-erythromycins were obtained by subsequent conversion of the corresponding methylrhamnosyl-erythronolide precursors using the S. erythraea strain SGT2 housing EryCIII, the desosaminyltransferase of the erythromycin pathway. 3-O-(2'-O-methylrhamnosyl)-erythromycin D was tested and found to be significantly active against a strain of erythromycin-sensitive Bacillus subtilis. PMID:11555300

  7. Distinction between the Cfr Methyltransferase Conferring Antibiotic Resistance and the Housekeeping RlmN Methyltransferase

    DEFF Research Database (Denmark)

    Atkinson, Gemma C; Hansen, Lykke H; Tenson, Tanel;

    2013-01-01

    The cfr gene encodes the Cfr methyltransferase that primarily methylates C-8 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to six classes of antibiotics of clinical and veterinary importance. The rlmN gene encodes the Rlm...

  8. Targeting histone methyltransferases and demethylases in clinical trials for cancer therapy.

    Science.gov (United States)

    Morera, Ludovica; Lübbert, Michael; Jung, Manfred

    2016-01-01

    The term epigenetics is defined as heritable changes in gene expression that are not due to alterations of the DNA sequence. In the last years, it has become more and more evident that dysregulated epigenetic regulatory processes have a central role in cancer onset and progression. In contrast to DNA mutations, epigenetic modifications are reversible and, hence, suitable for pharmacological interventions. Reversible histone methylation is an important process within epigenetic regulation, and the investigation of its role in cancer has led to the identification of lysine methyltransferases and demethylases as promising targets for new anticancer drugs. In this review, we describe those enzymes and their inhibitors that have already reached the first stages of clinical trials in cancer therapy, namely the histone methyltransferases DOT1L and EZH2 as well as the demethylase LSD1. PMID:27222667

  9. Aflatoxin B1 induced upregulation of protein arginine methyltransferase 5 in human cell lines.

    Science.gov (United States)

    Ghufran, Md Sajid; Ghosh, Krishna; Kanade, Santosh R

    2016-09-01

    The exposure of naturally occurring mycotoxins affects human health and play a vital role in cancer initiation and progression. Aflatoxin B1 is a difuranocoumarin mycotoxin, classified as a group I carcinogen. The present study was conducted to assess the effect of aflatoxin B1 on epigenetic regulatory proteins. The protein arginine methyltransferase 5 expression was induced upon aflatoxin B1 treatment in a dose and time dependent manner. Further global arginine methylation was also increased in the same manner. This is the first report showing the induction of epigenetic regulatory protein, protein arginine methyltransferase 5 upon aflatoxin B1 treatment. Further study is required to establish the detailed pathway of PRMT5 induction. PMID:27242039

  10. A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

    Science.gov (United States)

    Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

    2014-05-01

    The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium. PMID:24573606

  11. Cytoplasmic sequestration of an O6-methylguanine-DNA methyltransferase enhancer binding protein in DNA repair-deficient human cells

    OpenAIRE

    Frank Y. Chen; Harris, Linda C.; Joanna S Remack; Brent, Thomas P.

    1997-01-01

    O6-Methylguanine-DNA methyltransferase (MGMT), an enzyme that repairs adducts at O6 of guanine in DNA, is a major determinant of susceptibility to simple methylating carcinogens or of tumor response to anticancer chloroethylating drugs. To investigate the mechanisms underlying cellular expression of this DNA repair enzyme, we focused on the role of a 59-bp enhancer of the human MGMT gene in the regulation of its expression. By using chloramphenicol acetyltransferase reporter assays, we found ...

  12. DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

    International Nuclear Information System (INIS)

    Highlights: ► CDA-II inhibits myogenic differentiation in a dose-dependent manner. ► CDA-II repressed expression of muscle transcription factors and structural proteins. ► CDA-II inhibited proliferation and migration of C2C12 myoblasts. -- Abstract: CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.

  13. DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zirong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China); Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Jin, Guorong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China); Lin, Shuibin [Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Lin, Xiumei [Department of Hematology, Guangzhou First Municipal People' s Hospital, Guangzhou 510180 (China); Gu, Yumei [Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Zhu, Yujuan; Hu, Chengbin; Zhang, Qingjiong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China); Wu, Lizi [Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Shen, Huangxuan, E-mail: shenhx@mail.sysu.edu.cn [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer CDA-II inhibits myogenic differentiation in a dose-dependent manner. Black-Right-Pointing-Pointer CDA-II repressed expression of muscle transcription factors and structural proteins. Black-Right-Pointing-Pointer CDA-II inhibited proliferation and migration of C2C12 myoblasts. -- Abstract: CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.

  14. Human TRMU encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase is a putative nuclear modifier gene for the phenotypic expression of the deafness-associated 12S rRNA mutations

    International Nuclear Information System (INIS)

    Nuclear modifier genes have been proposed to modulate the phenotypic manifestation of human mitochondrial 12S rRNA A1491G mutation associated with deafness in many families world-wide. Here we identified and characterized the putative nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. A 1937 bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The human TRMU gene containing 11 exons encodes a 421 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs. TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, kidney, and brain. Immunofluorescence analysis of human 143B cells expressing TRMU-GFP fusion protein demonstrated that the human Trmu localizes and functions in mitochondrion. Furthermore, we show that in families with the deafness-associated 12S rRNA A1491G mutation there is highly suggestive linkage and linkage disequilibrium between microsatellite markers adjacent to TRMU and the presence of deafness. These observations suggest that human TRMU may modulate the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations

  15. 胶质母细胞瘤MGMT基因启动子甲基化与蛋白表达%Heterogeneity of O6-methylguanine-DNA methyltransferase protein expression and gene promoter methylation in glioblastoma

    Institute of Scientific and Technical Information of China (English)

    潘强; 杨学军; 纪延伟; 孙健; 韩建国; 高松; 李罡; 张文高

    2010-01-01

    Objective To study the correlation of MGMT gene promoter methylation and protein expression and their regional variation in different specimens obtained from different regions within the tumor in patients with newly diagnosed glioblastoma. Methods Two to four samples in the same tumor were collected from different regions in 30 patients with newly diagnosed glioblastoma patients. In five patients among them,mutispecimens were obtained under assistance of neuronavigation system during the operation. In all samples,MGMT promoter profile were analyzed by Methylation - specific polymerase - Chain - reaction analysis, MSP ,while MGMT protein expression was detected in tissue sections by immunohistochemistry,IHC. Results MGMT promoter methylation was detected in 43. 56% (44/101) specimens. MGMT protein expression in tissue sections was assessed and scored:(1 :no or positive tumor cells 50%) ,The rate of MGMT staining with a score 1,2,3 in all of tumor sections was 32. 67% ,43.56% ,23. 76% respectively. No significant correlation between MGMT protein expression and promoter methylation(x2 =2. 905, P =0.088) was found. The regional heterogeneity of MGMT protein expression within the same tumor was in 57% (17/30) patients ;and the regional heterogeneity of gene promoter methylation was in37%(11/30)patients. Conclusions MGMT promoter methylation is probably not the only modulating element in MGMT protein expression. The heterogeneity of MGMT protein expression and its promoter methylation in the same tumor questions their guiding significance in making therapeutic scheme for individual patients with malignant glioma in clinical practice.%目的 研究新发胶质母细胞瘤中肿瘤不同部位MGMT基因启动子甲基化及其蛋白表达关系及区域差异性.方法 在30例新发胶质母细胞瘤肿瘤不同部位采取2~4块标本,其中5例在术中神经导航引导下采取.甲基化特异性PCR(MSP)法检测标本中MGMT基因启动子甲基化状况,免疫组

  16. miR-29 Represses the Activities of DNA Methyltransferases and DNA Demethylases

    Directory of Open Access Journals (Sweden)

    Izuho Hatada

    2013-07-01

    Full Text Available Members of the microRNA-29 (miR-29 family directly target the DNA methyltransferases, DNMT3A and DNMT3B. Disturbances in the expression levels of miR-29 have been linked to tumorigenesis and tumor aggressiveness. Members of the miR-29 family are currently thought to repress DNA methylation and suppress tumorigenesis by protecting against de novo methylation. Here, we report that members of the miR-29 family repress the activities of DNA methyltransferases and DNA demethylases, which have opposing roles in control of DNA methylation status. Members of the miR-29 family directly inhibited DNA methyltransferases and two major factors involved in DNA demethylation, namely tet methylcytosine dioxygenase 1 (TET1 and thymine DNA glycosylase (TDG. Overexpression of miR-29 upregulated the global DNA methylation level in some cancer cells and downregulated DNA methylation in other cancer cells, suggesting that miR-29 suppresses tumorigenesis by protecting against changes in the existing DNA methylation status rather than by preventing de novo methylation of DNA.

  17. Recombinant rat liver guanidinoacetate methyltransferase: Reactivity and function of sulfhydryl groups

    International Nuclear Information System (INIS)

    Rat liver guanidinoacetate methyltransferase, produced in Escherichia coli by recombinant DNA technique, possesses five cysteine residues per molecule. No disulfide bond is present. Analysis of the chymotryptic peptides derived from the iodo[14C]acetate-modified enzyme shows that Cys-90, Cys-15, Cys-219, and Cys-207 are alkylated by the reagent in order of decreasing reactivity. Incubation of the enzyme with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in the absence and presence of cystamine [2,2'-dithiobis(ethylamine)] causes the appearance of 4 and 5 mol of 2-nitro-5-mercaptobenzoate/mol of enzyme, respectively. Reaction of the methyltransferase with an equimolar amount of DTNB results in an almost quantitative disulfide cross-linking of Cys-15 and Cys-90 with loss of a large portion of the activity. The methyltransferase is completely inactivated by iodoacetate following nonlinear kinetics. Comparison of the extent of inactivation with that of modification of cysteine residues and the experiment with the enzyme whose Cys-15 and Cys-90 are cross-linked suggest that alkylation of Cys-15 and Cys-90 results in a partially active enzyme and that carboxymethylation of Cys-219 completely eliminates enzyme activity. The inactivation of guanidinoacetate methyltransferase by iodoacetate or DTNB is not protected by substrates. Furthermore, disulfide cross-linking of Cys-15 and Cys-90 or carboxymethylation of Cys-219 does not impair the enzyme's capacity to bind S-adenosylmethionine. Thus, these cysteine residues appear to occur outside the active-site region, but their integrity is crucial for the expression of enzyme activity

  18. Monolignol 4-O-methyltransferases and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chang-Jun; Bhuiya, Mohammad-Wadud; Zhang, Kewei

    2014-11-18

    Modified (iso)eugenol 4-O-methyltransferase enzymes having novel capacity for methylation of monolignols and reduction of lignin polymerization in plant cell wall are disclosed. Sequences encoding the modified enzymes are disclosed.

  19. The inheritance of human erythrocyte catechol-O-methyltransferase activity

    International Nuclear Information System (INIS)

    Erythrocyte catechol-O-methyltransferase (COMT) activity was analyzed in 107 individuals from 23 families. The observed activities were consistent with an autosomal codominant inheritance of the gene coding for erythrocyte COMT activity. (author)

  20. Hypnotizability and Catechol-O-Methyltransferase (COMT) polymorphysms in Italians

    OpenAIRE

    Alessandro Gialluisi; Serena Barbuti; Giancarlo Carli; Enrica Laura Santarcangelo

    2014-01-01

    Higher brain dopamine content depending on lower activity of Catechol-O-Methyltransferase (COMT) in subjects with high hypnotisability scores (highs) has been considered responsible for their attentional characteristics. However, the results of the previous genetic studies on association between hypnotisability and the Catechol-O-Methyltransferase (COMT) single nucleotide polymorphism (SNP) rs4680 (Val158Met) were inconsistent. Here, we used a selective genotyping approach to re-evaluate the ...

  1. Identification and functional characterization of lysine methyltransferases of Entamoeba histolytica.

    Science.gov (United States)

    Borbolla-Vázquez, Jessica; Orozco, Esther; Medina-Gómez, Christian; Martínez-Higuera, Aarón; Javier-Reyna, Rosario; Chávez, Bibiana; Betanzos, Abigail; Rodríguez, Mario A

    2016-07-01

    Lysine methylation of histones, a posttranslational modification catalyzed by lysine methyltransferases (HKMTs), plays an important role in the epigenetic regulation of transcription. Lysine methylation of non-histone proteins also impacts the biological function of proteins. Previously it has been shown that lysine methylation of histones of Entamoeba histolytica, the protozoan parasite that infects 50 million people worldwide each year and causing up to 100,000 deaths annually, is implicated in the epigenetic machinery of this microorganism. However, the identification and characterization of HKMTs in this parasite had not yet been determined. In this work we identified four HKMTs in E. histolytica (EhHKMT1 to EhHKMT4) that are expressed by trophozoites. Enzymatic assays indicated that all of them are able to transfer methyl groups to commercial histones. EhHKMT1, EhHKMT2 and EhHKMT4 were detected in nucleus and cytoplasm of trophozoites. In addition EhHKMT2 and EhHKMT4 were located in vesicles containing ingested cells during phagocytosis, and they co-immunoprecipitated with EhADH, a protein involved in the phagocytosis of this parasite. Results suggest that E. histolytica uses its HKMTs to regulate transcription by epigenetic mechanisms, and at least two of them could also be implicated in methylation of proteins that participate in phagocytosis. PMID:27062489

  2. Inhibition of SUV39H1 Methyltransferase Activity by DBC1*

    OpenAIRE

    Li, Zhenyu; Chen, Lihong; Kabra, Neha; Wang, Chuangui; Fang, Jia; Chen, Jiandong

    2009-01-01

    SUV39H1 is a histone H3K9-specific methyltransferase important for heterochromatin formation, regulation of gene expression, and induction of senescence in premalignant cells. SUV39H1 forms a complex with SirT1, and its activity is stimulated by SirT1 binding. Here we present evidence that the product of the DBC1 (deleted in breast cancer 1) gene disrupts the SUV39H1-SirT1 complex. Furthermore, DBC1 binds to the SUV39H1 catalytic domain and inhibits its ability to ...

  3. Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation

    OpenAIRE

    Zhang, Guoqiang; Estève, Pierre-Olivier; Chin, Hang Gyeong; Terragni, Jolyon; Dai, Nan; Corrêa, Ivan R.; Pradhan, Sriharsa

    2015-01-01

    Mammalian cells contain copious amounts of RNA including both coding and noncoding RNA (ncRNA). Generally the ncRNAs function to regulate gene expression at the transcriptional and post-transcriptional level. Among ncRNA, the long ncRNA and small ncRNA can affect histone modification, DNA methylation targeting and gene silencing. Here we show that endogenous DNA methyltransferase 1 (DNMT1) co-purifies with inhibitory ncRNAs. MicroRNAs (miRNAs) bind directly to DNMT1 with high affinity. The bi...

  4. Queen pheromones modulate DNA methyltransferase activity in bee and ant workers.

    Science.gov (United States)

    Holman, Luke; Trontti, Kalevi; Helanterä, Heikki

    2016-01-01

    DNA methylation is emerging as an important regulator of polyphenism in the social insects. Research has concentrated on differences in methylation between queens and workers, though we hypothesized that methylation is involved in mediating other flexible phenotypes, including pheromone-dependent changes in worker behaviour and physiology. Here, we find that exposure to queen pheromone affects the expression of two DNA methyltransferase genes in Apis mellifera honeybees and in two species of Lasius ants, but not in Bombus terrestris bumblebees. These results suggest that queen pheromones influence the worker methylome, pointing to a novel proximate mechanism for these key social signals. PMID:26814223

  5. DNA methyltransferase-dependent transcription of the phage Mu mom gene.

    OpenAIRE

    Hattman, S

    1982-01-01

    The phage Mu mom gene controls an unusual DNA modification. Expression of the mom function requires an active host (dam+) DNA adenine methylase [S-adenosyl-L-methionine:DNA (6-aminopurine)-methyltransferase]; in dam- hosts, Mu development is normal except that the viral DNA does not undergo the mom modification. The present communication compares transcription of the mom gene in dam+ versus dam- cells. 32P-labeled probes were prepared by nick-translation of a purified mom gene-containing rest...

  6. Genome-wide identification and comparative analysis of cytosine-5 DNA methyltransferases and demethylase families in wild and cultivated peanut

    Directory of Open Access Journals (Sweden)

    Pengfei eWang

    2016-02-01

    Full Text Available AbstractDNA methylation plays important roles in genome protection, regulation of gene expression and was associated with plants development. Plant DNA methylation pattern was mediated by cytosine-5 DNA methyltransferases and demethylase. Although the genomes of AA and BB wild peanuts have been fully sequence, these two gene families have not been studied. In this study we report the identification and analysis of putative cytosine-5 DNA methyltransferases (C5-MTases and demethylase in AA and BB wild peanuts. Cytosine-5 DNA methyltransferases in AA and BB wild peanuts could be classified in known MET, CMT and DRM2 groups based on their domain organization. This result was supported by the gene and protein structural characteristics and phylogenetic analysis. We found that some wild peanut DRM2 numbers didn’t contain UBA domain which was different from other plants such as Arabidopsis, maize, soybean. Five DNA demethylase were found in AA genome and five in BB genome. The selective pressure analysis showed that wild peanut C5-MTases gene mainly underwent purifying selection but many positive selection sites can be detected. Conversely, DNA demethylase genes mainly underwent positive selection during evolution. Additionally, the expression dynamic of cytosine-5 DNA methyltransferases and demethylase genes in different cultivated peanut tissues were analyzed. Expression result showed that cold, heat or drought stress could influence the expression level of C5-MTases and DNA demethylase genes in cultivated peanut. These results are useful for better understanding the complexity of these two gene families, and will facilitate epigenetic studies in peanut.

  7. O6-Methylguanine-DNA methyltransferase status in neuroendocrine tumours: prognostic relevance and association with response to alkylating agents

    OpenAIRE

    Walter, T.; van Brakel, B; Vercherat, C; Hervieu, V; Forestier, J.; Chayvialle, J-A; Molin, Y.; Lombard-Bohas, C; Joly, M-O; Scoazec, J-Y

    2015-01-01

    Background: O6-Methylguanine-DNA methyltransferase (MGMT) loss of expression has been suggested to be predictive of response to temozolomide in neuroendocrine tumours (NETs), but so far, only limited data are available. We evaluated the prognostic and predictive value of MGMT status, assessed by two molecular methods and immunohistochemistry, in a large series of NETs of different origins. Methods: A total of 107 patients, including 53 treated by alkylants (temozolomide, dacarbazine or strept...

  8. Methylation pattern of the O6-methylguanine-DNA methyltransferase gene in colon during progressive colorectal tumorigenesis

    OpenAIRE

    NAGASAKA, Takeshi; Goel, Ajay; Notohara, Kenji; Takahata, Takaomi; Sasamoto, Hiromi; Uchida, Takuyuki; Nishida, Naoshi; Tanaka, Noriaki; Boland, Clement Richard; Matsubara, Nagahide

    2008-01-01

    O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair gene which is frequently methylated in colorectal cancer (CRC). However, it remains controversial whether methylation of specific CpG sequences within MGMT promoter leads to loss of its protein expression, and if MGMT methylation correlates with G to A transition mutations in KRAS. Two methylation sensitive regions (Mp and Eh region) of MGMT promoter were investigated in 593 specimens of colorectal tissue: 233 CRCs, 104 adenomatous...

  9. A Small-Molecule Probe of the Histone Methyltransferase G9a Induces Cellular Senescence in Pancreatic Adenocarcinoma

    OpenAIRE

    Wang, Qiu; Paulk, Joshiawa Lanair James; Kubicek, Stefan; Kemp, Melissa M.; Adams, Drew J.; Shamji, Alykhan Farid; Wagner, Bridget K.; Schreiber, Stuart L.; Yuan, Yuan

    2012-01-01

    Post-translational modifications of histones alter chromatin structure and play key roles in gene expression and specification of cell states. Small molecules that target chromatin-modifying enzymes selectively are useful as probes and have promise as therapeutics, although very few are currently available. G9a (also named euchromatin histone methyltransferase 2 (EHMT2)) catalyzes methylation of lysine 9 on histone H3 (H3K9), a modification linked to aberrant silencing of tumor-suppressor gen...

  10. Activating and inhibitory functions for the histone lysine methyltransferase G9a in T helper cell differentiation and function

    OpenAIRE

    Lehnertz, Bernhard; Northrop, Jeffrey P.; Antignano, Frann; Burrows, Kyle; Hadidi, Sima; Mullaly, Sarah C.; Rossi, Fabio M.V.; Zaph, Colby

    2010-01-01

    Accumulating evidence suggests that the regulation of gene expression by histone lysine methylation is crucial for several biological processes. The histone lysine methyltransferase G9a is responsible for the majority of dimethylation of histone H3 at lysine 9 (H3K9me2) and is required for the efficient repression of developmentally regulated genes during embryonic stem cell differentiation. However, whether G9a plays a similar role in adult cells is still unclear. We identify a critical role...

  11. The Putative Protein Methyltransferase LAE1 of Trichoderma atroviride Is a Key Regulator of Asexual Development and Mycoparasitism

    OpenAIRE

    Aghcheh, Razieh Karimi; Irina S. Druzhinina; Kubicek, Christian P

    2013-01-01

    In Ascomycota the protein methyltransferase LaeA is a global regulator that affects the expression of secondary metabolite gene clusters, and controls sexual and asexual development. The common mycoparasitic fungus Trichoderma atroviride is one of the most widely studied agents of biological control of plant-pathogenic fungi that also serves as a model for the research on regulation of asexual sporulation (conidiation) by environmental stimuli such as light and/or mechanical injury. In order ...

  12. The histone methyltransferase SDG8 mediates the epigenetic modification of light and carbon responsive genes in plants

    OpenAIRE

    Ying LI; Mukherjee, Indrani; Thum, Karen E; Tanurdzic, Milos; Katari, Manpreet S.; Obertello, Mariana; Edwards, Molly B; McCombie, W Richard; Martienssen, Robert A.; Coruzzi, Gloria M.

    2015-01-01

    Background Histone methylation modifies the epigenetic state of target genes to regulate gene expression in the context of developmental and environmental changes. Previously, we used a positive genetic screen to identify an Arabidopsis mutant, cli186, which was impaired in carbon and light signaling. Here, we report a deletion of the Arabidopsis histone methyltransferase SDG8 in this mutant (renamed sdg8-5), which provides a unique opportunity to study the global function of a specific histo...

  13. DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish

    OpenAIRE

    Liu, Xiaohui; Jia, Xiaoe; Yuan, Hao; Ma, Ke; Chen, Yi; Jin, Yi; Deng, Min; Pan, Weijun; Chen, Saijuan; Chen, Zhu; de The, Hugues; Zon, Leonard; Zhou, Yi; Zhou, Jun; Zhu, Jun

    2015-01-01

    Background: DNA methyltransferase 1 (Dnmt1) regulates expression of many critical genes through maintaining parental DNA methylation patterns on daughter DNA strands during mitosis. It is essential for embryonic development and diverse biological processes, including maintenance of hematopoietic stem and progenitor cells (HSPCs). However, the precise molecular mechanism of how Dnmt1 is involved in HSPC maintenance remains unexplored. Methods: An N-ethyl-N-nitrosourea (ENU)-based genetic scree...

  14. Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus.

    Directory of Open Access Journals (Sweden)

    Matthew B Kilgore

    Full Text Available Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer's disease. This compound is primarily isolated from daffodil (Narcissus spp., snowdrop (Galanthus spp., and summer snowflake (Leucojum aestivum. Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4'-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4'-O-methyltransferase (NpN4OMT of the proposed galanthamine biosynthetic pathway.

  15. CpG methyltransferase induced down-regulation of claudin-7,-8 and its effects on proliferation and apoptosis of human colorectal cancer HT-29 cells

    Institute of Scientific and Technical Information of China (English)

    王文辉

    2013-01-01

    Objective To explore the regulatory effect of CpG methyltransferase (M.SssI) on expression of claudin-7and claudin-8,promoting apoptosis and inhibiting proliferation of human colorectal cancer HT-29 cells.Methods HT-29 cells were treated with M.SssI (50 U/ml) for

  16. Purification and properties of thioether methyltransferase

    International Nuclear Information System (INIS)

    A method to assay activity was developed which measures acceptance of methyl groups from [methyl-3H]-S-adenosylmethionine by dimethyl selenide. The product, [3H]trimethylselenonium ion, is separated by HPLC and quantitated by scintillation counting. Thioether methyltransferase from mouse liver and lung resides primarily in the cytosol. In terms of specific activity the enzyme is most active in the lung and liver. Purification from lung cytosol requires a three-step process of DEAE and gel filtration column chromatographies followed by chromatofocusing. SDS-Polyacrylamide gel electrophoresis shows a single homogeneous band with a molecular mass of 28,000 daltons. Vmax and Km values for dimethyl selenide as a substrate are 15. 7 pmol/min and 0.44 μM, respectively. Our studies have also shown that this purified enzyme is capable of methylating a wide range of compounds. To further test the enzyme's role in detoxification, in vivo studies were performed by injecting mice with substrate and [methyl-3H]methionine and analyzing tissue extracts and urine for [methyl-3H]sulfonium

  17. Isolation of DNA methyltransferase from plants

    International Nuclear Information System (INIS)

    DNA methyltransferases (DMT) were isolated from nuclei of cauliflower, soybean, and pea by extraction with 0.35 M NaCl. Assays were performed on hemimethylated Micrococcus luteus DNA or on M. luteus DNA to test for maintenance or de novo methylase activity, respectively. Fully methylated DNA was used as a substrate to determine background levels of methylation. Based on these tests, yields of maintenance DMT activity in the crude extract from pea hypocotyl, soybean hypocotyl, and cauliflower inflorescence were 2.8, 0.9, and 1.6 units per g wet tissue (one unit equals 1 pmol of methyl from [3H]AdoMet incorporated into acid precipitable material per h at 300). Two peaks of DMT activity were detected in the soybean nuclear extract following phosphocellulose chromatography. One eluted at 0.4 M and the other at 0.8 M KCl. With both fractions maintenance activity was approximately 2 times that of the de novo activity. Using gel filtration the DMT eluted at 220,000 Daltons. The optimal pH for activity was between 6.5 and 7.0, and the optimal temperature was 300

  18. Purification, crystallization and diffraction studies of the methyltransferases BT-2972 and BVU-3255 from antibiotic-resistant pathogens of the genus Bacteroides from the human intestine

    International Nuclear Information System (INIS)

    The expression, purification, crystallization and diffraction of two methyltransferases BT-2972 and BVU-3255 from two Bacteroides species of antibiotic-resistant pathogens from the human intestine are reported. The methyltransferases BT-2972 and BVU-3255 from two different Bacteroides species that are antibiotic-resistant pathogens from the human intestine were cloned, overexpressed and purified, yielding approximately 120 mg of each protein from 1 l culture. Apo BT-2972 and BVU-3255 and their complexes with S-adenosylmethionine or S-adenosylhomocysteine were crystallized in four different crystal forms using the hanging-drop vapour-diffusion method. These crystals diffracted to resolutions ranging from 2.8 to 2.2 Å. Sequence analysis suggested that the two proteins are homologous small-molecule methyltransferases

  19. The Critical Role of Protein Arginine Methyltransferase prmt8 in Zebrafish Embryonic and Neural Development Is Non-Redundant with Its Paralogue prmt1

    OpenAIRE

    Lin, Yu-Ling; Tsai, Yun-Jung; Liu, Yu-Fang; Cheng, Yi-Chuan; Hung, Chuan-Mao; Lee, Yi-Jen; Pan, Huichin; Li, Chuan

    2013-01-01

    Protein arginine methyltransferase (PRMT) 1 is the most conserved and widely distributed PRMT in eukaryotes. PRMT8 is a vertebrate-restricted paralogue of PRMT1 with an extra N-terminal sequence and brain-specific expression. We use zebrafish (Danio rerio) as a vertebrate model to study PRMT8 function and putative redundancy with PRMT1. The transcripts of zebrafish prmt8 were specifically expressed in adult zebrafish brain and ubiquitously expressed from zygotic to early segmentation stage be...

  20. Characterization of Leishmania major phosphatidylethanolamine methyltransferases LmjPEM1 and LmjPEM2 and their inhibition by choline analogs

    Science.gov (United States)

    Bibis, Stergios S.; Dahlstrom, Kelly; Zhu, Tongtong; Zufferey, Rachel

    2014-01-01

    Phosphatidylcholine (PC) is the most abundant phospholipid in the membranes of the human parasite Leishmania. It is synthesized via two metabolic routes, the de novo pathway that starts with the uptake of choline, and the threefold methylation of phosphatidylethanolamine. Choline was shown to be dispensable for Leishmania; thus, the methylation pathway likely represents the primary route for PC production. Here, we have identified and characterized two phosphatidylethanolamine methyltransferases, LmjPEM1 and LmjPEM2. Both enzymes are expressed in promastigotes as well as in the vertebrate form amastigotes, suggesting that these methyltransferases are important for the development of the parasite throughout its life cycle. These enzymes are maximally expressed during the log phase of growth which correlates with the demand of PC synthesis during cell multiplication. Immunofluorescence studies combined with cell fractionation have shown that both methyltransferases are localized at the endoplasmic reticulum membrane. Heterologous expression in yeast has demonstrated that LmjPEM1 and LmjPEM2 complement the choline auxotrophy phenotype of a yeast double null mutant lacking phosphatidylethanolamine methyltransferase activity. LmjPEM1 catalyzes the first, and to a lesser extent, the second methylation reaction. In contrast, LmjPEM2 has the capacity to add the second and third methyl group onto phosphatidylethanolamine to yield (lyso)PC; it can also add the first methyl group, albeit with very low efficiency. Finally, we have demonstrated using inhibition studies with choline analogs that miltefosine and octadecyltrimethylammonium bromide are potent inhibitors of this metabolic pathway. PMID:25176160

  1. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR).

    Science.gov (United States)

    Kanno, Yuichiro; Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-03-27

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. PMID:25721668

  2. PML-RARA requires DNA methyltransferase 3A to initiate acute promyelocytic leukemia.

    Science.gov (United States)

    Cole, Christopher B; Verdoni, Angela M; Ketkar, Shamika; Leight, Elizabeth R; Russler-Germain, David A; Lamprecht, Tamara L; Demeter, Ryan T; Magrini, Vincent; Ley, Timothy J

    2016-01-01

    The DNA methyltransferases DNMT3A and DNMT3B are primarily responsible for de novo methylation of specific cytosine residues in CpG dinucleotides during mammalian development. While loss-of-function mutations in DNMT3A are highly recurrent in acute myeloid leukemia (AML), DNMT3A mutations are almost never found in AML patients with translocations that create oncogenic fusion genes such as PML-RARA, RUNX1-RUNX1T1, and MLL-AF9. Here, we explored how DNMT3A is involved in the function of these fusion genes. We used retroviral vectors to express PML-RARA, RUNX1-RUNX1T1, or MLL-AF9 in bone marrow cells derived from WT or DNMT3A-deficient mice. Additionally, we examined the phenotypes of hematopoietic cells from Ctsg-PML-RARA mice, which express PML-RARA in early hematopoietic progenitors and myeloid precursors, with or without DNMT3A. We determined that the methyltransferase activity of DNMT3A, but not DNMT3B, is required for aberrant PML-RARA-driven self-renewal ex vivo and that DNMT3A is dispensable for RUNX1-RUNX1T1- and MLL-AF9-driven self-renewal. Furthermore, both the PML-RARA-driven competitive transplantation advantage and development of acute promyelocytic leukemia (APL) required DNMT3A. Together, these findings suggest that PML-RARA requires DNMT3A to initiate APL in mice. PMID:26595813

  3. Crystallization and preliminary X-ray crystallographic studies of O-methyltransferase from Anabaena PCC 7120

    International Nuclear Information System (INIS)

    The O-methyltransferase (OMT) from the Anabaena PCC 7120 has been overexpressed in a soluble form in E. coli, purified and crystallized. The crystals belonged to space group C2221 and diffracted to 2.4 Å resolution. O-Methyltransferase (OMT) is a ubiquitous enzyme that exists in bacteria, plants and humans and catalyzes a methyl-transfer reaction using S-adenosyl-l-methionine as a methyl donor and a wide range of phenolics as acceptors. To investigate the structure and function of OMTs, omt from Anabaena PCC 7120 was cloned into expression vector pET21a and expressed in a soluble form in Escherichia coli strain BL21 (DE3). The recombinant OMT protein was purified to homogeneity using a two-step strategy. Crystals of OMT that diffracted to a resolution of 2.4 Å were obtained using the hanging-drop vapour-diffusion method. The crystals belonged to space group C2221, with unit-cell parameters a = 131.620, b = 227.994, c = 150.777 Å, α = β = γ = 90°. There are eight molecules per asymmetric unit

  4. Biochemical characterization of maintenance DNA methyltransferase DNMT-1 from silkworm, Bombyx mori.

    Science.gov (United States)

    Mitsudome, Takumi; Mon, Hiroaki; Xu, Jian; Li, Zhiqing; Lee, Jae Man; Patil, Anandrao Ashok; Masuda, Atsushi; Iiyama, Kazuhiro; Morokuma, Daisuke; Kusakabe, Takahiro

    2015-03-01

    DNA methylation is an important epigenetic mechanism involved in gene expression of vertebrates and invertebrates. In general, DNA methylation profile is established by de novo DNA methyltransferases (DNMT-3A, -3B) and maintainance DNA methyltransferase (DNMT-1). DNMT-1 has a strong substrate preference for hemimethylated DNA over the unmethylated one. Because the silkworm genome lacks an apparent homologue of de novo DNMT, it is still unclear that how silkworm chromosome establishes and maintains its DNA methylation profile. As the first step to unravel this enigma, we purified recombinant BmDNMT-1 using baculovirus expression system and characterized its DNA-binding and DNA methylation activity. We found that the BmDNMT-1 preferentially methylates hemimethylated DNA despite binding to both unmethylated and hemimethylated DNA. Interestingly, BmDNMT-1 formed a complex with DNA in the presence or absence of methyl group donor, S-Adenosylmethionine (AdoMet) and the AdoMet-dependent complex formation was facilitated by Zn(2+) and Mn(2+). Our results provide clear evidence that BmDNMT-1 retained the function as maintenance DNMT but its sensitivity to metal ions is different from mammalian DNMT-1. PMID:25623240

  5. Multiple lysine methylation of PCAF by Set9 methyltransferase

    International Nuclear Information System (INIS)

    The molecular functions of several non-histone proteins are regulated through lysine modification by histone methyltransferases. The p300/CBP-associated factor (PCAF) is an acetyltransferase that has been implicated in many cellular processes. Here, we report that PCAF is a novel substrate of Set9 methyltransferase. In vitro mapping experiments revealed six lysine residues could be methylated by Set9. A comparison of amino acid sequences of target sites revealed the novel consensus motif which differs from previously identified Set9-consensus sequence. Further methyltransferase assays focusing on the six lysine residues showed that K78 and K89 are preferentially methylated in full-length PCAF in vitro. Using specific antibodies recognizing mono-methylated K89, in vivo PCAF methylation and its nuclear localization were demonstrated. Our data may lead to a new insight into PCAF functions and provide additional information to identify unknown targets of Set9.

  6. Sex-specific dysregulation of cysteine oxidation and the methionine and folate cycles in female cystathionine gamma-lyase null mice: a serendipitous model of the methylfolate trap

    Directory of Open Access Journals (Sweden)

    Hua Jiang

    2015-09-01

    Full Text Available In addition to its role in the endogenous synthesis of cysteine, cystathionine gamma-lyase (CGL is a major physiological source of the vasorelaxant hydrogen sulfide. Cgl null mice are potentially useful for studying the influence of this compound upon vascular tone and endothelial function. Here, we confirm a previous report that female Cgl null mice exhibit an approximate 45-fold increase in plasma total homocysteine compared to wild type controls. This level of homocysteine is approximately 3.5-fold higher than that observed in male Cgl null mice and is essentially equivalent to that observed in mouse models of cystathionine beta synthase deficient homocystinuria. Cgl null mice of both sexes exhibited decreased expression of methylenetetrahydrofolate reductase and cysteinesulfinate decarboxylase compared to WT controls. Female Cgl null mice exhibited a sex-specific induction of betaine homocysteine S-methyltransferase and methionine adenosyltransferase 1, alpha and a 70% decrease in methionine synthase expression accompanied by significantly decreased plasma methionine. Decreased plasma cysteine levels in female Cgl null mice were associated with sex-specific dysregulation of cysteine dioxygenase expression. Comparative histological assessment between cystathionine beta-synthase and Cgl null mice indicated that the therapeutic potential of cystathionine against liver injury merits possible further investigation. Collectively, our data demonstrates the importance of considering sex when investigating mouse models of inborn errors of metabolism and indicate that while female Cgl null mice are of questionable utility for studying the physiological role of hydrogen sulfide, they could serve as a useful model for studying the consequences of methionine synthase deficiency and the methylfolate trap.

  7. O6-methylguanine-DNA methyltransferase as a prognostic and predictive marker for basal-like breast cancer treated with cyclophosphamide-based chemotherapy

    OpenAIRE

    ISONO, SAYURI; FUJISHIMA, MAKOTO; AZUMI, TATSUYA; HASHIMOTO, YUKIHIKO; Komoike, Yoshifumi; YUKAWA, MASAO; WATATANI, MASAHIRO

    2014-01-01

    The O6-methylguanine-DNA methyltransferase (MGMT) protein protects cells from alkylating agents by removing alkyl groups from the O6-position of guanine. However, its effect on DNA damage induced by cyclophosphamide (CPM) is unclear. The present study investigated whether MGMT expression was correlated with prognosis in patients with breast cancer that was managed according to a common therapeutic protocol or treated with CPM-based chemotherapy. The intrinsic subtypes and MGMT protein express...

  8. Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity

    OpenAIRE

    Hegi, M E; L. Liu; Herman, J G; Stupp, R; Wick, W.; Weller, M.; Mehta, M P; Gilbert, M. R.

    2008-01-01

    Resistance to alkylating agents via direct DNA repair by O(6)-methylguanine methyltransferase (MGMT) remains a significant barrier to the successful treatment of patients with malignant glioma. The relative expression of MGMT in the tumor may determine response to alkylating agents, and epigenetic silencing of the MGMT gene by promoter methylation plays an important role in regulating MGMT expression in gliomas. MGMT promoter methylation is correlated with improved progression-free and overal...

  9. Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells

    DEFF Research Database (Denmark)

    Freiesleben, Ulrik Von

    1996-01-01

    Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome. The...

  10. Label-free electrochemical detection of human methyltransferase from tumors.

    Science.gov (United States)

    Furst, Ariel L; Muren, Natalie B; Hill, Michael G; Barton, Jacqueline K

    2014-10-21

    The role of abnormal DNA methyltransferase activity in the development and progression of cancer is an essential and rapidly growing area of research, both for improved diagnosis and treatment. However, current technologies for the assessment of methyltransferase activity, particularly from crude tumor samples, limit this work because they rely on radioactivity or fluorescence and require bulky instrumentation. Here, we report an electrochemical platform that overcomes these limitations for the label-free detection of human DNA(cytosine-5)-methyltransferase1 (DNMT1) methyltransferase activity, enabling measurements from crude cultured colorectal cancer cell lysates (HCT116) and biopsied tumor tissues. Our multiplexed detection system involving patterning and detection from a secondary electrode array combines low-density DNA monolayer patterning and electrocatalytically amplified DNA charge transport chemistry to measure selectively and sensitively DNMT1 activity within these complex and congested cellular samples. Based on differences in DNMT1 activity measured with this assay, we distinguish colorectal tumor tissue from healthy adjacent tissue, illustrating the effectiveness of this two-electrode platform for clinical applications. PMID:25288757

  11. The Histone Methyltransferase Activity of MLL1 Is Dispensable for Hematopoiesis and Leukemogenesis

    Directory of Open Access Journals (Sweden)

    Bibhu P. Mishra

    2014-05-01

    Full Text Available Despite correlations between histone methyltransferase (HMT activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4 methyltransferase critical for maintaining hematopoietic stem cells (HSCs. Here, we show that the SET domain, and thus HMT activity of MLL1, is dispensable for maintaining HSCs and supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16 acetylation is selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 is sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function.

  12. Characterization of a multifunctional caffeoyl-CoA O-methyltransferase activated in grape berries upon drought stress.

    Science.gov (United States)

    Giordano, Debora; Provenzano, Sofia; Ferrandino, Alessandra; Vitali, Marco; Pagliarani, Chiara; Roman, Federica; Cardinale, Francesca; Castellarin, Simone D; Schubert, Andrea

    2016-04-01

    Drought stress affects anthocyanin accumulation and modification in vegetative and reproductive plant tissues. Anthocyanins are the most abundant flavonoids in grape (Vitis vinifera L.) coloured berry genotypes and are essential markers of grape winemaking quality. They are mostly mono- and di-methylated, such modifications increase their stability and improve berry quality for winemaking. Anthocyanin methylation in grape berries is induced by drought stress. A few caffeoyl-CoA O-methyltransferases (CCoAOMTs) active on anthocyanins have been described in grape. However, no drought-activated O-methyltransferases have been described in grape berries yet. In this study, we characterized VvCCoAOMT, a grapevine gene known to induce methylation of CoA esters in cultured grape cells. Transcript accumulation of VvCCoAOMT was detected in berry skins, and increased during berry ripening on the plant, and in cultured berries treated with ABA, concomitantly with accumulation of methylated anthocyanins, suggesting that anthocyanins may be substrates of this enzyme. Contrary as previously observed in cell cultures, biotic stress (Botrytis cinerea inoculation) did not affect VvCCoAOMT gene expression in leaves or berries, while drought stress increased VvCCoAOMT transcript in berries. The recombinant VvCCoAOMT protein showed in vitro methylating activity on cyanidin 3-O-glucoside. We conclude that VvCCoAOMT is a multifunctional O-methyltransferase that may contribute to anthocyanin methylation activity in grape berries, in particular under drought stress conditions. PMID:26851572

  13. The betaine/GABA transporter and betaine: roles in brain, kidney and liver

    Directory of Open Access Journals (Sweden)

    Stephen A Kempson

    2014-04-01

    Full Text Available The physiological roles of the betaine/GABA transporter (BGT1; slc6a12 are still being debated. BGT1 is a member of the solute carrier family 6 (the neurotransmitter, sodium symporter transporter family and mediates cellular uptake of betaine and GABA in a sodium- and chloride- dependent process. Most of the studies of BGT1 concern its function and regulation in the kidney medulla where its role is best understood. The conditions here are hostile due to hyperosmolarity and significant concentrations of NH4Cl and urea. To withstand the hyperosmolarity, cells trigger osmotic adaptation, involving concentration of a transcriptional factor TonEBP/NFAT5 in the nucleus, and accumulate betaine and other osmolytes. Data from renal cells in culture, primarily MDCK, revealed that transcriptional regulation of BGT1 by TonEBP/NFAT5 is relatively slow. To allow more acute control of the abundance of BGT1 protein in the plasma membrane, there is also post-translation regulation of BGT1 protein trafficking which is dependent on intracellular calcium and ATP. Further, betaine may be important in liver metabolism as a methyl donor. In fact, in the mouse the liver is the organ with the highest content of BGT1. Hepatocytes express high levels of both BGT1 and the only enzyme that can metabolize betaine, namely betaine:homocysteine –S-methyltransferase (BHMT1. The BHMT1 enzyme removes a methyl group from betaine and transfers it to homocysteine, a potential risk factor for cardiovascular disease. Finally, BGT1 has been proposed to play a role in controlling brain excitability and thereby represents a target for anticonvulsive drug development. The latter hypothesis is controversial due to very low expression levels of BGT1 relative to other GABA transporters in brain, and also the primary location of BGT1 at the surface of the brain in the leptomeninges. These issues are discussed in detail.

  14. Structure and Function of Flavivirus NS5 Methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Zhou,Y.; Ray, D.; Zhao, Y.; Dong, H.; Ren, S.; Li, Z.; Guo, Y.; Bernard, K.; Shi, P.; Li, H.

    2007-01-01

    The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m{sup 7}GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA {yields} m{sup 7}GpppA {yields} m{sup 7}GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m{sup 7}GpppA-RNA can be readily methylated to m{sup 7}GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 {angstrom} resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K{sub 61}-D{sub 146}-K{sub 182}-E{sub 218} motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.

  15. Engineering Monolignol 4-O-Methyltransferases to Modulate Lignin Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Bhuiya, M.W.; Liu, C.

    2010-01-01

    Lignin is a complex polymer derived from the oxidative coupling of three classical monolignols. Lignin precursors are methylated exclusively at the meta-positions (i.e. 3/5-OH) of their phenyl rings by native O-methyltransferases, and are precluded from substitution of the para-hydroxyl (4-OH) position. Ostensibly, the para-hydroxyls of phenolics are critically important for oxidative coupling of phenoxy radicals to form polymers. Therefore, creating a 4-O-methyltransferase to substitute the para-hydroxyl of monolignols might well interfere with the synthesis of lignin. The phylogeny of plant phenolic O-methyltransferases points to the existence of a batch of evolutionarily 'plastic' amino acid residues. Following one amino acid at a time path of directed evolution, and using the strategy of structure-based iterative site-saturation mutagenesis, we created a novel monolignol 4-O-methyltransferase from the enzyme responsible for methylating phenylpropenes. We show that two plastic residues in the active site of the parental enzyme are vital in dominating substrate discrimination. Mutations at either one of these separate the evolutionarily tightly linked properties of substrate specificity and regioselective methylation of native O-methyltransferase, thereby conferring the ability for para-methylation of the lignin monomeric precursors, primarily monolignols. Beneficial mutations at both sites have an additive effect. By further optimizing enzyme activity, we generated a triple mutant variant that may structurally constitute a novel phenolic substrate binding pocket, leading to its high binding affinity and catalytic efficiency on monolignols. The 4-O-methoxylation of monolignol efficiently impairs oxidative radical coupling in vitro, highlighting the potential for applying this novel enzyme in managing lignin polymerization in planta.

  16. G9a, a multipotent regulator of gene expression

    OpenAIRE

    Shankar, Shilpa Rani; Bahirvani, Avinash G.; Rao, Vinay Kumar; Bharathy, Narendra; Ow, Jin Rong; Taneja, Reshma

    2013-01-01

    Lysine methylation of histone and non-histone substrates by the methyltransferase G9a is mostly associated with transcriptional repression. Recent studies, however, have highlighted its role as an activator of gene expression through mechanisms that are independent of its methyltransferase activity. Here we review the growing repertoire of molecular mechanisms and substrates through which G9a regulates gene expression. We also discuss emerging evidence for its wide-ranging functions in develo...

  17. Physiology and posttranscriptional regulation of methanol:coenzyme M methyltransferase isozymes in Methanosarcina acetivorans C2A.

    Science.gov (United States)

    Opulencia, Rina B; Bose, Arpita; Metcalf, William W

    2009-11-01

    Methanosarcina species possess three operons (mtaCB1, mtaCB2, and mtaCB3) encoding methanol-specific methyltransferase 1 (MT1) isozymes and two genes (mtaA1 and mtaA2) with the potential to encode a methanol-specific methyltransferase 2 (MT2). Previous genetic studies showed that these genes are differentially regulated and encode enzymes with distinct levels of methyltransferase activity. Here, the effects of promoter strength on growth and on the rate of methane production were examined by constructing strains in which the mtaCB promoters were exchanged. When expressed from the strong PmtaC1 or PmtaC2 promoter, each of the MtaC and MtaB proteins supported growth and methane production at wild-type levels. In contrast, all mtaCB operons exhibited poorer growth and lower rates of methane production when PmtaC3 controlled their expression. Thus, previously observed phenotypic differences can be attributed largely to differences in promoter activity. Strains carrying various combinations of mtaC, mtaB, and mtaA expressed from the strong, tetracycline-regulated PmcrB(tetO1) promoter exhibited similar growth characteristics on methanol, showing that all combinations of MtaC, MtaB, and MtaA can form functional MT1/MT2 complexes. However, an in vitro assay of coupled MT1/MT2 activity showed significant variation between the strains. Surprisingly, these variations in activity correlated with differences in protein abundance, despite the fact that all the encoding genes were expressed from the same promoter. Quantitative reverse transcriptase PCR and reporter gene fusion data suggest that the mtaCBA transcripts show different stabilities, which are strongly influenced by the growth substrate. PMID:19767431

  18. 亚砷酸钠对HaCaT细胞中DNMT1、HDAC1基因mRNA转录及蛋白表达的影响%Effects of Sodium Arsenite on Transcription and Expression of DNA Methyltransferase 1 (DNMT1) and Histone Deacetylase 1 (HDAC1) Gene in HaCaT Cells

    Institute of Scientific and Technical Information of China (English)

    潘雪莉; 张爱华

    2011-01-01

    Objective To observe the influences of different doses of sodium .arsenite on mRNA transcription and protein expression of DNA methyltransferase 1 (DNMTl) and histone deacetylase 1 (HDA Cl) gene in HaCaT cells. Methods HaCaT cells were treated with NaAsO2 at the doses of 0, 3.13, 6.25, 12.5,25 μmol/L, every other day, 24 h one time, for three times.The mRNA transcription of DNMT1 and HDA Cl was detected by real-time quantitative PCR, the protein expression of DNMT1 and HDAC1 was detected by Western blot. Human epidermal squamous carcinoma cell line A431 cells were set as the positive control group. Results Among the groups of HaCaT cells treated by 0, 3.13, 6.25, 12.5 and 25μmol/L NaAsO2, the level of DNMT1 mRNA transcription were 0.650 90±0.174 05, 0.930 53±0.145 56, 0.752 93±0.244 62, 0.558 1l ±0.051 02 and 0.370 17±0.08 84, the differences were significant (F=6.539, P<0.05); the level of HDAC1 mRNA transcription were 2.02180±0.179 57,1.496 98±0.107 55, 1.303 24±0.152 08, 1.037 75±0.204 88 and 0.816 74±0.153 12, the differences were significant(F=17.914,P<0.05). The level of DNMT1 protein expression were 0.000 87±0.000 10, 0.026 04±0.002 63, 0.283 19±0.072 62, 0.842 61±0.082 39 and 1.579 87±0.200 83, the differences were significant(F=27.799, P<0.05); the level of HDACl protein expression were 0.034 65±0.012 03, 0.273 65±0.012 02, 0.634 90±0.239 76, 0.775 03±0.126 51 and 1.126 16±0.167 52, the differences were significant(F=9.820, P<0.05). Conclusion DNMT1 and HDACl protein high expression is the important molecular event of arsenic-induced skin lesion, which may provide the possibility for using its inhibitors to explore the arsenic poisoning prevention and treatment.%目的 了解不同剂量的亚砷酸钠(NaAsO2)对人皮肤角质形成细胞(HaCaT细胞)中DNA甲基转移酶1(DNMT1)、组蛋白去乙酰化酶1(HDAC1)基因mRNA转录和蛋白表达的影响.方法 分别以0、3.13、6.25、12.5和25μmol/L NaAsO2溶液

  19. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    International Nuclear Information System (INIS)

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR

  20. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    Energy Technology Data Exchange (ETDEWEB)

    Kanno, Yuichiro, E-mail: ykanno@phar.toho-u.ac.jp; Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-03-27

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR.

  1. Screening of hepatocyte proteins binding to complete S protein of hepatitis B virus by yeast-two hybrid system

    Institute of Scientific and Technical Information of China (English)

    Gui-Qin Bai; Jun Cheng; Shu-Lin Zhang; Yan-Ping Huang; Lin Wang; Yan Liu; Shu-Mei Lin

    2005-01-01

    AIM: To investigate the biological function of complete S protein and to look for proteins interacting with complete S protein in hepatocytes.METHODS: We constructed bait plasmid expressing complete S protein of HBV by cloning the gene of complete S protein into pGBKT7, then the recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After extracting and sequencing of plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics.RESULTS: Nineteen colonies were selected and sequenced.Among them, five colonies were Homo sapiens solute carrier family 25, member 23 (SLC25A23), one was Homo sapiens calreticulin, one was human serum albumin (ALB)gene, one was Homo sapiens metallothionein 2A, two were Homo sapiens betaine-homocysteine methyltransferase,three were Homo sapiensNa+ and H+ coupled amino acid transport system N, one was Homo sapiens CD81 antigen (target of anti-proliferative antibody 1) (CD81), three were Homo sapiens diazepam binding inhibitor, two colonies were new genes with unknown function.CONCLUSION: The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with complete S protein of HBV. The complete S protein may bind to different proteins i.e., its multiple functions in vivo.

  2. Effects of sodium arsenite on hypermethylation, transcription and expression of O6-methylguanine-DNA methyltransferase gene in HaCaT cells%亚砷酸钠对HaCaT细胞MGMT基因甲基化和mRNA及蛋白表达水平的影响

    Institute of Scientific and Technical Information of China (English)

    潘雪莉; 张爱华

    2011-01-01

    Objective To investigate the DNA methylation feature and DNA methylation regulation to its transcription and expression of O6-methylguanine-DNA methyltransferase gene (MGMT) in NaAsO2-treated HaCaT cells. Methods HaCaT cells were treated 72 hours at intervals and repeatedly by 3.13, 6.25,12.50, and 25.00 μmol/L NaAsO2, MGMT gene promoter region was amplified in the transcription initiation site - 329 - + 93 region by bisulfate-sequencing polymerase chain reaction (BSP), the mRNA transcription and the protein expression of MGMT was detected by real-time quantitative PCR and Western blotting. NaAsO2-untreated HaCaT cell was set as a blank control, and human epidermal squamous carcinoma cell strain A431 was set as a positive control. Results Among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, the positive rates of the DNA methylation of promoter region in MGMT gene were 0.63%(l/160), 6.25% (10/160), 10.63%( 17/160) and 18.75% (30/160), respectively, and methylated CpG sites were mainly located in - 249--146 region relative to transcription start site. There was no DNA methylation in the blank control. There were significant differences between the blank control and the NaAsO2-treated cells (x2 = 76.687, P< 0.05). Average levels of MGMT mRNA were 1.518 31 ± 0.180 54, 1.425 22 ± 0.180 39, 1.014 54 ± 0.096 79 and 0.887 72 ± 0.020 00, respectively among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, compared with the blank control cells(1.198 29 ± 0.159 97), there were significant differences(F = 37.359, P < 0.05). Average levels of MGMT protein were 1.174 47 ± 0.064 75, 0.848 83 ± 0.057 01, 0.471 63 ± 0.023 34 and 0.240 34 ± 0.014 43, respectively among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, compared with the blank control cells (1.066 19 ± 0.061 24), there were significant differences(F = 20.687, P < 0.05). Conclusions Arsenic can cause Cp

  3. The potential role of juvenile hormone acid methyltransferase in methyl farnesoate (MF) biosynthesis in the swimming crab, Portunus trituberculatus.

    Science.gov (United States)

    Xie, Xi; Tao, Tian; Liu, Mingxin; Zhou, Yanqi; Liu, Zhiye; Zhu, Dongfa

    2016-05-01

    Juvenile hormone (JH) and methyl farnesoate (MF) play essential roles in the development and reproduction of insects and crustaceans respectively. Juvenile hormone acid methyltransferase (JHAMT) catalyzes the methyl esterification in insect JH biosynthesis, while the corresponding step in crustacean MF biosynthesis was long thought to be catalyzed by farnesoic acid O-methyltransferase (FAMeT). However, the new discovery of JHAMT orthologs in crustaceans indicates that JHAMT may also play essential role in the MF biosynthesis in crustaceans. Here we cloned and characterized the full-length cDNA encoding JHAMT in the swimming crab Portunus trituberculatus (PtJHAMT). Sequence and structure analysis of PtJHAMT revealed that it was composed of a 6-stranded β sheet with 9 α helices, and contained a signature Sadenosyl-l-methionine (SAM) binding motif, which is the hallmark in all SAM dependent methyltransferases (SAM-MTs). Several active sites that are critical for the interaction of SAM and JH/FA substrate were also conserved in PtJHAMT. The gene expression of PtJHAMT was highly specific to the mandibular organ, which is the sole site of MF synthesis. PtJHAMT expression significantly increased in the late-vitellogenic stage and mature stage, which suggests a possible role of PtJHAMT in modulating ovarian development. The role of PtJHAMT and PtFAMeT in MF biosynthesis was further investigated by RNA interfering (RNAi). Injection of PtJHAMT and PtFAMeT dsRNA both led to a decrease in hemolymph MF titers. Injection of PtHMGR dsRNA caused the decrease in PtJHAMT expression, but had no effect on mRNA level of PtFAMeT. Together these results suggested that JHAMT and FAMeT are both involved in the MF biosynthesis in crustaceans, while the JHAMT is highly specific to FA substrate, and FAMeT may have more catalytic functions. PMID:26952760

  4. Epigenetic Regulation of Autophagy by the Methyltransferase G9a

    OpenAIRE

    Artal-Martinez de Narvajas, Amaia; Gomez, Timothy S.; Zhang, Jin-San; Mann, Alexander O.; Taoda, Yoshiyuki; Gorman, Jacquelyn A.; Herreros-Villanueva, Marta; Gress, Thomas M; Ellenrieder, Volker; Bujanda, Luis; Kim, Do-Hyung; Kozikowski, Alan P.; Koenig, Alexander; Billadeau, Daniel D.

    2013-01-01

    Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the cytoplasmic machinery that orchestrates autophagy induction during starvation, hypoxia, or receptor stimulation has been widely studied, the key epigenetic events that initiate and maintain the autophagy process remain unknown. Here we show that the methyltransferase G9a coordinates the transcriptional activation of key regulators of autophagosome formation by remo...

  5. Histamine N-methyltransferase Modulates Human Bronchial Smooth Muscle Contraction

    OpenAIRE

    Tamaoki, J.; Chiyotani, A.; Tagaya, E; Isono, K; Konno, K

    1994-01-01

    To elucidate the modulatory role of histamine-degrading enzymes in airway constrictor responses, human bronchial strips were studied under isometric conditions in vitro. Pretreatment of tissues with the histamine N-methyltransferase (HMT) inhibitor SKF 91488 specifically potentiated the contractile responses to histamine, causing a leftward displacement of the concentration response curves, whereas the diamine oxidase inhibitor aminoguanidine had no effect. This potentiation was attenuated by...

  6. Plant isoflavone and isoflavanone O-methyltransferase genes

    Science.gov (United States)

    Broeckling, Bettina E.; Liu, Chang-Jun; Dixon, Richard A.

    2014-08-19

    The invention provides enzymes that encode O-methyltransferases (OMTs) from Medicago truncatula that allow modification to plant (iso)flavonoid biosynthetic pathways. In certain aspects of the invention, the genes encoding these enzymes are provided. The invention therefore allows the modification of plants for isoflavonoid content. Transgenic plants comprising such enzymes are also provided, as well as methods for improving disease resistance in plants. Methods for producing food and nutraceuticals, and the resulting compositions, are also provided.

  7. Crystallization of the novel S-adenosyl-l-methionine-dependent C-methyltransferase CouO from Streptomyces rishiriensis and preliminary diffraction data analysis

    International Nuclear Information System (INIS)

    Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-adenosyl-l-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified. Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-adenosyl-l-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified. CouO was crystallized from a single condition in the Morpheus crystallization screen. A vitrified crystal diffracted to 2.05 Å resolution and belonged to space group P21, with unit-cell parameters a = 33.02, b = 82.87, c = 76.77 Å, β = 96.93°

  8. Imatinib causes epigenetic alterations of PTEN gene via upregulation of DNA methyltransferases and polycomb group proteins

    International Nuclear Information System (INIS)

    We have recently reported the possible imatinib-resistant mechanism; long-term exposure of leukemia cells to imatinib downregulated levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) via hypermethylation of its promoter region (Leukemia 2010; 24: 1631). The present study explored the molecular mechanisms by which imatinib caused methylation on the promoter region of this tumor suppressor gene in leukemia cells. Real-time reverse transcription PCR found that long-term exposure of chronic eosinophilic leukemia EOL-1 cells expressing FIP1L1/platelet-derived growth factor receptor-α to imatinib induced expression of DNA methyltransferase 3A (DNMT3A) and histone-methyltransferase enhancer of zeste homolog 2 (EZH2), a family of polycomb group, thereby increasing methylation of the gene. Immunoprecipitation assay found the increased complex formation of DNMT3A and EZH2 proteins in these cells. Moreover, chromatin immunoprecipitation assay showed that amounts of both DNMT3A and EZH2 proteins bound around the promoter region of PTEN gene were increased in EOL-1 cells after exposure to imatinib. Furthermore, we found that levels of DNMT3A and EZH2 were strikingly increased in leukemia cells isolated from individuals with chronic myelogenous leukemia (n=1) and Philadelphia chromosome-positive acute lymphoblastic leukemia (n=2), who relapsed after treatment with imatinib compared with those isolated at their initial presentation. Taken together, imatinib could cause drug-resistance via recruitment of polycomb gene complex to the promoter region of the PTEN and downregulation of this gene's transcripts in leukemia patients

  9. Molecular and biochemical characterization of the jasmonic acid methyltransferase gene from black cottonwood (Populus trichocarpa)

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Nan [ORNL; Yao, Jianzhuang [University of Tennessee, Knoxville (UTK); Chaiprasongsuk, Minta [University of Tennessee, Knoxville (UTK); Li, Guanglin [University of Tennessee, Knoxville (UTK); Guan, Ju [University of Tennessee, Knoxville (UTK); Tschaplinski, Timothy J [ORNL; Guo, Hong [University of Tennessee, Knoxville (UTK); Chen, Feng [University of Tennessee, Knoxville (UTK)

    2013-01-01

    Methyl jasmonate is a metabolite known to be produced by many plants and has roles in diverse biological processes. It is biosynthesized by the action of S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase (JMT), which belongs to the SABATH family of methyltransferases. Herein is reported the isolation and biochemical characterization of a JMT gene from black cottonwood (Populus trichocarpa). The genome of P. trichocarpa contains 28 SABATH genes (PtSABATH1 to PtSABATH28). Recombinant PtSABATH3 expressed in Escherichia coli showed the highest level of activity with jasmonic acid (JA) among carboxylic acids tested. It was therefore renamed PtJMT1. PtJMT1 also displayed activity with benzoic acid (BA), with which the activity was about 22% of that with JA. PtSABATH2 and PtSABATH4 were most similar to PtJMT1 among all PtSABATHs. However, neither of them had activity with JA. The apparent Km values of PtJMT1 using JA and BA as substrate were 175 lM and 341 lM, respectively. Mutation of Ser-153 and Asn-361, two residues in the active site of PtJMT1, to Tyr and Ser respectively, led to higher specific activity with BA than with JA. Homology-based structural modeling indicated that substrate alignment, in which Asn-361 is involved, plays a role in determining the substrate specificity of PtJMT1. In the leaves of young seedlings of black cottonwood, the expression of PtJMT1 was induced by plant defense signal molecules methyl jasmonate and salicylic acid and a fungal elicitor alamethicin, suggesting that PtJMT1 may have a role in plant defense against biotic stresses. Phylogenetic analysis suggests that PtJMT1 shares a common ancestor with the Arabidopsis JMT, and functional divergence of these two apparent JMT orthologs has occurred since the split of poplar and Arabidopsis lineages.

  10. Age-Associated Decrease of the Histone Methyltransferase SUV39H1 in HSC Perturbs Heterochromatin and B Lymphoid Differentiation

    OpenAIRE

    Dounia Djeghloul; Klaudia Kuranda; Isabelle Kuzniak; Daniela Barbieri; Irina Naguibneva; Caroline Choisy; Jean-Christophe Bories; Christine Dosquet; Marika Pla; Valérie Vanneaux; Gérard Socié; Françoise Porteu; David Garrick; Michele Goodhardt

    2016-01-01

    The capacity of hematopoietic stem cells (HSC) to generate B lymphocytes declines with age, contributing to impaired immune function in the elderly. Here we show that the histone methyltransferase SUV39H1 plays an important role in human B lymphoid differentiation and that expression of SUV39H1 decreases with age in both human and mouse HSC, leading to a global reduction in H3K9 trimethylation and perturbed heterochromatin function. Further, we demonstrate that SUV39H1 is a target of microRNA...

  11. The histone methyltransferase SUV420H2 and Heterochromatin Proteins HP1 interact but show different dynamic behaviours

    OpenAIRE

    Souza Patricia P; Völkel Pamela; Trinel Dave; Vandamme Julien; Rosnoblet Claire; Héliot Laurent; Angrand Pierre-Olivier

    2009-01-01

    Abstract Background Histone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions. In particular, trimethylation at lysine 9 of histone H3 (H3K9) and at lysine 20 of histone H4 (H4K20) governed by the histone methyltransferases SUV39H1/2 and SUV420H1/2 respectively, have emerged as a hallmark of pericentric heterochromatin. Controlled chromatin organization is crucial for gene expression regulation and genome stability. Therefore, it is ess...

  12. Age-Associated Decrease of the Histone Methyltransferase SUV39H1 in HSC Perturbs Heterochromatin and B Lymphoid Differentiation

    OpenAIRE

    Djeghloul, Dounia; Kuranda, Klaudia; Kuzniak, Isabelle; Barbieri, Daniela; Naguibneva, Irina; Choisy, Caroline; Bories, Jean-Christophe; Dosquet, Christine; Pla, Marika; Vanneaux, Valérie; Socié, Gérard; Porteu, Françoise; Garrick, David; Goodhardt, Michele

    2016-01-01

    Summary The capacity of hematopoietic stem cells (HSC) to generate B lymphocytes declines with age, contributing to impaired immune function in the elderly. Here we show that the histone methyltransferase SUV39H1 plays an important role in human B lymphoid differentiation and that expression of SUV39H1 decreases with age in both human and mouse HSC, leading to a global reduction in H3K9 trimethylation and perturbed heterochromatin function. Further, we demonstrate that SUV39H1 is a target of ...

  13. H3K9 methyltransferase G9a negatively regulates UHRF1 transcription during leukemia cell differentiation

    OpenAIRE

    Kim, Kee-Beom; Son, Hye-Ju; Choi, Sulji; Hahm, Ja Young; Jung, Hyeonsoo; Baek, Hee Jo; Kook, Hoon; Hahn, Yoonsoo; Kook, Hyun; Seo, Sang-Beom

    2015-01-01

    Histone H3K9 methyltransferase (HMTase) G9a-mediated transcriptional repression is a major epigenetic silencing mechanism. UHRF1 (ubiquitin-like with PHD and ring finger domains 1) binds to hemimethylated DNA and plays an essential role in the maintenance of DNA methylation. Here, we provide evidence that UHRF1 is transcriptionally downregulated by H3K9 HMTase G9a. We found that increased expression of G9a along with transcription factor YY1 specifically represses UHRF1 transcription during T...

  14. Structural characterization of the mitomycin 7-O-methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Shanteri; Chang, Aram; Goff, Randal D.; Bingman, Craig A.; Grüschow, Sabine; Sherman, David H.; Phillips, Jr., George N.; Thorson, Jon S. (Michigan); (UW)

    2014-10-02

    Mitomycins are quinone-containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9{beta}- and C9{alpha}-configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR-S-adenosylhomocysteine (SAH) binary complex and MmcR-SAH-mitomycin A (MMA) ternary complex at resolutions of 1.9 and 2.3 {angstrom}, respectively. The study revealed MmcR to adopt a common S-adenosyl-L-methionine-dependent O-methyltransferase fold and the presence of a structurally conserved active site general acid-base pair is consistent with a proton-assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins.

  15. Molecular Cloning and Characterization of O-Methyltransferase from Mango Fruit (Mangifera indica cv. Alphonso).

    Science.gov (United States)

    Chidley, Hemangi G; Oak, Pranjali S; Deshpande, Ashish B; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

    2016-05-01

    Flavour of ripe Alphonso mango is invariably dominated by the de novo appearance of lactones and furanones during ripening. Of these, furanones comprising furaneol (4-hydroxy-2,5-dimethyl-3(2H)-furanone) and mesifuran (2,5-dimethyl-4-methoxy-3(2H)-furanone) are of particular importance due to their sweet, fruity caramel-like flavour characters and low odour detection thresholds. We isolated a 1056 bp complete open reading frame of a cDNA encoding S-adenosyl-L-methionine-dependent O-methyltransferase from Alphonso mango. The recombinantly expressed enzyme, MiOMTS showed substrate specificity towards furaneol and protocatechuic aldehyde synthesizing mesifuran and vanillin, respectively, in an in vitro assay reaction. A semi-quantitative PCR analysis showed fruit-specific expression of MiOMTS transcripts. Quantitative real-time PCR displayed ripening-related expression pattern of MiOMTS in both pulp and skin of Alphonso mango. Also, early and significantly enhanced accumulation of its transcripts was detected in pulp and skin of ethylene-treated fruits. Ripening-related and fruit-specific expression profile of MiOMTS and substrate specificity towards furaneol is a suggestive of its involvement in the synthesis of mesifuran in Alphonso mango. Moreover, a significant trigger in the expression of MiOMTS transcripts in ethylene-treated fruits point towards the transcriptional regulation of mesifuran biosynthesis by ethylene. PMID:27039187

  16. Protein arginine methyltransferase 5 is associated with malignant phenotype and peritoneal metastasis in gastric cancer.

    Science.gov (United States)

    Kanda, Mitsuro; Shimizu, Dai; Fujii, Tsutomu; Tanaka, Haruyoshi; Shibata, Masahiro; Iwata, Naoki; Hayashi, Masamichi; Kobayashi, Daisuke; Tanaka, Chie; Yamada, Suguru; Nakayama, Goro; Sugimoto, Hiroyuki; Koike, Masahiko; Fujiwara, Michitaka; Kodera, Yasuhiro

    2016-09-01

    Identification of novel gastric cancer (GC)-related molecules is necessary to improve management of patients with GC in both diagnostic and therapeutic aspects. The aim of the present study was to determine whether protein arginine methyltransferase 5 (PRMT5) acts as an oncogene in the progression of GC and whether it serves as a novel diagnostic marker and therapeutic target. We conducted global expression profiling of GC cell lines and RNA interference experiments to evaluate the effect of PRMT5 expression on the phenotype of GC cells. We analysed tissues of 179 patients with GC to assess the association of PRMT5 mRNA levels with clinicopathological factors. Differential expression of PRMT5 mRNA by GC cell lines correlated positively with the levels of GEMIN2, STAT3 and TGFB3. PRMT5 knockdown reduced the proliferation, invasion and migration of a GC cell line. PRMT5 mRNA levels were significantly higher in GC tissues than the corresponding adjacent normal tissues and were independent of tumour depth, differentiation and lymph node metastasis. High PRMT5 expression was an independent risk factor of positive peritoneal lavage cytology (odds ratio 3.90, P=0.003) and decreased survival. PRMT5 enhances the malignant phenotype of GC cell lines and its expression in gastric tissues may serve as a biomarker for patient stratification and a potential target of therapy. PMID:27315569

  17. DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents.

    OpenAIRE

    Glassner, Brian; Weeda, Geert; Allan, James; Broekhof, Jose'; Carls, Nick; Donker, Ingrid; Engelward, Bevin; Hampson, Richard; Hersmus, Remko; Hickman, Mark; Roth, Richard; Warren, Henry; Wu, Mavis; Hoeijmakers, Jan; Samson, Leona

    1999-01-01

    textabstractWe have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA methyltransferase activity, suggesting that Mgmt constitutes the major, if not the only, O6-methylguanine DNA methyltransferase. Primary mouse embryo fibroblasts and bone marrow cells from Mgmt -/- mice were s...

  18. Molecular Mechanism of Action of Plant DRM De Novo DNA Methyltransferases

    OpenAIRE

    Zhong, Xuehua; Du, Jiamu; Hale, Christopher J.; Gallego-Bartolome, Javier; Feng, Suhua; Vashisht, Ajay A.; Chory, Joanne; Wohlschlegel, James A.; Patel, Dinshaw J.; Jacobsen, Steven E.

    2014-01-01

    DNA methylation is a conserved epigenetic gene regulation mechanism. DOMAINS REARRANGED METHYLTRANSFERASE (DRM) is a key de novo methyltransferase in plants, but how DRM acts mechanistically is poorly understood. Here, we report the crystal structure of the methyltransferase domain of tobacco DRM (NtDRM) and reveal a molecular basis for its rearranged structure. NtDRM forms a functional homo-dimer critical for catalytic activity. We also show that Arabidopsis DRM2 exists in complex with the s...

  19. Reprogramming metabolism by histone methyltransferase NSD2 drives endocrine resistance via coordinated activation of pentose phosphate pathway enzymes.

    Science.gov (United States)

    Wang, Junjian; Duan, Zhijian; Nugent, Zoann; Zou, June X; Borowsky, Alexander D; Zhang, Yanhong; Tepper, Clifford G; Li, Jian Jian; Fiehn, Oliver; Xu, Jianzhen; Kung, Hsing-Jien; Murphy, Leigh C; Chen, Hong-Wu

    2016-08-10

    Metabolic reprogramming such as the aerobic glycolysis or Warburg effect is well recognized as a common feature of tumorigenesis. However, molecular mechanisms underlying metabolic alterations for tumor therapeutic resistance are poorly understood. Through gene expression profiling analysis we found that histone H3K36 methyltransferase NSD2/MMSET/WHSC1 expression was highly elevated in tamoxifen-resistant breast cancer cell lines and clinical tumors. IHC analysis indicated that NSD2 protein overexpression was associated with the disease recurrence and poor survival. Ectopic expression of NSD2 wild type, but not the methylase-defective mutant, drove endocrine resistance in multiple cell models and xenograft tumors. Mechanistically, NSD2 was recruited to and methylated H3K36me2 at the promoters of key glucose metabolic enzyme genes. Its overexpression coordinately up-regulated hexokinase 2 (HK2) and glucose-6-phosphate dehydrogenase (G6PD), two key enzymes of glycolysis and the pentose phosphate pathway (PPP), as well as TP53-induced glycolysis regulatory phosphatase TIGAR. Consequently, NSD2-driven tamoxifen-resistant cells and tumors displayed heightened PPP activity, elevated NADPH production, and reduced ROS level, without significantly altered glycolysis. These results illustrate a coordinated, epigenetic activation of key glucose metabolic enzymes in therapeutic resistance and nominate methyltransferase NSD2 as a potential therapeutic target for endocrine resistant breast cancer. PMID:27164560

  20. A human tRNA methyltransferase 9-like protein prevents tumour growth by regulating LIN9 and HIF1-α.

    Science.gov (United States)

    Begley, Ulrike; Sosa, Maria Soledad; Avivar-Valderas, Alvaro; Patil, Ashish; Endres, Lauren; Estrada, Yeriel; Chan, Clement T Y; Su, Dan; Dedon, Peter C; Aguirre-Ghiso, Julio A; Begley, Thomas

    2013-03-01

    Emerging evidence points to aberrant regulation of translation as a driver of cell transformation in cancer. Given the direct control of translation by tRNA modifications, tRNA modifying enzymes may function as regulators of cancer progression. Here, we show that a tRNA methyltransferase 9-like (hTRM9L/KIAA1456) mRNA is down-regulated in breast, bladder, colorectal, cervix and testicular carcinomas. In the aggressive SW620 and HCT116 colon carcinoma cell lines, hTRM9L is silenced and its re-expression and methyltransferase activity dramatically suppressed tumour growth in vivo. This growth inhibition was linked to decreased proliferation, senescence-like G0/G1-arrest and up-regulation of the RB interacting protein LIN9. Additionally, SW620 cells re-expressing hTRM9L did not respond to hypoxia via HIF1-α-dependent induction of GLUT1. Importantly, hTRM9L-negative tumours were highly sensitive to aminoglycoside antibiotics and this was associated with altered tRNA modification levels compared to antibiotic resistant hTRM9L-expressing SW620 cells. Our study links hTRM9L and tRNA modifications to inhibition of tumour growth via LIN9 and HIF1-α-dependent mechanisms. It also suggests that aminoglycoside antibiotics may be useful to treat hTRM9L-deficient tumours. PMID:23381944

  1. 亚砷酸钠对人皮肤角质形成细胞MGMT基因组蛋白乙酰化及转录与表达的影响%Effects of Sodium Arsenite on Histone Acetylation, Transcription and Expression of O6-Methylguanine-DNA Methyltransferase Gene in HaCaT Cells

    Institute of Scientific and Technical Information of China (English)

    潘雪莉; 张爱华

    2011-01-01

    [Objective]To observe the influences of different doses of sodium arsenite on histone acetylation regulation, mRNA transcription and protein expression of O6-methylguanine-DNA methyltransferase gene( MGMT)in HaCaT cells.[Methods]HaCaT cells were treated with 25.00, 12.50, 6.25 and 3.13 μmol/L NaAsO2 for 72h at intervals and repeatedly.Histone acetylation modifications in two transcription regulatory region ( ChIP1, ChIP2 region ) and in coding region ( ChIP3 region, the control region ) of MGMT gene were detected by chromatin immuno-precipitation combined with quantitative PCR, the mRNA transcription and the protein expression of MGMT were detected by real-time quantitative PCR and Western blot.HaCaT cells untreated with NaAsO2( 0.00 μmol/L )were set as the blank control group, human epidermal squamous carcinoma cell line A31 cells were set as the positive control group.[Results]Among the groups of HaCaT cells treated with 0.00, 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, the levels of histone acetylation of H3K9 in ChIP1 transcription regulatory region of MGMT gene were 176.68 ± 8.50, 175.71 ± 18.14, 161.26 ± 16.28, 146.23 ±24.00 and 82.64 ± 33.87 respectively, the differences were significant( F= 28.809, P < 0.05 ).The levels of histone acetylation of H4 in ChIP1 transcription regulatory region were 183.59 ± 11.98, 180.84 ± 24.10, 166.52 ± 5.48, 156.87 ± 10.64 and 103.42 ± 7.04, the differences were significant ( F= 36.493, P < 0.05 ).The levels of histone acetylation of H3K9 in ChIP2 transcription regulatory region were 171.11 ± 16.54, 167.55 ± 8.97, 156.51 ± 8.59, 135.88 ± 16.55 and 82.01 ± 3.96, the differences were significant( F=49.626,P<0.05 ).The levels of histone acetylation of H4 in ChIP2 transcription regulatory region were 117.23 ± 16.21, 143.29 ± 10.59, 135.87 ±7.44, 105.48 ± 7.56 and 78.79 ± 6.92, the differences were significant( F=25.438, P<0.05 ).The levels of histone acetylation of H3K9 in ChIP3 coding region were 37

  2. Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

    Energy Technology Data Exchange (ETDEWEB)

    Mizuno, Kouichi, E-mail: koumno@akita-pu.ac.jp [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Matsuzaki, Masahiro [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Kanazawa, Shiho [Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610 (Japan); Tokiwano, Tetsuo; Yoshizawa, Yuko [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Kato, Misako [Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610 (Japan)

    2014-10-03

    Graphical abstract: Trigonelline synthase catalyzes the conversion of nicotinic acid to trigonelline. We isolated and characterized trigonelline synthase gene(s) from Coffea arabica. - Highlights: • Trigonelline is a major compound in coffee been same as caffeine is. • We isolated and characterized trigonelline synthase gene. • Coffee trigonelline synthases are highly homologous with coffee caffeine synthases. • This study contributes the fully understanding of pyridine alkaloid metabolism. - Abstract: Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-{sup 14}C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or

  3. Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

    International Nuclear Information System (INIS)

    Graphical abstract: Trigonelline synthase catalyzes the conversion of nicotinic acid to trigonelline. We isolated and characterized trigonelline synthase gene(s) from Coffea arabica. - Highlights: • Trigonelline is a major compound in coffee been same as caffeine is. • We isolated and characterized trigonelline synthase gene. • Coffee trigonelline synthases are highly homologous with coffee caffeine synthases. • This study contributes the fully understanding of pyridine alkaloid metabolism. - Abstract: Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-14C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with

  4. Role of microRNAs and DNA methyltransferases in transmitting induced genomic instability between cell generations

    Directory of Open Access Journals (Sweden)

    Katriina eHuumonen

    2014-09-01

    Full Text Available There is limited understanding of how radiation or chemicals induce genomic instability, and how the instability is epigenetically transmitted to the progeny of exposed cells or organisms. Here we measured the expression of microRNAs (miRNAs and DNA methyltransferases (DNMTs in murine embryonal fibroblasts exposed to ionizing radiation or 2,3,7,8 -tetrachlorodibenzo-p-dioxin (TCDD, which were previously shown to induce genomic instability in this cell line. Cadmium was used as a reference agent that does not induce genomic instability in our experimental model. Measurements at 8 and 15 days after exposure did not identify any such persistent changes that could be considered as signals transmitting genomic instability to the progeny of exposed cells. However, measurements at 2 days after exposure revealed findings that may reflect initial stages of genomic instability. Changes that were common to TCDD and two doses of radiation (but not to cadmium included 5 candidate signature miRNAs and general up-regulation of miRNA expression. Expression of DNMT3a, DNMT3b and DNMT2 were suppressed by cadmium but not by TCDD or radiation, consistently with the hypothesis that sufficient expression of DNMTs is necessary in the initial phase of induced genomic instability.

  5. DNA methyltransferase inhibition increases efficacy of adoptive cellular immunotherapy of murine breast cancer.

    Science.gov (United States)

    Terracina, Krista P; Graham, Laura J; Payne, Kyle K; Manjili, Masoud H; Baek, Annabel; Damle, Sheela R; Bear, Harry D

    2016-09-01

    Adoptive T cell immunotherapy is a promising approach to cancer treatment that currently has limited clinical applications. DNA methyltransferase inhibitors (DNAMTi) have known potential to affect the immune system through multiple mechanisms that could enhance the cytotoxic T cell responses, including: upregulation of tumor antigen expression, increased MHC class I expression, and blunting of myeloid derived suppressor cells (MDSCs) expansion. In this study, we have investigated the effect of combining the DNAMTi, decitabine, with adoptive T cell immunotherapy in the murine 4T1 mammary carcinoma model. We found that expression of neu, MHC class I molecules, and several murine cancer testis antigens (CTA) was increased by decitabine treatment of 4T1 cells in vitro. Decitabine also increased expression of multiple CTA in two human breast cancer cell lines. Decitabine-treated 4T1 cells stimulated greater IFN-gamma release from tumor-sensitized lymphocytes, implying increased immunogenicity. Expansion of CD11b + Gr1 + MDSC in 4T1 tumor-bearing mice was significantly diminished by decitabine treatment. Decitabine treatment improved the efficacy of adoptive T cell immunotherapy in mice with established 4T1 tumors, with greater inhibition of tumor growth and an increased cure rate. Decitabine may have a role in combination with existing and emerging immunotherapies for breast cancer. PMID:27416831

  6. Protective effect of O6-methylguanine-DNA-methyltransferase on mammalian cells

    Institute of Scientific and Technical Information of China (English)

    LI Dong-bo; WANG Ji-shi; FANG Qin; SUN Hai-yang; XU Wei; LI Wei-da

    2007-01-01

    Background O6-methylguanine-DNA-methyltransferase (MGMT) is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells.Methods Mammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells (PBMCs) via liposome, then assayed for gene expression at RNA and protein levels. MTT assay was used to check the drug resistance of cells transfected with MGMT gene.Results MGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC were 13.4 and 4.0 times that of the empty vector transfected groups respectively.Results of Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC50 values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC.Conclusion The alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.

  7. Binding induced RNA conformational changes control substrate recognition and catalysis by the thiostrepton resistance methyltransferase (Tsr).

    Science.gov (United States)

    Kuiper, Emily G; Conn, Graeme L

    2014-09-19

    Ribosomal RNA (rRNA) post-transcriptional modifications are essential for ribosome maturation, translational fidelity, and are one mechanism used by both antibiotic-producing and pathogenic bacteria to resist the effects of antibiotics that target the ribosome. The thiostrepton producer Streptomyces azureus prevents self-intoxication by expressing the thiostrepton-resistance methyltransferase (Tsr), which methylates the 2'-hydroxyl of 23 S rRNA nucleotide adenosine 1067 within the thiostrepton binding site. Tsr is a homodimer with each protomer containing an L30e-like amino-terminal domain (NTD) and a SPOUT methyltransferase family catalytic carboxyl-terminal domain (CTD). We show that both enzyme domains are required for high affinity RNA substrate binding. The Tsr-CTD has intrinsic, weak RNA affinity that is necessary to direct the specific high-affinity Tsr-RNA interaction via NTDs, which have no detectable RNA affinity in isolation. RNA structure probing experiments identify the Tsr footprint on the RNA and structural changes in the substrate, induced specifically upon NTD binding, which are necessary for catalysis by the CTD. Additionally, we identify a key amino acid in each domain responsible for CTD-RNA binding and the observed NTD-dependent RNA structural changes. These studies allow us to develop a model for Tsr-RNA interaction in which the coordinated substrate recognition of each Tsr structural domain is an obligatory pre-catalytic recognition event. Our findings underscore the complexity of substrate recognition by RNA modification enzymes and the potential for direct involvement of the RNA substrate in controlling the process of its modification. PMID:25086036

  8. Knock-down of protein L-isoaspartyl O-methyltransferase increases β-amyloid production by decreasing ADAM10 and ADAM17 levels

    Institute of Scientific and Technical Information of China (English)

    Narkhyun BAE; Se Eun BYEON; Jihyuk SONG; Sang-Jin LEE; Moosik KWON; Inhee MOOK-JUNG; Jae Youl CHO; Sungyoul HONG

    2011-01-01

    Aim: To examine the role of protein L-isoaspartyl O-methyltransferase (PIMT; EC 2.1.1.77) on the secretion of Aβ peptides.Methods: HEK293 APPsw cells were treated with PIMT siRNA or adenosine dialdehyde (AdOX), a broad-spectrum methyltransferase inhibitor. Under the conditions, the level of Aβ secretion and regulatory mechanism by PIMT were examined.Results: Knock-down of PIMT and treatment with AdOX significantly increased Aβ40 secretion. Reductions in levels of PIMT decreased the secretion of soluble amyloid precursor protein alpha (sAPPα) without altering the total expression of APP or its membrane-bound C83 fragment. However, the levels of the C99 fragment generated by β-secretase were enhanced. Moreover, the decreased secretion of sAPPα resulting from PIMT knock-down seemed to be linked with the suppression of the expression of α-secretase gene products,α-disintegrin and metalloprotease 10 (ADAM10) and ADAM17, as indicated by Western blot analysis. In contrast, ADAM10 was not down-regulated in response to treatment with the protein arginine methyltransferase (PRMT) inhibitor, AMI-1.Conclusion: This study demonstrates a novel role for PIMT, but not PRMT, as a negative regulator of Aβ peptide formation and a potential protective factor in the pathogenesis of AD.

  9. Inhibition of histone methyltransferases SUV39H1 and G9a leads to neuroprotection in an in vitro model of cerebral ischemia.

    Science.gov (United States)

    Schweizer, Sophie; Harms, Christoph; Lerch, Heike; Flynn, Jennifer; Hecht, Jochen; Yildirim, Ferah; Meisel, Andreas; Märschenz, Stefanie

    2015-10-01

    Cerebral ischemia induces a complex transcriptional response with global changes in gene expression. It is essentially regulated by transcription factors as well as epigenetic players. While it is well known that the inhibition of transcriptionally repressive histone deacetylases leads to neuroprotection, the role of histone methyltransferases in the postischemic transcriptional response remains elusive. We investigated the effects of inhibition of the repressive H3K9 histone methyltransferases SUV39H1 and G9a on neuronal survival, H3K9 promoter signatures and gene expression. Their inhibition either with the specific blocker chaetocin or by use of RNA interference promoted neuronal survival in oxygen glucose deprivation (OGD). Brain-derived neurotrophic factor (BDNF) was upregulated and BDNF promoter regions showed an increase in histone marks characteristic for active transcription. The BDNF blockade with K252a abrogated the protective effect of chaetocin treatment. In conclusion, inhibition of histone methyltransferases SUV39H1 and G9a confers neuroprotection in a model of hypoxic metabolic stress, which is at least in part mediated by BDNF. PMID:25966950

  10. Archease from Pyrococcus abyssi improves substrate specificity and solubility of a tRNA m5C methyltransferase

    DEFF Research Database (Denmark)

    Auxilien, Sylvie; El Khadali, Fatima; Rasmussen, Anette;

    2007-01-01

    reading frame (PAB1947), which is shown here to encode a tRNA m(5)C methyltransferase. In vitro, the purified recombinant methyltransferase catalyzes m(5)C formation at several cytosines within tRNAs with preference for C49. The specificity of the methyltransferase is increased by the archease...

  11. Human placental DNA methyltransferase: DNA substrate and DNA binding specificity.

    OpenAIRE

    Wang, R.Y.; Huang, L. H.; Ehrlich, M

    1984-01-01

    We have partially purified a DNA methyltransferase from human placenta using a novel substrate for a highly sensitive assay of methylation of hemimethylated DNA. This substrate was prepared by extensive nick translation of bacteriophage XP12 DNA, which normally has virtually all of its cytosine residues replaced by 5-methylcytosine (m5C). Micrococcus luteus DNA was just as good a substrate if it was first similarly nick translated with m5dCTP instead of dCTP in the polymerization mixture. At ...

  12. Expanding Cofactor Repertoire of Protein Lysine Methyltransferase for Substrate Labeling

    OpenAIRE

    Islam, Kabirul; Zheng, Weihong; Yu, Haiqiang; Deng, Haiteng; Luo, Minkui

    2011-01-01

    Protein lysine methyltransferases (PKMTs) play crucial roles in normal physiology and disease processes. Profiling PKMT targets is an important but challenging task. With cancer-relevant G9a as a target, we have demonstrated the success in developing S-adenosyl-L-methionine (SAM) analogues, particularly (E)-hex-2-en-5-ynyl SAM (Hey-SAM), as cofactors for engineered G9a. Hey-SAM analogue in combination with G9a Y1154A mutant modifies the same set of substrates as their native counterparts with...

  13. Purification and characterization of DNA methyltransferases from Neisseria gonorrhoeae.

    OpenAIRE

    Piekarowicz, A; Yuan, R.; Stein, D C

    1988-01-01

    Three DNA methyltransferases, M.NgoAI, and M.NgoBI and M.NgoBII, free of any nuclease activities were isolated from Neisseria gonorrhoeae strains WR220 and MUG116 respectively. M.NgoAI recognizes the sequence 5' GGCC 3' and methylates the first 5' cytosine on both strands. M.NgoBI and M.NgoBII recognize 5' TCACC 3' and 5' GTAN5CTC 3' respectively. M.NgoBII methylates cytosine on only one strand to produce 5' GTAN5mCTC 3'.

  14. Novel Protein Arginine Methyltransferase 8 Isoform Is Essential for Cell Proliferation.

    Science.gov (United States)

    Hernandez, Sarah; Dominko, Tanja

    2016-09-01

    Identification of molecular mechanisms that regulate cellular replicative lifespan is needed to better understand the transition between a normal and a neoplastic cell phenotype. We have previously reported that low oxygen-mediated activity of FGF2 leads to an increase in cellular lifespan and acquisition of regeneration competence in human dermal fibroblasts (iRC cells). Though cells display a more plastic developmental phenotype, they remain non-tumorigenic when injected into SCID mice (Page et al. [2009] Cloning Stem Cells 11:417-426; Page et al. [2011] Eng Part A 17:2629-2640) allowing for investigation of mechanisms that regulate increased cellular lifespan in a non-tumorigenic system. Analysis of chromatin modification enzymes by qRT-PCR revealed a 13.3-fold upregulation of the arginine methyltransferase PRMT8 in iRC cells. Increased protein expression was confirmed in both iRC and human embryonic stem cells-the first demonstration of endogenous human PRMT8 expression outside the brain. Furthermore, iRC cells express a novel PRMT8 mRNA variant. Using siRNA-mediated knockdown we demonstrated that this novel variant was required for proliferation of human dermal fibroblasts (hDFs) and grade IV glioblastomas. PRMT8 upregulation in a non-tumorigenic system may offer a potential diagnostic biomarker and a therapeutic target for cells in pre-cancerous and cancerous states. J. Cell. Biochem. 117: 2056-2066, 2016. © 2016 Wiley Periodicals, Inc. PMID:26851891

  15. miR-221/222 Target the DNA Methyltransferase MGMT in Glioma Cells

    Science.gov (United States)

    Roscigno, Giuseppina; Romano, Giulia; Diaz-Lagares, Angel; Iaboni, Margherita; Donnarumma, Elvira; Fiore, Danilo; De Marinis, Pasqualino; Soini, Ylermi; Esteller, Manel; Condorelli, Gerolama

    2013-01-01

    Glioblastoma multiforme (GBM) is one of the most deadly types of cancer. To date, the best clinical approach for treatment is based on administration of temozolomide (TMZ) in combination with radiotherapy. Much evidence suggests that the intracellular level of the alkylating enzyme O6-methylguanine–DNA methyltransferase (MGMT) impacts response to TMZ in GBM patients. MGMT expression is regulated by the methylation of its promoter. However, evidence indicates that this is not the only regulatory mechanism present. Here, we describe a hitherto unknown microRNA-mediated mechanism of MGMT expression regulation. We show that miR-221 and miR-222 are upregulated in GMB patients and that these paralogues target MGMT mRNA, inducing greater TMZ-mediated cell death. However, miR-221/miR-222 also increase DNA damage and, thus, chromosomal rearrangements. Indeed, miR-221 overexpression in glioma cells led to an increase in markers of DNA damage, an effect rescued by re-expression of MGMT. Thus, chronic miR-221/222-mediated MGMT downregulation may render cells unable to repair genetic damage. This, associated also to miR-221/222 oncogenic potential, may poor GBM prognosis. PMID:24147153

  16. MiR-221/222 target the DNA methyltransferase MGMT in glioma cells.

    Directory of Open Access Journals (Sweden)

    Cristina Quintavalle

    Full Text Available Glioblastoma multiforme (GBM is one of the most deadly types of cancer. To date, the best clinical approach for treatment is based on administration of temozolomide (TMZ in combination with radiotherapy. Much evidence suggests that the intracellular level of the alkylating enzyme O(6-methylguanine-DNA methyltransferase (MGMT impacts response to TMZ in GBM patients. MGMT expression is regulated by the methylation of its promoter. However, evidence indicates that this is not the only regulatory mechanism present. Here, we describe a hitherto unknown microRNA-mediated mechanism of MGMT expression regulation. We show that miR-221 and miR-222 are upregulated in GMB patients and that these paralogues target MGMT mRNA, inducing greater TMZ-mediated cell death. However, miR-221/miR-222 also increase DNA damage and, thus, chromosomal rearrangements. Indeed, miR-221 overexpression in glioma cells led to an increase in markers of DNA damage, an effect rescued by re-expression of MGMT. Thus, chronic miR-221/222-mediated MGMT downregulation may render cells unable to repair genetic damage. This, associated also to miR-221/222 oncogenic potential, may poor GBM prognosis.

  17. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  18. Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Wenyu [Univ. of Toronto, ON (Canada); Chory, Emma J. [Dana-Farber Cancer Inst., Boston, MA (United States); Northeastern Univ., Boston, MA (United States); Wernimont, Amy K. [Univ. of Toronto, ON (Canada); Tempel, Wolfram [Univ. of Toronto, ON (Canada); Scopton, Alex [Univ. of Toronto, ON (Canada); Federation, Alexander [Dana-Farber Cancer Inst., Boston, MA (United States); Marineau, Jason J. [Dana-Farber Cancer Inst., Boston, MA (United States); Qi, Jun [Dana-Farber Cancer Inst., Boston, MA (United States); Barsyte-Lovejoy, Dalia [Univ. of Toronto, ON (Canada); Yi, Joanna [Dana-Farber Cancer Inst., Boston, MA (United States); Marcellus, Richard [Ontario Inst. for Cancer Research, Toronto, ON (Canada); Iacob, Roxana E. [Northeastern Univ., Boston, MA (United States); Engen, John R. [Northeastern Univ., Boston, MA (United States); Griffin, Carly [Ontario Inst. for Cancer Research, Toronto, ON (Canada); Aman, Ahmed [Ontario Inst. for Cancer Research, Toronto, ON (Canada); Wienholds, Erno [Univ. of Toronto, ON (Canada); Li, Fengling [Univ. of Toronto, ON (Canada); Pineda, Javier [Dana-Farber Cancer Inst., Boston, MA (United States); Univ. of Notre Dame, IN (United States); Estiu, Guillermina [Univ. of Notre Dame, IN (United States); Shatseva, Tatiana [Univ. of Toronto, ON (Canada); Hajian, Taraneh [Univ. of Toronto, ON (Canada); Al-awar, Rima [Ontario Inst. for Cancer Research, Toronto, ON (Canada); Dick, John E. [Univ. of Toronto, ON (Canada); Vedadi, Masoud [Univ. of Toronto, ON (Canada); Brown, Peter J. [Univ. of Toronto, ON (Canada); Arrowsmith, Cheryl H. [Univ. of Toronto, ON (Canada); Bradner, James E. [Dana-Farber Cancer Inst., Boston, MA (United States); Harvard Medical School, Boston, MA (United States); Schapira, Matthieu [Univ. of Toronto, ON (Canada)

    2012-12-18

    Selective inhibition of protein methyltransferases is a promising new approach to drug discovery. An attractive strategy towards this goal is the development of compounds that selectively inhibit binding of the cofactor, S-adenosylmethionine, within specific protein methyltransferases. Here we report the three-dimensional structure of the protein methyltransferase DOT1L bound toEPZ004777, the first S-adenosylmethionine-competitive inhibitor of a protein methyltransferase with in vivo efficacy. This structure and those of four new analogues reveal remodelling of the catalytic site. EPZ004777 and a brominated analogue, SGC0946, inhibit DOT1L in vitro and selectively kill mixed lineage leukaemia cells, in which DOT1L is aberrantly localized via interaction with an oncogenic MLL fusion protein. These data provide important new insight into mechanisms of cell-active S-adenosylmethionine-competitive protein methyltransferase inhibitors, and establish a foundation for the further development of drug-like inhibitors of DOT1L for cancer therapy.

  19. A novel arsenic methyltransferase gene of Westerdykella aurantiaca isolated from arsenic contaminated soil: phylogenetic, physiological, and biochemical studies and its role in arsenic bioremediation.

    Science.gov (United States)

    Verma, Shikha; Verma, Pankaj Kumar; Meher, Alok Kumar; Dwivedi, Sanjay; Bansiwal, Amit Kumar; Pande, Veena; Srivastava, Pankaj Kumar; Verma, Praveen Chandra; Tripathi, Rudra Deo; Chakrabarty, Debasis

    2016-03-01

    Elevated arsenic concentration in the environment and agricultural soil is a serious concern to crop production and human health. Among different detoxification mechanisms, the methylation of arsenic is a widespread phenomenon in nature. A number of microorganisms are able to methylate arsenic, but less is known about the arsenic metabolism in fungi. We identified a novel arsenic methyltransferase (WaarsM) gene from a soil fungus, Westerdykella aurantiaca. WaarsM showed sequence homology with all known arsenic methyltransferases having three conserved SAM binding motifs. The expression of WaarsM enhanced arsenic resistance in E. coli (Δars) and S. cerevisiae (Δacr2) strains by biomethylation and required endogenous reductants, preferably GSH, for methyltransferase activity. The purified WaarsM catalyzes the production of methylated arsenicals from both AsIII and AsV, and also displays AsV reductase activity. It displayed higher methyltransferase activity and lower KM 0.1945 ± 0.021 mM and KM 0.4034 ± 0.078 mM for AsIII and AsV, respectively. S. cerevisiae (Δacr2) cells expressing WaarsM produced 2.2 ppm volatile arsenic and 0.64 ppm DMA(v) with 0.58 ppm volatile arsenicals when exposed to 20 ppm AsV and 2 ppm AsIII, respectively. Arsenic tolerance in rice after co-culture with genetically engineered yeast suggested its potential role in arsenic bioremediation. Thus, characterization of WaarsM provides a potential strategy to reduce arsenic concentration in soil with reduced arsenic accumulation in crops grown in arsenic contaminated areas, and thereby alleviating human health risks. PMID:26776948

  20. Lipid substrate specificity of phosphatidylethanolamine N-methyltransferase of Tetrahymena

    International Nuclear Information System (INIS)

    The ciliate protozoan Tetrahymena thermophila forms about 60% of its phosphatidylcholine by methylation of phosphatidylethanolamine with S-adenosylmethionine using the enzyme phosphatidylethanolamine N-methyltransferase. Analogues of ethanolamine or of ethanolamine phosphate are incorporated into the phospholipids of Tetrahymena when cells are cultured in their presence. These compounds, 3-amino-1-propanol, 2-aminoethylphosphonate, 3-aminopropylphosphonate and N,N-dimethylaminoethylphosphonate replace from 50 to 75% of the ethanolamine phosphate in phosphatidylethanolamine. However, analysis of the phospholipids of lipid-altered Tetrahymena showed that none of the phosphatidylethanolamine analogues had been converted to the corresponding phosphatidylcholine analogue. No incorration of [14C-CH3]methionine into the phosphatidylcholine analogues could be demonstrated in vivo, nor was label from [3H-CH3]S-adenosylmethionine incorporated in virto. Thus, only phosphatidylethanolamine and its monomethyl and dimethyl derivatives have been found to be substrates for the phosphatidylethanoiamine N-methyltransferase. The enzyme therefore requires a phospholipid substrate containing an ester linkage between the alkylamine and phosphorus, with the amino group required to be β to the alcohol

  1. Hypnotizability and Catechol-O-Methyltransferase (COMT polymorphysms in Italians

    Directory of Open Access Journals (Sweden)

    Alessandro Gialluisi

    2014-01-01

    Full Text Available Higher brain dopamine content depending on lower activity of Catechol-O-Methyltransferase (COMT in subjects with high hypnotisability scores (highs has been considered responsible for their attentional characteristics. However, the results of the previous genetic studies on association between hypnotisability and the Catechol-O-Methyltransferase (COMT single nucleotide polymorphism (SNP rs4680 (Val158Met were inconsistent. Here, we used a selective genotyping approach to re-evaluate the association between hypnotisability and COMT in the context of a two-SNP haplotype analysis, considering not only the Val158Met polymorphism, but also the closely located rs4818 SNP. An Italian sample of 53 highs, 49 low hypnotizable subjects (lows and 57 controls, were genotyped for a segment of 805 bp of the COMT gene, including Val158Met and the closely located rs4818 SNP. Our selective genotyping approach had 97.1% power to detect the previously reported strongest association at the significance level of 5%. We found no evidence of association at the SNP, haplotype and diplotype levels. Thus, our results challenge the dopamine-based theory of hypnosis and indirectly support recent neuropsychological and neurophysiological findings reporting the lack of any association between hypnotisability and focused attention abilities.

  2. Structural biology of human H3K9 methyltransferases.

    Directory of Open Access Journals (Sweden)

    Hong Wu

    Full Text Available UNLABELLED: SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  3. Distinct roles for histone methyltransferases G9a and GLP in cancer germline antigen gene regulation in human cancer cells and murine ES cells

    OpenAIRE

    Link, Petra A.; Gangisetty, Omkaram; James, Smitha R.; Woloszynska-Read, Anna; Tachibana, Makoto; Shinkai, Yoichi; Karpf, Adam R.

    2009-01-01

    The H3K9me2 histone methyltransferases G9a and GLP repress Mage-a class cancer germline (CG) antigen gene expression in murine ES cells but the role of these enzymes in CG antigen gene regulation in human cancer cells is unknown. Here we show that while independent or dual knockdown of G9a and GLP in human cancer cells leads to reduced global and CG antigen promoter-associated H3K9me2 levels it does not activate CG antigen gene expression. Moreover, CG antigen gene repression is maintained fo...

  4. Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule

    OpenAIRE

    van der Gun, Bernardina T. F.; Maluszynska-Hoffman, Maria; Kiss, Antal; Arendzen, Alice J.; Ruiters, Marcel H.J.; McLaughlin, Pamela M. J.; Weinhold, Elmar; Rots, Marianne G.

    2010-01-01

    The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that has been identified as a marker of cancer-initiating cells. EpCAM is highly expressed on most carcinomas, and transient silencing of EpCAM expression leads to reduced oncogenic potential. To silence the EpCAM gene in a persistent manner via targeted DNA methylation, a low activity mutant (C141S) of the CpG-specific DNA methyltransferase M.SssI was coupled to a triple-helix-forming oligonucleotide (TFO−C141S) specifi...

  5. Deregulation of histone lysine methyltransferases contributes to oncogenic transformation of human bronchoepithelial cells

    Directory of Open Access Journals (Sweden)

    Yoda Satoshi

    2008-11-01

    Full Text Available Abstract Background Alterations in the processing of the genetic information in carcinogenesis result from stable genetic mutations or epigenetic modifications. It is becoming clear that nucleosomal histones are central to proper gene expression and that aberrant DNA methylation of genes and histone methylation plays important roles in tumor progression. To date, several histone lysine methyltransferases (HKMTs have been identified and histone lysine methylation is now considered to be a critical regulator of transcription. However, still relatively little is known about the role of HKMTs in tumorigenesis. Results We observed differential HKMT expression in a lung cancer model in which normal human bronchial epithelial (NHBE cells expressing telomerase, SV40 large T antigen, and Ras were immortal, formed colonies in soft agar, and expressed specific HKMTs for H3 lysine 9 and 27 residues but not for H3 lysine 4 residue. Modifications in the H3 tails affect the binding of proteins to the histone tails and regulate protein function and the position of lysine methylation marks a gene to be either activated or repressed. In the present study, suppression by siRNA of HKMTs (EZH2, G9A, SETDB1 and SUV39H1 that are over-expressed in immortalized and transformed cells lead to reduced cell proliferation and much less anchorage-independent colony growth. We also found that the suppression of H3-K9, G9A and SUV39H1 induced apoptosis and the suppression of H3-K27, EZH2 caused G1 arrest. Conclusion Our results indicate the potential of these HKMTs in addition to the other targets for epigenetics such as DNMTs and HDACs to be interesting therapeutic targets.

  6. The methyltransferase Setdb1 is essential for meiosis and mitosis in mouse oocytes and early embryos.

    Science.gov (United States)

    Eymery, Angeline; Liu, Zichuan; Ozonov, Evgeniy A; Stadler, Michael B; Peters, Antoine H F M

    2016-08-01

    Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 downregulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a newly discovered meiotic and embryonic competence factor safeguarding genome integrity at the onset of life. PMID:27317807

  7. Identification and characterization of DNAzymes targeting DNA methyltransferase I for suppressing bladder cancer proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xiangbo; Zhang, Lu; Ding, Nianhua; Yang, Xinghui; Zhang, Jin; He, Jiang; Li, Zhi; Sun, Lun-Quan, E-mail: lunquansun@csu.edu.cn

    2015-05-29

    Epigenetic inactivation of genes plays a critical role in many important human diseases, especially in cancer. A core mechanism for epigenetic inactivation of the genes is methylation of CpG islands in genome DNA, which is catalyzed by DNA methyltransferases (DNMTs). The inhibition of DNMTs may lead to demethylation and expression of the silenced tumor suppressor genes. Although DNMT inhibitors are currently being developed as potential anticancer agents, only limited success is achieved due to substantial toxicity. Here, we utilized a multiplex selection system to generate efficient RNA-cleaving DNAzymes targeting DNMT1. The lead molecule from the selection was shown to possess efficient kinetic profiles and high efficiency in inhibiting the enzyme activity. Transfection of the DNAzyme caused significant down-regulation of DNMT1 expression and reactivation of p16 gene, resulting in reduced cell proliferation of bladder cancers. This study provides an alternative for targeting DNMTs for potential cancer therapy. - Highlights: • Identified DNMT1-targeted DNAzymes by multiplex selection system. • Biochemically characterized a lead DNAzyme with high kinetic efficiency. • Validated DNMT1-targeted DNAzyme in its enzymatic and cellular activities.

  8. Role of type II protein arginine methyltransferase 5 in the regulation of Circadian Per1 gene.

    Directory of Open Access Journals (Sweden)

    Jungtae Na

    Full Text Available Circadian clocks are the endogenous oscillators that regulate rhythmic physiological and behavioral changes to correspond to daily light-dark cycles. Molecular dissections have revealed that transcriptional feedback loops of the circadian clock genes drive the molecular oscillation, in which PER/CRY complexes inhibit the transcriptional activity of the CLOCK/BMAL1 heterodimer to constitute a negative feedback loop. In this study, we identified the type II protein arginine methyltransferase 5 (PRMT5 as an interacting molecule of CRY1. Although the Prmt5 gene was constitutively expressed, increased interaction of PRMT5 with CRY1 was observed when the Per1 gene was repressed both in synchronized mouse liver and NIH3T3 cells. Moreover, rhythmic recruitment of PRMT5 and CRY1 to the Per1 gene promoter was found to be associated with an increased level of histone H4R3 dimethylation and Per1 gene repression. Consistently, decreased histone H4R3 dimethylation and altered rhythmic Per1 gene expression were observed in Prmt5-depleted cells. Taken together, these findings provide an insight into the link between histone arginine methylation by PRMT5 and transcriptional regulation of the circadian Per1 gene.

  9. Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation.

    Science.gov (United States)

    Zhang, Guoqiang; Estève, Pierre-Olivier; Chin, Hang Gyeong; Terragni, Jolyon; Dai, Nan; Corrêa, Ivan R; Pradhan, Sriharsa

    2015-07-13

    Mammalian cells contain copious amounts of RNA including both coding and noncoding RNA (ncRNA). Generally the ncRNAs function to regulate gene expression at the transcriptional and post-transcriptional level. Among ncRNA, the long ncRNA and small ncRNA can affect histone modification, DNA methylation targeting and gene silencing. Here we show that endogenous DNA methyltransferase 1 (DNMT1) co-purifies with inhibitory ncRNAs. MicroRNAs (miRNAs) bind directly to DNMT1 with high affinity. The binding of miRNAs, such as miR-155-5p, leads to inhibition of DNMT1 enzyme activity. Exogenous miR-155-5p in cells induces aberrant DNA methylation of the genome, resulting in hypomethylation of low to moderately methylated regions. And small shift of hypermethylation of previously hypomethylated region was also observed. Furthermore, hypomethylation led to activation of genes. Based on these observations, overexpression of miR-155-5p resulted in aberrant DNA methylation by inhibiting DNMT1 activity, resulting in altered gene expression. PMID:25990724

  10. Insufficiency of phosphatidylethanolamine N-methyltransferase is risk for lean non-alcoholic steatohepatitis

    Science.gov (United States)

    Nakatsuka, Atsuko; Matsuyama, Makoto; Yamaguchi, Satoshi; Katayama, Akihiro; Eguchi, Jun; Murakami, Kazutoshi; Teshigawara, Sanae; Ogawa, Daisuke; Wada, Nozomu; Yasunaka, Tetsuya; Ikeda, Fusao; Takaki, Akinobu; Watanabe, Eijiro; Wada, Jun

    2016-01-01

    Although obesity is undoubtedly major risk for non-alcoholic steatohepatitis (NASH), the presence of lean NASH patients with normal body mass index has been recognized. Here, we report that the insufficiency of phosphatidylethanolamine N-methyltransferase (PEMT) is a risk for the lean NASH. The Pemt−/− mice fed high fat-high sucrose (HFHS) diet were protected from diet-induced obesity and diabetes, while they demonstrated prominent steatohepatitis and developed multiple liver tumors. Pemt exerted inhibitory effects on p53-driven transcription by forming the complex with clathrin heavy chain and p53, and Pemt−/− mice fed HFHS diet demonstrated prominent apoptosis of hepatocytes. Furthermore, hypermethylation and suppressed mRNA expression of F-box protein 31 and hepatocyte nuclear factor 4α resulted in the prominent activation of cyclin D1. PEMT mRNA expression in liver tissues of NASH patients was significantly lower than those with simple steatosis and we postulated the distinct clinical entity of lean NASH with insufficiency of PEMT activities. PMID:26883167

  11. Discovery and Optimization of Novel, Selective Histone Methyltransferase SET7 Inhibitors by Pharmacophore- and Docking-Based Virtual Screening.

    Science.gov (United States)

    Meng, Fanwang; Cheng, Sufang; Ding, Hong; Liu, Shien; Liu, Yan; Zhu, Kongkai; Chen, Shijie; Lu, Junyan; Xie, Yiqian; Li, Linjuan; Liu, Rongfeng; Shi, Zhe; Zhou, Yu; Liu, Yu-Chih; Zheng, Mingyue; Jiang, Hualiang; Lu, Wencong; Liu, Hong; Luo, Cheng

    2015-10-22

    Histone methyltransferases are involved in various biological functions, and these methylation regulating enzymes' abnormal expression or activity has been noted in several human cancers. Within this context, SET domain-containing (lysine methyltransferase) 7 (SET7, also called KMT7, SETD7, SET9) is of increasing significance due to its diverse roles in biological functions and diseases, such as diabetes, cancers, alopecia areata, atherosclerotic vascular disease, HIV, and HCV. In this study, DC-S100, which was discovered by pharmacophore- and docking-based virtual screening, was identified as the hit compound of SET7 inhibitor. Structure-activity relationship (SAR) analysis was performed on analogs of DC-S100 and according to the putative binding mode of DC-S100, structure modifications were made to improve its activity. Of note, compounds DC-S238 and DC-S239, with IC50 values of 4.88 and 4.59 μM, respectively, displayed selectivity for DNMT1, DOT1L, EZH2, NSD1, SETD8, and G9a. Taken together, DC-S238 and DC-S239 can serve as leads for further investigation as SET7 inhibitors and the chemical toolkits for functional biology studies of SET7. PMID:26390175

  12. Ribosomal protein methylation in Escherichia coli: the gene prmA, encoding the ribosomal protein L11 methyltransferase, is dispensable.

    Science.gov (United States)

    Vanet, A; Plumbridge, J A; Guérin, M F; Alix, J H

    1994-12-01

    The prmA gene, located at 72 min on the Escherichia coli chromosome, is the genetic determinant of ribosomal protein L11-methyltransferase activity. Mutations at this locus, prmA1 and prmA3, result in a severely undermethylated form of L11. No effect, other than the lack of methyl groups on L11, has been ascribed to these mutations. DNA sequence analysis of the mutant alleles prmA1 and prmA3 detected point mutations near the C-terminus of the protein and plasmids overproducing the wild-type and the two mutant proteins have been constructed. The wild-type PrmA protein could be crosslinked to its radiolabelled substrate, S-adenosyl-L-methionine (SAM), by u.v. irradiation indicating that it is the gene for the methyltransferase rather than a regulatory protein. One of the mutant proteins, PrmA3, was also weakly crosslinked to SAM. Both mutant enzymes when expressed from the overproducing plasmids were capable of catalysing the incorporation of 3H-labelled methyl groups from SAM to L11 in vitro. This confirmed the observation that the mutant proteins possess significant residual activity which could account for their lack of growth phenotype. However, a strain carrying an in vitro-constructed null mutation of the prmA gene, transferred to the E. coli chromosome by homologous recombination, was perfectly viable. PMID:7715456

  13. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    International Nuclear Information System (INIS)

    Research highlights: → Successful fusion of GFP to M.EcoKI DNA methyltransferase. → GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. → FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

  14. Histopathological changes in rat liver in hyper-and hypothyroidism are associated with DNA methyltransferase activity

    Institute of Scientific and Technical Information of China (English)

    Zaporozhan VM; Mescheryakova NV

    2013-01-01

    [Objective] There is evidence that thyroid hormones (TH) could affect on collagen development.Also there is some data that methylation play a significant role in fibrogenesis.That's why the aim of the article was to find out if such pathomorphological changes in liver as fibrosis that were caused by TH could be associated with the changes of DNA methyltransferase activity.[Methods] Experimental study was presented on 89 mature white rats.3 experimental groups were performed:1-experimental hyperthyroidism,2-experimental hypothyroidism,3-control group.The thyroid profile for T3,T4,TTH and profile activity for DNA methyltransferase (DNMT) were determined.The weight of liver was examined.Microscopy and morphometry were performed.[Results] In experimental hyperthyroidism and hypothyroidism the respective changes in TH concentrations occur with a normal level of TTH.It was revealed gender differences in TH levels under impaired thyroid status.In hypothyroidism a decrease of TH concentration occurs in females and males,but it is more expressed in males.In hyperthyroidism the increased concentrations of TH is observed in all rodents,but it is more significant in males.The weight of liver gain in hyperthyroid rodents and decreased of liver weight in hypothyroid rats.In experimentally hyperthyroidism the pathomorphological changed in liver were characterized as an acute vascular insufficiency,hypertension,inflammation,toxic effects of hormones,an initial stage of hepatitis with a high activity,traces of old hemorrhage and a marked fibrosis in the liver.Hypothyroidism also effects on the liver with the signs of acute vascular insufficiency,hypoxia,toxic effects of hormones,necrosis and mild fibrotic changes in the liver tissue.The enzymatic activity of DNMT was increased both in hyperthyroidism and in hypothyroidism.[Conclusions] From results that were've obtained we could conclude that persistent shifts of TH cause pathomorphological changes in the liver.They were more

  15. Expression of O6-methylguanine-DNA methyltransferase in human gliomas and therapeutic evaluation of STZ and ACNU combination chemotherapy%MGMT在神经胶质瘤中表达的临床意义及联合应用STZ对愈后的影响

    Institute of Scientific and Technical Information of China (English)

    胡苹; 娄晋宁; 章扬培; 马雄君; 赵奎明

    2006-01-01

    目的:通过观察神经胶质瘤中O6-甲基鸟嘌呤DNA甲基转移酶(O6-methylguanine-DNA methyltransferase MGMT)的表达状态,探讨MGMT在胶质瘤中的表达与亚硝脲(ACNU/BCNU)耐药之间的关系;同时对于部分MGMT阳性患者给予链脲菌素(STZ)联合ACNU/BCNU治疗,观察疗效.方法:采用免疫组化法对神经胶质瘤石蜡标本检测MGMT,并与临床亚硝脲的随访结果进行比较;对8例MGMT阳性率高且对亚硝脲治疗无效的难治性脑瘤患者,开展STZ联合ACNU治疗.结果:68例神经胶质瘤,MGMT表达的总阳性率为55.9%.在使用亚硝脲治疗的患者中,除8例加用STZ联合治疗外,余60例中,27例MGMT(-)者,使用ACNU治疗绝大部分有效;30例MGMT(+~++)者,使用ACNU治疗后绝大部分复发或死亡,表明MGMT表达与亚硝脲化疗呈负相关;另外8例MGMT阳性患者在进行了STZ联合ACNU治疗后,1例可测肿瘤全部消失,2例肿瘤体积缩小在50%以上,4例肿瘤体积消退<50%,1例无效.结论:MGMT阳性的胶质瘤患者比MGMT阴性的患者明显耐药,且耐药程度与MGMT表达强度无关,仅与其是否阳性有关.对于MGMT阳性患者,可选择ACNU/BCNU联合STZ使用,以提高亚硝脲的疗效.因此,化疗前检测胶质瘤MGMT的表达情况,预测化疗敏感性,对指导临床化疗具有重要意义.

  16. Relationship between the Expression of O6-methylguanine DNA methyltransferase in Glioma and the Survival Time of Patients%MGMT在脑胶质瘤组织中的表达及其与患者生存期的关系

    Institute of Scientific and Technical Information of China (English)

    孙彦辉; 张亚卓; 王忠诚; 孙梅珍; 赵东海

    2004-01-01

    背景与目的:目前的研究已经证实 DNA修复酶-- 6 氧甲基鸟嘌呤 DNA甲基转移酶( O6 methylguanine DNA methyltransferase,MGMT)在脑胶质瘤组织中的表达与肿瘤的耐药性有一定的关系,并且能够影响肿瘤的化疗效果.本研究通过分析 MGMT在脑胶质瘤组织中的表达及其与患者生存期的关系,为基于耐药机制上的脑胶质瘤分子分类提供参考资料.方法:用组织芯片技术和免疫组织化学方法检测 311例脑胶质瘤石蜡标本中 MGMT的表达情况,并对所有患者进行手术后的 5年随访.结果: MGMT表达阳性者 126例,占 40.51% (126/311).其中,星形细胞瘤中阳性率为 50.41% (61/121),少枝胶质细胞瘤中为 25.71% (18/70),少枝星形细胞瘤中为 28.13% (18/64),胶质母细胞瘤中为 51.79% (29/56);在Ⅰ~Ⅱ级胶质瘤中 , MGMT表达的阳性率为 36.56% (68/186),而在Ⅲ~Ⅳ级胶质瘤中为 46.40% (58/125),经χ 2检验分析,两者之间有显著性差异 (P< 0.001);将 MGMT的表达与患者生存期的关系绘制成 Kaplan Meier生存曲线,并进行 log rank 分析, MGMT表达阳性者与阴性者之间的差异有显著性 (P< 0.05).结论: MGMT在脑胶质瘤的异常表达与肿瘤的组织类型、病理级别有关, MGMT表达阳性患者的生存期明显低于表达阴性的患者.

  17. Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity.

    Science.gov (United States)

    Kraus, Daniel; Yang, Qin; Kong, Dong; Banks, Alexander S; Zhang, Lin; Rodgers, Joseph T; Pirinen, Eija; Pulinilkunnil, Thomas C; Gong, Fengying; Wang, Ya-chin; Cen, Yana; Sauve, Anthony A; Asara, John M; Peroni, Odile D; Monia, Brett P; Bhanot, Sanjay; Alhonen, Leena; Puigserver, Pere; Kahn, Barbara B

    2014-04-10

    In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respective controls. NNMT methylates nicotinamide (vitamin B3) using S-adenosylmethionine (SAM) as a methyl donor. Nicotinamide is a precursor of NAD(+), an important cofactor linking cellular redox states with energy metabolism. SAM provides propylamine for polyamine biosynthesis and donates a methyl group for histone methylation. Polyamine flux including synthesis, catabolism and excretion, is controlled by the rate-limiting enzymes ornithine decarboxylase (ODC) and spermidine-spermine N(1)-acetyltransferase (SSAT; encoded by Sat1) and by polyamine oxidase (PAO), and has a major role in energy metabolism. We report that NNMT expression is increased in WAT and liver of obese and diabetic mice. Nnmt knockdown in WAT and liver protects against diet-induced obesity by augmenting cellular energy expenditure. NNMT inhibition increases adipose SAM and NAD(+) levels and upregulates ODC and SSAT activity as well as expression, owing to the effects of NNMT on histone H3 lysine 4 methylation in adipose tissue. Direct evidence for increased polyamine flux resulting from NNMT inhibition includes elevated urinary excretion and adipocyte secretion of diacetylspermine, a product of polyamine metabolism. NNMT inhibition in adipocytes increases oxygen consumption in an ODC-, SSAT- and PAO-dependent manner. Thus, NNMT is a novel regulator of histone methylation, polyamine flux and NAD(+)-dependent SIRT1 signalling, and is a unique and attractive target for treating obesity and type 2 diabetes. PMID

  18. Identification of white campion (Silene latifolia guaiacol O-methyltransferase involved in the biosynthesis of veratrole, a key volatile for pollinator attraction

    Directory of Open Access Journals (Sweden)

    Gupta Alok K

    2012-08-01

    Full Text Available Abstract Background Silene latifolia and its pollinator, the noctuid moth Hadena bicruris, represent an open nursery pollination system wherein floral volatiles, especially veratrole (1, 2-dimethoxybenzene, lilac aldehydes, and phenylacetaldehyde are of key importance for floral signaling. Despite the important role of floral scent in ensuring reproductive success in S. latifolia, the molecular basis of scent biosynthesis in this species has not yet been investigated. Results We isolated two full-length cDNAs from S. latifolia that show similarity to rose orcinol O-methyltransferase. Biochemical analysis showed that both S. latifolia guaiacol O-methyltransferase1 (SlGOMT1 &S. latifolia guaiacol O-methyltransferase2 (SlGOMT2 encode proteins that catalyze the methylation of guaiacol to form veratrole. A large Km value difference between SlGOMT1 (~10 μM and SlGOMT2 (~501 μM resulted that SlGOMT1 is 31-fold more catalytically efficient than SlGOMT2. qRT-PCR expression analysis showed that the SlGOMT genes are specifically expressed in flowers and male S. latifolia flowers had 3- to 4-folds higher level of GOMT gene transcripts than female flower tissues. Two related cDNAs, S. dioica O-methyltransferase1 (SdOMT1 and S. dioica O-methyltransferase2 (SdOMT2, were also obtained from the sister species Silene dioica, but the proteins they encode did not methylate guaiacol, consistent with the lack of veratrole emission in the flowers of this species. Our evolutionary analysis uncovered that SlGOMT1 and SlGOMT2 genes evolved under positive selection, whereas SdOMT1 and SdOMT2 genes show no evidence for selection. Conclusions Altogether, we report the identification and functional characterization of the gene, SlGOMT1 that efficiently catalyzes veratrole formation, whereas another copy of this gene with only one amino acid difference, SlGOMT2 was found to be less efficient for veratrole synthesis in S. latifolia.

  19. Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N6-adenine DNA methyltransferases of Neisseria meningitidis

    Science.gov (United States)

    Seib, Kate L.; Jen, Freda E.-C.; Tan, Aimee; Scott, Adeana L.; Kumar, Ritesh; Power, Peter M.; Chen, Li-Tzu; Wu, Hsing-Ju; Wang, Andrew H.-J.; Hill, Dorothea M. C.; Luyten, Yvette A.; Morgan, Richard D.; Roberts, Richard J.; Maiden, Martin C. J.; Boitano, Matthew; Clark, Tyson A.; Korlach, Jonas; Rao, Desirazu N.; Jennings, Michael P.

    2015-01-01

    Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N6-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N6-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5′-CGYm6AG-3′), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5′-ACm6ACC-3′) and ModD1 (exemplified by M.Nme579I) (5′-CCm6AGC-3′). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5′-CGYm6AG-3′. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5′-GCGCm6AGG-3′ sites, to 100% at 5′-ACGTm6AGG-3′ sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching. PMID:25845594

  20. EXPRESS

    International Nuclear Information System (INIS)

    This paper presents EXPRESS, an expert system developed for the automation of reliability studies. The first part consists in the description of the method for static thermohydraulic systems. In this step, the authors define the knowledge representation based on the two inference engines - ALOUETTE and LCR developed by EDF. They explain all the process to construct a fault tree from a topological and functional description of the system. Numerous examples are exhibited in illustration of the method. This is followed by the lessons derived from the studies performed on some safety systems of the PALUEL nuclear plant. The development of the same approach for electric power systems is described, insisting on the difference resulting from the sequential nature of these systems. Finally, they show the main advantages identified during the studies

  1. An audit of thiopurine methyltransferase genotyping and phenotyping before intended azathioprine treatment for dermatological conditions

    DEFF Research Database (Denmark)

    Vestergaard, T; Bygum, A

    2009-01-01

    Summary Background. Determining thiopurine methyltransferase (TPMT) genotype and phenotype before azathioprine treatment predicts which patients are most likely to develop myelosuppression. Aim. To evaluate the course of azathioprine treatment in people with TPMT heterozygosity and whether this d...

  2. Arsenic (+3 oxidation state) methyltransferase and the methylation of arsenicals in the invertebrate chordate Ciona intestinalis

    Science.gov (United States)

    Biotransformation of inorganic arsenic (iAs) involves methylation catalyzed by arsenic (+3 oxidation state) methyltransferase (As3mt), yielding mono- , di- , and trimethylated arsenicals. To investigate the evolution of molecular mechanisms that mediate arsenic biotransformation,...

  3. Erythrocuyte catechol-O-methyltransferase activity in related families with schizophrenia

    International Nuclear Information System (INIS)

    Erythrocyte catechol-O-methyltransferase (COMT) activity has been analyzed in 72 individuals from 17 related families with 18 schizophrenic members. No association with any of the three COMT genotypes could be found in this pedigree. (author)

  4. “MGMT for pt mgmt”: Is Methylguanine-DNA Methyltransferase Testing Ready for Patient Management?

    OpenAIRE

    Iafrate, A. John; Louis, David N.

    2008-01-01

    This Commentary reports on a robust quantitative assay for the interpretation of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation assays that should facilitate the comparison and implementation of such assays across laboratories.

  5. The RNA–Methyltransferase Misu (NSun2) Poises Epidermal Stem Cells to Differentiate

    OpenAIRE

    Sandra Blanco; Agata Kurowski; Jennifer Nichols; Watt, Fiona M.; Salvador Aznar Benitah; Michaela Frye

    2011-01-01

    Homeostasis of most adult tissues is maintained by balancing stem cell self-renewal and differentiation, but whether post-transcriptional mechanisms can regulate this process is unknown. Here, we identify that an RNA methyltransferase (Misu/Nsun2) is required to balance stem cell self-renewal and differentiation in skin. In the epidermis, this methyltransferase is found in a defined sub-population of hair follicle stem cells poised to undergo lineage commitment, and its depletion results in e...

  6. Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA

    OpenAIRE

    Lukinavičius, Gražvydas; Lapinaitė, Audronė; Urbanavičiūtė, Giedrė; Gerasimaitė, Rūta; Klimašauskas, Saulius

    2012-01-01

    DNA methyltransferases catalyse the transfer of a methyl group from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto specific target sites on DNA and play important roles in organisms from bacteria to humans. AdoMet analogs with extended propargylic side chains have been chemically produced for methyltransferase-directed transfer of activated groups (mTAG) onto DNA, although the efficiency of reactions with synthetic analogs remained low. We performed steric engineering of the co...

  7. Structure–function analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase

    OpenAIRE

    Zheng, Sushuang; Shuman, Stewart

    2008-01-01

    The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit and a stimulatory subunit. Structure–function analysis of the catalytic subunit by alanine scanning and conservative substitutions (49 mutations at 25 amino acids) identified 12 functional groups essential for methyltransferase activity in vivo, most of which were essential for cap methylation in vitro. Defects in cap binding were demonstrated for a subset of lethal m...

  8. Identification and characterization of arsenite methyltransferase from an archaeon, Methanosarcina acetivorans C2A.

    Science.gov (United States)

    Wang, Pei-Pei; Sun, Guo-Xin; Zhu, Yong-Guan

    2014-11-01

    Arsenic is a ubiquitous toxic contaminant in the environment. The methylation of arsenic can affect its toxicity and is primarily mediated by biological processes. Few studies have focused on the mechanism of arsenic methylation in archaea although archaea are widespread in the environment. Here, an arsenite [As(III)] methyltransferase (ArsM) was identified and characterized from an archaeon Methanosarcina acetivorans C2A. Heterologous expression of MaarsM was shown to confer As(III) resistance to an arsenic-sensitive strain of E. coli through arsenic methylation and subsequent volatilization. Purified MaArsM protein was further identified the function in catalyzing the formation of various methylated products from As(III) in vitro. Methylation of As(III) by MaArsM is highly dependent on the characteristics of the thiol cofactors used, with some of them (coenzyme M, homocysteine, and dithiothreitol) more efficient than GSH. Site-directed mutagenesis demonstrated that three conserved cysteine (Cys) residues (Cys62, Cys150, and Cys200) in MaArsM were necessary for As(III) methylation, of which only Cys150 and Cys200 were required for the methylation of monomethylarsenic. These results present a molecular pathway for arsenic methylation in archaea and provide some insight into the role of archaea in As biogeochemistry. PMID:25295694

  9. Methylation mediated by an anthocyanin, O-methyltransferase, is involved in purple flower coloration in Paeonia.

    Science.gov (United States)

    Du, Hui; Wu, Jie; Ji, Kui-Xian; Zeng, Qing-Yin; Bhuiya, Mohammad-Wadud; Su, Shang; Shu, Qing-Yan; Ren, Hong-Xu; Liu, Zheng-An; Wang, Liang-Sheng

    2015-11-01

    Anthocyanins are major pigments in plants. Methylation plays a role in the diversity and stability of anthocyanins. However, the contribution of anthocyanin methylation to flower coloration is still unclear. We identified two homologous anthocyanin O-methyltransferase (AOMT) genes from purple-flowered (PsAOMT) and red-flowered (PtAOMT) Paeonia plants, and we performed functional analyses of the two genes in vitro and in vivo. The critical amino acids for AOMT catalytic activity were studied by site-directed mutagenesis. We showed that the recombinant proteins, PsAOMT and PtAOMT, had identical substrate preferences towards anthocyanins. The methylation activity of PsAOMT was 60 times higher than that of PtAOMT in vitro. Interestingly, this vast difference in catalytic activity appeared to result from a single amino acid residue substitution at position 87 (arginine to leucine). There were significant differences between the 35S::PsAOMT transgenic tobacco and control flowers in relation to their chromatic parameters, which further confirmed the function of PsAOMT in vivo. The expression levels of the two homologous AOMT genes were consistent with anthocyanin accumulation in petals. We conclude that AOMTs are responsible for the methylation of cyanidin glycosides in Paeonia plants and play an important role in purple coloration in Paeonia spp. PMID:26208646

  10. ANTITUMOR EFFECT OF SARCNU IN A 06-METHYLGUANINE-DNA METHYLTRANSFERASE POSITIVE HUMAN GLIOMA XENOGRAFT MODEL

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To assess whether novel analogue of nitrosoureas, 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU), has antitumor effect to 06-methylguanine-DNA methyltransferase (MGMT) positive tumors in vivo. Methods: MGMT positive human glioma cell line SF-767 xenografts in nude mice were treated with SarCNU. The antitumor efficacy of SarCNU was compared with the results of 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment with or without 06-benzylguanine (06-BG) preadministration. Results: Since the SF-767 is MGMT strongly positive, BCNU treatment alone did not result in a satisfactory anticancer effect. As expected, 06-BG by depleting MGMT activity, significantly enhanced BCNU antitumor efficacy (p<0.001). More interestingly, SarCNU treatment alone had a better antitumor effect than 06-BG plus BCNU treatment (F=51.7, p=0.00036). Conclusion: Since SarCNU enters cells via extraneuronal monoamine transporter (EMT), the enhanced antitumor activity of SarCNU in this MGMT positive human tumor xenograft model may be due to the presence of EMT in SF-767.SarCNU may be used as an alternative treatment for MGMT positive tumors, specifically for tumors expressing EMT.

  11. Tools for DNA adenine methyltransferase identification analysis of nuclear organization during C. elegans development.

    Science.gov (United States)

    Sharma, Rahul; Ritler, Dominic; Meister, Peter

    2016-04-01

    C. elegans has recently emerged as a valuable model to understand the link between nuclear organization and cell fate, by combining microscopy approaches, genome-wide mapping techniques with advanced genetics. Crucial to these analyses are techniques to determine the genome-wide interaction pattern of proteins with DNA. Chromatin immunoprecipitation has proven valuable but it requires considerable amounts of starting material. This is sometimes difficult to achieve, in particular for specific genotypes (balanced strains, different sexes, severe phenotypes…). As an alternative to ChIP, DNA adenine methyltransferase identification by sequencing (DamID-seq) was recently shown to be able to characterize binding sites in single mammalian cells. Additionally, DamID can be achieved for cell-type specific analysis by expressing Dam fusion proteins under tissue specific promoters in a controlled manner. In this report, we present a user-friendly pipeline to analyse DamID-seq data in C. elegans. Based upon this pipeline, we provide a comparative analysis of libraries generated with different starting material and discuss important library features. Moreover, we introduce an adaptation of an imaging based tool to visualize in vivo the cell-specific tridimensional binding pattern of any protein of interest. genesis 54:151-159, 2016. © 2016 Wiley Periodicals, Inc. PMID:26845390

  12. Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Yanqi; Zhang, Xing; Horton, John R.; Upadhyay, Anup K.; Spannhoff, Astrid; Liu, Jin; Synder, James P.; Bedford, Mark T.; Cheng, Xiaodong; (Emory-MED); (Emory); (Texas)

    2009-03-26

    Histone lysine methylation is an important epigenetic mark that regulates gene expression and chromatin organization. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by methylating histone H3 Lys9. BIX-01294 was originally identified as a G9a inhibitor during a chemical library screen of small molecules and has previously been used in the generation of induced pluripotent stem cells. Here we present the crystal structure of the catalytic SET domain of GLP in complex with BIX-01294 and S-adenosyl-L-homocysteine. The inhibitor is bound in the substrate peptide groove at the location where the histone H3 residues N-terminal to the target lysine lie in the previously solved structure of the complex with histone peptide. The inhibitor resembles the bound conformation of histone H3 Lys4 to Arg8, and is positioned in place by residues specific for G9a and GLP through specific interactions.

  13. Characterization and structure of DhpI, a phosphonate O-methyltransferase involved in dehydrophos biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jin-Hee; Bae, Brian; Kuemin, Michael; Circello, Benjamin T.; Metcalf, William W.; Nair, Satish K.; van der Donk, Wilfred A. (UIUC)

    2012-03-15

    Phosphonate natural products possess a range of biological activities as a consequence of their ability to mimic phosphate esters or tetrahedral intermediates formed in enzymatic reactions involved in carboxyl group metabolism. The dianionic form of these compounds at pH 7 poses a drawback with respect to their ability to mimic carboxylates and tetrahedral intermediates. Microorganisms producing phosphonates have evolved two solutions to overcome this hurdle: biosynthesis of monoanionic phosphinates containing two P-C bonds or esterification of the phosphonate group. The latter solution was first discovered for the antibiotic dehydrophos that contains a methyl ester of a phosphonodehydroalanine group. We report here the expression, purification, substrate scope, and structure of the O-methyltransferase from the dehydrophos biosynthetic gene cluster. The enzyme utilizes S-adenosylmethionine to methylate a variety of phosphonates including 1-hydroxyethylphosphonate, 1,2-dihydroxyethylphosphonate, and acetyl-1-aminoethylphosphonate. Kinetic analysis showed that the best substrates are tripeptides containing as C-terminal residue a phosphonate analog of alanine suggesting the enzyme acts late in the biosynthesis of dehydrophos. These conclusions are corroborated by the X-ray structure that reveals an active site that can accommodate a tripeptide substrate. Furthermore, the structural studies demonstrate a conformational change brought about by substrate or product binding. Interestingly, the enzyme has low substrate specificity and was used to methylate the clinical antibiotic fosfomycin and the antimalaria clinical candidate fosmidomycin, showing its promise for applications in bioengineering.

  14. Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

    International Nuclear Information System (INIS)

    Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain and examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.

  15. Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

    International Nuclear Information System (INIS)

    Highlights: ► Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ► PRMT5 augments the EBNA2-dependent transcription. ► PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ► PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection

  16. Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Jungeun; Shin, Bongjin; Park, Eui-Soon; Yang, Sujeong; Choi, Seunga [Department of Microbiology, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); BK21 Bio Brain Center, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); Kang, Misun [Department of Microbiology, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); Rho, Jaerang, E-mail: jrrho@cnu.ac.kr [Department of Microbiology, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); BK21 Bio Brain Center, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); GRAST, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of)

    2010-01-01

    Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain and examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.

  17. Catechol-O-Methyltransferase Gene Polymorphisms in Specific Obsessive-Compulsive Disorder Patients' Subgroups.

    Science.gov (United States)

    Melo-Felippe, Fernanda Brito; de Salles Andrade, Juliana Braga; Giori, Isabele Gomes; Vieira-Fonseca, Tamiris; Fontenelle, Leonardo Franklin; Kohlrausch, Fabiana Barzotti

    2016-01-01

    Pharmacological data and animal models support the hypothesis that the dopaminergic (DA) system is implicated in obsessive-compulsive disorder (OCD). Therefore, this case-control study assessed whether genetics variations in catechol-O-methyltransferase gene (COMT) could influence susceptibility to OCD and OCD features in a Brazilian sample. A sample of 199 patients with OCD and 200 healthy individuals was genotyped for -287A > G (rs2075507) and Val158Met (rs4680) single nucleotide polymorphisms (SNPs) by TaqMan(®) or restriction mapping. We observed a statistically significant predominance of the Met low-activity allele in the male patient group as compared to the male healthy control group. The -287A > G polymorphism's genotypes and alleles were significantly overrepresented among male individuals with ordering and female subjects with washing symptoms. We also found female hoarders to exhibit a significant higher frequency of the low activity Met/Met genotype of Val158Met polymorphism compared to female patients who did not express this dimension. Our data suggest an influence of COMT polymorphisms on OCD and OCD patients' features, such as gender, and ordering, washing, and hoarding symptom dimensions. Further studies to confirm the clinical importance of COMT SNPs in OCD are warranted. PMID:26687156

  18. Cloning, functional characterization and catalytic mechanism of a bergaptol O-methyltransferase from Peucedanum praeruptorum Dunn

    Directory of Open Access Journals (Sweden)

    Yucheng eZhao

    2016-05-01

    Full Text Available Coumarins are main active components of Peucedanum praeruptorum Dunn. Among them, methoxylated coumarin compound, such as bergapten, xanthotoxin and isopimpinellin, has high officinal value and plays an important role in medicinal field. However, major issues associated with the biosynthesis mechanism of coumarins remain unsolved and no corresponding enzyme has been cloned from P. praeruptorum. In this study, a local BLASTN program was conducted to find the candidate genes from P. praeruptorum transcriptome database using the nucleotide sequence of Ammi majus bergaptol O-methyltransferase (AmBMT, GenBank accession No: AY443006 as a template. As a result, a 1335 bp full-length of cDNA sequence which contains an open reading frame of 1080 bp encoding a BMT polypeptide of 359 amino acids was obtained. The recombinant protein was functionally expressed in Escherichia coli and displayed an observed activity to bergaptol. In vitro experiments show that the protein has narrow substrate specificity for bergaptol. Expression profile indicated that the cloned gene had a higher expression level in roots and can be induced by methyl jasmonate (MeJA. Subcellular localization analysis showed that the BMT protein was located in cytoplasm in planta. Homology modeling and docking based site-directed mutagenesis have been employed to investigate the amino acid residues in BMT required for substrate binding and catalysis. Conservative amino acid substitutions at residue H264 affected BMT catalysis, whereas substitutions at residues F171, M175, D226 and L312 affected substrate binding. The systemic study summarized here will enlarge our knowledge on OMTs and provide useful information in investigating the coumarins biosynthesis mechanism in P. praeruptorum.

  19. DNA methyltransferase inhibitor 5-aza-CdR enhances the radiosensitivity of gastric cancer cells

    International Nuclear Information System (INIS)

    The National Comprehensive Cancer Network guidelines recommend radiotherapy as a standard treatment for patients with a high risk of recurrence in gastric cancer. Because radiation is harmful to the surrounding organs, a radiation sensitizer might therefore be useful to decrease the side effects of patients with advanced gastric carcinoma. The aim of the current study was to clarify the effect of a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (CdR), on radiation sensitivity in gastric cancer cells. Five gastric cancer cell lines, OCUM-2M, OCUM-12, KATO-III, MKN-45, and MKN-74, were used. The effects of 5-aza-CdR with irradiation on the growth activity, cell-cycle distribution, apoptosis, and apoptosis-associated gene expression were examined. 5-aza-CdR sensitized three of five gastric cancer cell lines to radiation. A combination of irradiation and 5-aza-CdR significantly (P2-M phase and the apoptotic rate with irradiation in combination with 5-aza-CdR were increased in OCUM-2M, OCUM-12, and MKN-45 cells compared with irradiation alone, but not in KATO-III and MKN-74 cells. 5-aza-CdR increased the expression of p53, RASSF1, and death-associated protein kinases (DAPK) genes compared with the control or irradiation alone. These findings suggest that 5-aza-CdR might therefore be useful as a radiation sensitizer to treat some types of gastric carcinoma. The arrest at G2-M phase and increased apoptotic rate might be partly mediated by enhanced expression of the p53, RASSF1, or DAPK gene families by 5-aza-CdR. (author)

  20. Cloning, Functional Characterization, and Catalytic Mechanism of a Bergaptol O-Methyltransferase from Peucedanum praeruptorum Dunn

    Science.gov (United States)

    Zhao, Yucheng; Wang, Nana; Zeng, Zhixiong; Xu, Sheng; Huang, Chuanlong; Wang, Wei; Liu, Tingting; Luo, Jun; Kong, Lingyi

    2016-01-01

    Coumarins are main active components of Peucedanum praeruptorum Dunn. Among them, methoxylated coumarin compound, such as bergapten, xanthotoxin, and isopimpinellin, has high officinal value and plays an important role in medicinal field. However, major issues associated with the biosynthesis mechanism of coumarins remain unsolved and no corresponding enzyme has been cloned from P. praeruptorum. In this study, a local BLASTN program was conducted to find the candidate genes from P. praeruptorum transcriptome database using the nucleotide sequence of Ammi majus bergaptol O-methyltransferase (AmBMT, GenBank accession No: AY443006) as a template. As a result, a 1335 bp full-length of cDNA sequence which contains an open reading frame of 1080 bp encoding a BMT polypeptide of 359 amino acids was obtained. The recombinant protein was functionally expressed in Escherichia coli and displayed an observed activity to bergaptol. In vitro experiments show that the protein has narrow substrate specificity for bergaptol. Expression profile indicated that the cloned gene had a higher expression level in roots and can be induced by methyl jasmonate (MeJA). Subcellular localization analysis showed that the BMT protein was located in cytoplasm in planta. Homology modeling and docking based site-directed mutagenesis have been employed to investigate the amino acid residues in BMT required for substrate binding and catalysis. Conservative amino acid substitutions at residue H264 affected BMT catalysis, whereas substitutions at residues F171, M175, D226, and L312 affected substrate binding. The systemic study summarized here will enlarge our knowledge on OMTs and provide useful information in investigating the coumarins biosynthesis mechanism in P. praeruptorum. PMID:27252733

  1. Genomewide identification of target genes of histone methyltransferase dG9a during Drosophila embryogenesis.

    Science.gov (United States)

    Shimaji, Kouhei; Konishi, Takahiro; Tanaka, Shintaro; Yoshida, Hideki; Kato, Yasuko; Ohkawa, Yasuyuki; Sato, Tetsuya; Suyama, Mikita; Kimura, Hiroshi; Yamaguchi, Masamitsu

    2015-11-01

    Post-translational modification of the histone plays important roles in epigenetic regulation of various biological processes. Among the identified histone methyltransferases (HMTases), G9a is a histone H3 Lys 9 (H3K9)-specific example active in euchromatic regions. Drosophila G9a (dG9a) has been reported to feature H3K9 dimethylation activity in vivo. Here, we show that the time required for hatching of a homozygous dG9a null mutant and heteroallelic combination of dG9a null mutants is delayed, suggesting that dG9a is at least partially responsible for progression of embryogenesis. Immunocytochemical analyses of the wild-type and the dG9a null mutant flies indicated that dG9a localizes in cytoplasm up to nuclear division cycle 7 where it is likely responsible for di-methylation of nucleosome-free H3K9. From cycles 8-11, dG9a moves into the nucleus and is responsible for di-methylating H3K9 in nucleosomes. RNA-sequence analysis utilizing early wild-type and dG9a mutant embryos showed that dG9a down-regulates expression of genes responsible for embryogenesis. RNA fluorescent in situ hybridization analysis further showed temporal and spatial expression patterns of these mRNAs did not significantly change in the dG9a mutant. These results indicate that dG9a controls transcription levels of some zygotic genes without changing temporal and spatial expression patterns of the transcripts of these genes. PMID:26334932

  2. Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.

  3. DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity

    International Nuclear Information System (INIS)

    Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process. A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of γH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry. Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of γH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone. Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair

  4. Cloning and characterization of a benzoic acid/salicylic acid carboxyl methyltransferase gene involved in floral scent production from lily (Lilium 'Yelloween').

    Science.gov (United States)

    Wang, H; Sun, M; Li, L L; Xie, X H; Zhang, Q X

    2015-01-01

    In lily flowers, the volatile ester methyl benzoate is one of the major and abundant floral scent compounds; however, knowledge regarding the biosynthesis of methyl benzoate remains unknown for Lilium. In this study, we isolated a benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) gene, LiBSMT, from petals of Lilium 'Yelloween'. The gene has an open reading frame of 1083 base pairs (bp) and encodes a protein of 41.05 kDa. Sequence alignment and phylogenetic analyses of LiBSMT revealed 40-50% similarity with other known benzenoid carboxyl methyltransferases in other plant species, and revealed homology to BSMT of Oryza sativa. Heterologous expression of this gene in Escherichia coli yielded an enzyme responsible for catalyzing benzoic acid and salicylic acid to methyl benzoate and methyl salicylate, respectively. Quantitative real-time polymerase chain reaction analysis showed that LiBSMT was preferentially expressed in petals. Moreover, the expression of LiBSMT in petals was developmentally regulated. These expression patterns correlate well with the emission of methyl benzoate. Our results indicate that LiBSMT plays an important role in floral scent methyl benzoate production and emission in lily flowers. PMID:26600510

  5. Crystal structure of dengue virus methyltransferase without S-adenosyl-L-methionine.

    Science.gov (United States)

    Noble, Christian G; Li, Shi-Hua; Dong, Hongping; Chew, Sock Hui; Shi, Pei-Yong

    2014-11-01

    Flavivirus methyltransferase is a genetically-validated antiviral target. Crystal structures of almost all available flavivirus methyltransferases contain S-adenosyl-L-methionine (SAM), the methyl donor molecule that co-purifies with the enzymes. This raises a possibility that SAM is an integral structural component required for the folding of dengue virus (DENV) methyltransferase. Here we exclude this possibility by solving the crystal structure of DENV methyltransferase without SAM. The SAM ligand was removed from the enzyme through a urea-mediated denaturation-and-renaturation protocol. The crystal structure of the SAM-depleted enzyme exhibits a vacant SAM-binding pocket, with a conformation identical to that of the SAM-enzyme co-crystal structure. Functionally, equivalent enzymatic activities (N-7 methylation, 2'-O methylation, and GMP-enzyme complex formation) were detected for the SAM-depleted and SAM-containing recombinant proteins. These results clearly indicate that the SAM molecule is not an essential component for the correct folding of DENV methyltransferase. Furthermore, the results imply a potential antiviral approach to search for inhibitors that can bind to the SAM-binding pocket and compete against SAM binding. To demonstrate this potential, we have soaked crystals of DENV methyltransferase without a bound SAM with the natural product Sinefungin and show that preformed crystals are capable of binding ligands in this pocket. PMID:25241250

  6. 5-氮杂-2'-脱氧胞苷对涎腺腺样囊性癌细胞系细胞MGMT和hMLH1基因表达的影响%Effect of 5-Aza-CdR on O6-methylguanine-DNA methyltransferase and human homosapiens mutL homolog 1 genes expression in salivary adenoid cystic carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    韩一凡; 李江; 王旭; 张春叶; 田臻

    2012-01-01

    Objective To investigate the effect of 5-aza-2'-deoxycytidine (5-Aza-CdR),a methylation inhibitor,on O6-methylguanine-DNA methyltransferase (MGMT) and human homo sapiens mutL homolog 1 (hMLH1) gene expression in salivary adenoid cystic carcinoma(SACC) cell line.Methods Methyl thiazolyl tetraxolium (MTT) assay was used to test the cytotoxicity of 5-Aza-CdR treatment at different concentrations.And then the mRNA expression of hMLH1 and MGMT was detected by reverse trancriptase polymerase chain reaction (RT-PCR) and real time PCR.Results The half maximal inhibitory concentration of a substance(IC50) value of 5-Aza-CdR in SACC-83 and SACC-LM cells was (11.816 ± 0.023) μmoL/L and (5.751 ± 0.049) μmol/L,respectively.RT-PCR and real time PCR showed that mRNA expression level of MGMT and hMLH1 in SACC cells increased after treated by 5-Aza-CdR (P < 0.01).Conclusions 5-Aza-CdR can change cells morphology and up-regulate mRNA expression of MGMT and hMLH1,which may be correlated with the reversion of hypermethylation status on these gene promoters in tumor cells.%目的 观察5-氮杂-2’-脱氧胞苷(5-aza-2-deoxycytidine,5-Aza-CdR)对体外培养人涎腺腺样囊性癌(salivary adenoid cystic carcinoma,SACC)细胞系细胞O6-甲基鸟嘌呤-DNA甲基转移酶(O6-methylguanine-DNA methyhransferase,MGMT)和人类mutL同源物1(homo sapiens mutL homolog 1,hMLH1)基因表达的影响,探讨DNA甲基转移酶抑制剂应用于SACC治疗的可行性及机制.方法 用不同浓度5-Aza-CdR分别处理体外培养SACC-83和SACC-LM细胞作为药物处理组,以药物处理浓度0 μmol/L为对照组.甲基噻唑基四唑法确定5-Aza-CdR的半数抑制浓度(half maximal inhibitory concentration of a substance,IC50);实时聚合酶链反应和反转录聚合酶链反应检测用药后细胞中MGMT和hMLH1 mRNA表达水平.结果 药物处理细胞24 h后细胞形态发生变化,并且随时间延长变化愈加显著.5-Aza-CdR对SACC-83和SACC-LM细胞的IC50

  7. A Role for Widely Interspaced Zinc Finger (WIZ) in Retention of the G9a Methyltransferase on Chromatin.

    Science.gov (United States)

    Simon, Jeremy M; Parker, Joel S; Liu, Feng; Rothbart, Scott B; Ait-Si-Ali, Slimane; Strahl, Brian D; Jin, Jian; Davis, Ian J; Mosley, Amber L; Pattenden, Samantha G

    2015-10-23

    G9a and GLP lysine methyltransferases form a heterodimeric complex that is responsible for the majority of histone H3 lysine 9 mono- and di-methylation (H3K9me1/me2). Widely interspaced zinc finger (WIZ) associates with the G9a-GLP protein complex, but its role in mediating lysine methylation is poorly defined. Here, we show that WIZ regulates global H3K9me2 levels by facilitating the interaction of G9a with chromatin. Disrupting the association of G9a-GLP with chromatin by depleting WIZ resulted in altered gene expression and protein-protein interactions that were distinguishable from that of small molecule-based inhibition of G9a/GLP, supporting discrete functions of the G9a-GLP-WIZ chromatin complex in addition to H3K9me2 methylation. PMID:26338712

  8. Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

    Science.gov (United States)

    Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

    2009-03-01

    The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts. PMID:19206228

  9. Human Mitochondrial Transcription Factor B1 Interacts with the C-Terminal Activation Region of h-mtTFA and Stimulates Transcription Independently of Its RNA Methyltransferase Activity

    OpenAIRE

    McCulloch, Vicki; Shadel, Gerald S.

    2003-01-01

    A significant advancement in understanding mitochondrial gene expression is the recent identification of two new human mitochondrial transcription factors, h-mtTFB1 and h-mtTFB2. Both proteins stimulate transcription in collaboration with the high-mobility group box transcription factor, h-mtTFA, and are homologous to rRNA methyltransferases. In fact, the dual-function nature of h-mtTFB1 was recently demonstrated by its ability to methylate a conserved rRNA substrate. Here, we demonstrate tha...

  10. Protein Lysine Methyltransferase G9a Inhibitors: Design, Synthesis, and Structure Activity Relationships of 2,4-Diamino-7-aminoalkoxy-quinazolines.†

    OpenAIRE

    Liu, Feng; Chen, Xin; Allali-Hassani, Abdellah; Quinn, Amy M.; Wigle, Tim J.; Wasney, Gregory A.; Dong, Aiping; Senisterra, Guillermo; Chau, Irene; Siarheyeva, Alena; Norris, Jacqueline L.; Kireev, Dmitri B.; Jadhav, Ajit; Herold, J. Martin; Janzen, William P.

    2010-01-01

    Protein lysine methyltransferase G9a, which catalyzes methylation of lysine 9 of histone H3 (H3K9) and lysine 373 (K373) of p53, is over expressed in human cancers. Genetic knockdown of G9a inhibits cancer cell growth and the di-methylation of p53 K373 results in the inactivation of p53. Initial SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template represented by 3a (BIX01294), a selective small molecule inhibitor of G9a and GLP, led to the discovery of 10 (UNC0224) as a potent...

  11. Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells

    OpenAIRE

    Sharma Shikhar; Gerke Daniel S; Han Han F; Jeong Shinwu; Stallcup Michael R; Jones Peter A; Liang Gangning

    2012-01-01

    Abstract Background DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear. Results Here we show that G9a...

  12. Molecular cloning and characterization of the gene encoding the adenine methyltransferase M.CviRI from Chlorella virus XZ-6E.

    OpenAIRE

    Stefan, C; Xia, Y N; Van Etten, J L

    1991-01-01

    The gene encoding the DNA methyltransferase M.CviRI from Chlorella virus XZ-6E was cloned and expressed in Escherichia coli. M.CviRI methylates adenine in TGCA sequences. DNA containing the M.CviRI gene was sequenced and a single open reading frame of 1137 bp was identified which could code for a polypeptide of 379 amino acids with a predicted molecular weight of 42,814. Comparison of the M.CviRI predicted amino acid sequence with another Chlorella virus and 14 bacterial adenine methyltransfe...

  13. Identification of 1,2,3,4,6-Penta-O-galloyl-β-d-glucopyranoside as a Glycine N-Methyltransferase Enhancer by High-Throughput Screening of Natural Products Inhibits Hepatocellular Carcinoma

    OpenAIRE

    Rajni Kant; Chia-Hung Yen; Chung-Kuang Lu; Ying-Chi Lin; Jih-Heng Li; Yi-Ming Arthur Chen

    2016-01-01

    Glycine N-methyltransferase (GNMT) expression is vastly downregulated in hepatocellular carcinomas (HCC). High rates of GNMT knockout mice developed HCC, while overexpression of GNMT prevented aflatoxin-induced carcinogenicity and inhibited liver cancer cell proliferation. Therefore, in this study, we aimed for the identification of a GNMT inducer for HCC therapy. We established a GNMT promoter-driven luciferase reporter assay as a drug screening platform. Screening of 324 pure compounds and ...

  14. Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic alkylating agent.

    OpenAIRE

    R. Maze; Carney, J P; Kelley, M R; Glassner, B J; Williams, D.A.; Samson, L

    1996-01-01

    The chloroethylnitrosourea (CNU) alkylating agents are commonly used for cancer chemotherapy, but their usefulness is limited by severe bone marrow toxicity that causes the cumulative depletion of all hematopoietic lineages (pancytopenia). Bone marrow CNU sensitivity is probably due to the inefficient repair of CNU-induced DNA damage; relative to other tissues, bone marrow cells express extremely low levels of the O6-methylguanine DNA methyltransferase (MGMT) protein that repairs cytotoxic O6...

  15. Purification, crystallization and preliminary X-ray crystallographic analysis of 23S RNA m2G2445 methyltransferase RlmL from Escherichia coli

    International Nuclear Information System (INIS)

    RlmL, a 23S rRNA m2G2445 methyltransferase from Escherichia coli, was expressed, purified and crystallized, and crystals diffracted to 2.2 Å. The RlmL (YcbY) protein in Escherichia coli is an rRNA methyltransferase that is specific for m2G2445 modification of 23S RNA. The rlmL gene was cloned into the expression vector pET28a and expressed in the host E. coli strain BL21 (DE3). Recombinant protein with a six-histidine tag was purified by Ni2+-affinity chromatography followed by gel filtration. Crystals were grown using the hanging-drop vapour-diffusion method and a detergent was used as an additive to improve diffraction quality. The final crystals diffracted to 2.2 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 73.6, b = 140.8, c = 102.9 Å, β = 102.3°. The crystal has a most probable solvent content of 62.8% with two molecules in the asymmetric unit

  16. Theoretical insights into catalytic mechanism of protein arginine methyltransferase 1.

    Directory of Open Access Journals (Sweden)

    Ruihan Zhang

    Full Text Available Protein arginine methyltransferase 1 (PRMT1, the major arginine asymmetric dimethylation enzyme in mammals, is emerging as a potential drug target for cancer and cardiovascular disease. Understanding the catalytic mechanism of PRMT1 will facilitate inhibitor design. However, detailed mechanisms of the methyl transfer process and substrate deprotonation of PRMT1 remain unclear. In this study, we present a theoretical study on PRMT1 catalyzed arginine dimethylation by employing molecular dynamics (MD simulation and quantum mechanics/molecular mechanics (QM/MM calculation. Ternary complex models, composed of PRMT1, peptide substrate, and S-adenosyl-methionine (AdoMet as cofactor, were constructed and verified by 30-ns MD simulation. The snapshots selected from the MD trajectory were applied for the QM/MM calculation. The typical SN2-favored transition states of the first and second methyl transfers were identified from the potential energy profile. Deprotonation of substrate arginine occurs immediately after methyl transfer, and the carboxylate group of E144 acts as proton acceptor. Furthermore, natural bond orbital analysis and electrostatic potential calculation showed that E144 facilitates the charge redistribution during the reaction and reduces the energy barrier. In this study, we propose the detailed mechanism of PRMT1-catalyzed asymmetric dimethylation, which increases insight on the small-molecule effectors design, and enables further investigations into the physiological function of this family.

  17. Characterization of the phospholipid methyltransferase in RBC ghost preparations

    International Nuclear Information System (INIS)

    The activity of the phospholipid methyltransferase from human RBC ghosts was studied using radio-HPLC techniques to analyze the products. Both monomethyl phosphatidyl ethanolamine (MMPE) and dimethyl phosphatidyl ethanolamine (DMPE) were used as substrated. The reaction rate was linear for 45 min. Apparent K/sub M/s of 24-28 uM and 19-21 uM were measured for these two substrates, respectively. The reaction rate was not linear with protein. It appeared to increase logarithmic. An apparent K/sub M/ for S-adenosylmethionine was 36-45 uM. These K/sub M/ values are similar to those reported by others for liver. As the concentration of MMPE was increased, the ratio of DMPE/PC also increased due largely to a greater increase in DMPE formation. Optimal reaction rates for the formation of DMPE were 0.9-1.3 pmol/mg/min, and an optimal rate of about 1.7-2.4 pmol/min/mg was measured for the conversion of DMPE to phosphatidyl choline (PC). Freezing the ghost preparation did not affect the activity of the enzyme. When no exogenous phospholipid was added to the incubation, the sum of the formation rates of all three methylated products was about 26 pmol/mg/hr. The relative amount of each product was 46% MMPE, 32% DMPE and 22% PC. When either MMPE or DMPE was added as substrate, the formation of MMPE was reduced to less than 1%

  18. Thiopurine methyltransferase activity in red blood cells of dogs.

    Science.gov (United States)

    Kidd, Linda Benjamin; Salavaggione, Oreste E; Szumlanski, Carol L; Miller, Jackie L; Weinshilboum, Richard M; Trepanier, Lauren

    2004-01-01

    Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of thiopurine medications such as azathioprine. In humans, activity varies widely among individuals, primarily because of genetic polymorphisms. Low TPMT activity increases the risk of myelosuppression from azathioprine and 6-mercaptopurine, whereas high TPMT activity is associated with poor drug efficacy. The purpose of this study was to determine whether dogs also show a wide range of TPMT activity. Heparinized blood samples were obtained from 177 dogs associated with a veterinary teaching hospital. Red blood cell (RBC) TPMT activity was measured by means of a modification of a radiochemical method as established for use in people. TPMT activity varied across a 9-fold range (7.9-71.8 U of RBC per milliliter; median, 21.7). Variation in TPMT activity was not associated with age, sex, or neutering status. Giant Schnauzers had much lower TPMT activity (7.9-20 U of RBC per milliliter; median, 13.1; P dogs could affect thiopurine drug toxicity and efficacy in canine patients. PMID:15058773

  19. Human erythrocyte thiol methyltransferase: radiochemical microassay and biochemical properties

    International Nuclear Information System (INIS)

    A radiochemical microassay for the measurement of thiol methyltransferase (TMT) activity in human red blood cell (RBC) membranes has been developed. Both 2-mercaptoethanol and dithiothreitol were used as substrates for the enzyme. The pH optimum of the reaction was approximately 9.0 when glycine-NaOH was used as a buffer. The apparent Michaelis-Menten (Ksub(M)) value for the methyl donor for the reaction, S-adenosyl-L-methionine, was 43 μmol/l. Human RBC TMT activity was neither activated nor inhibited by Ca2+, Mg2+, or tropolone, but the enzyme was inhibited by SKF 525A and by reagents that react with sulfhydryl grcups. The mean TMT activity in blood from 289 randomly selected adult white subjects was 10.93 +- 3.22 units per mg protein (mean +- S.D.). The activity was the same in samples from men and women. The results of experiments in which TMT activity was measured in mixtures of RBC membranes with relatively ''low'' and relatively ''high'' activities provided no evidence that individual variations in the enzyme activity were due to variations in endogenous TMT activators or inhibitors. (Auth.)

  20. Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168

    OpenAIRE

    Anjum, Awais; Kelly, Brathwaite; Aidley, Jack B; Connerton, Phillippa L.; Cummings, Nicola J; Parkhill, Julian; Ian F Connerton; Bayliss, Christopher D

    2016-01-01

    Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system ...

  1. The Roles of Two miRNAs in Regulating the Immune Response of Sea Cucumber.

    Science.gov (United States)

    Zhang, Pengjuan; Li, Chenghua; Zhang, Ran; Zhang, Weiwei; Jin, Chunhua; Wang, Lingling; Song, Linsheng

    2015-12-01

    MicroRNAs (miRNAs) have emerged as key regulators in many pathological processes by suppressing the transcriptional and post-transcriptional expression of target genes. MiR-2008 was previously found to be significantly up-regulated in diseased sea cucumber Apostichopus japonicus by high-through sequencing, whereas the reads of miR-137, a well-documented tumor repressor, displayed no significant change. In the present study, we found that miR-137 expression was slightly attenuated and miR-2008 was significantly enhanced after Vibrio splendidus infection or Lipopolysaccharides application. Further target screening and dual-luciferase reporter assay revealed that the two important miRNAs shared a common target gene of betaine-homocysteine S-methyltransferase (AjBHMT), which exhibited noncorrelated messenger RNA and protein expression patterns after bacterial challenge. In order to fully understand their regulatory mechanisms, we conducted the functional experiments in vitro and in vivo. The overexpression of miR-137 in sea cucumber or primary coelomocytes significantly decreased, whereas the inhibition of miR-137 increased the mRNA and protein expression levels of AjBHMT. In contrast, miR-2008 overexpression and inhibition showed no effect on AjBHMT mRNA levels, but the concentration of AjBHMT protein displayed significant changes both in vitro and in vivo. Consistently, the homocysteine (Hcy) contents were also accordingly altered in the aberrant expression analysis of both miRNAs, consistent with the results of the AjBHMT silencing assay in vitro and in vivo. More importantly, small interfering RNA mediated AjBHMT knockdown and Hcy exposure analyses both significantly increased reactive oxygen species (ROS) production and decreased the number of surviving invasive pathogen in sea cucumber coelomocytes. Taken together, these findings confirmed the differential roles of sea cucumber miR-137 and miR-2008 in regulating the common target AjBHMT to promote ROS production

  2. Liver proteomics in progressive alcoholic steatosis

    International Nuclear Information System (INIS)

    Fatty liver is an early stage of alcoholic and nonalcoholic liver disease (ALD and NALD) that progresses to steatohepatitis and other irreversible conditions. In this study, we identified proteins that were differentially expressed in the livers of rats fed 5% ethanol in a Lieber–DeCarli diet daily for 1 and 3 months by discovery proteomics (two-dimensional gel electrophoresis and mass spectrometry) and non-parametric modeling (Multivariate Adaptive Regression Splines). Hepatic fatty infiltration was significantly higher in ethanol-fed animals as compared to controls, and more pronounced at 3 months of ethanol feeding. Discovery proteomics identified changes in the expression of proteins involved in alcohol, lipid, and amino acid metabolism after ethanol feeding. At 1 and 3 months, 12 and 15 different proteins were differentially expressed. Of the identified proteins, down regulation of alcohol dehydrogenase (− 1.6) at 1 month and up regulation of aldehyde dehydrogenase (2.1) at 3 months could be a protective/adaptive mechanism against ethanol toxicity. In addition, betaine-homocysteine S-methyltransferase 2 a protein responsible for methionine metabolism and previously implicated in fatty liver development was significantly up regulated (1.4) at ethanol-induced fatty liver stage (1 month) while peroxiredoxin-1 was down regulated (− 1.5) at late fatty liver stage (3 months). Nonparametric analysis of the protein spots yielded fewer proteins and narrowed the list of possible markers and identified D-dopachrome tautomerase (− 1.7, at 3 months) as a possible marker for ethanol-induced early steatohepatitis. The observed differential regulation of proteins have potential to serve as biomarker signature for the detection of steatosis and its progression to steatohepatitis once validated in plasma/serum. -- Graphical abstract: The figure shows the Hierarchial cluster analysis of differentially expressed protein spots obtained after ethanol feeding for 1 (1–3

  3. Liver proteomics in progressive alcoholic steatosis

    Energy Technology Data Exchange (ETDEWEB)

    Fernando, Harshica [Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Wiktorowicz, John E.; Soman, Kizhake V. [Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Kaphalia, Bhupendra S.; Khan, M. Firoze [Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Shakeel Ansari, G.A., E-mail: sansari@utmb.edu [Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555 (United States)

    2013-02-01

    Fatty liver is an early stage of alcoholic and nonalcoholic liver disease (ALD and NALD) that progresses to steatohepatitis and other irreversible conditions. In this study, we identified proteins that were differentially expressed in the livers of rats fed 5% ethanol in a Lieber–DeCarli diet daily for 1 and 3 months by discovery proteomics (two-dimensional gel electrophoresis and mass spectrometry) and non-parametric modeling (Multivariate Adaptive Regression Splines). Hepatic fatty infiltration was significantly higher in ethanol-fed animals as compared to controls, and more pronounced at 3 months of ethanol feeding. Discovery proteomics identified changes in the expression of proteins involved in alcohol, lipid, and amino acid metabolism after ethanol feeding. At 1 and 3 months, 12 and 15 different proteins were differentially expressed. Of the identified proteins, down regulation of alcohol dehydrogenase (− 1.6) at 1 month and up regulation of aldehyde dehydrogenase (2.1) at 3 months could be a protective/adaptive mechanism against ethanol toxicity. In addition, betaine-homocysteine S-methyltransferase 2 a protein responsible for methionine metabolism and previously implicated in fatty liver development was significantly up regulated (1.4) at ethanol-induced fatty liver stage (1 month) while peroxiredoxin-1 was down regulated (− 1.5) at late fatty liver stage (3 months). Nonparametric analysis of the protein spots yielded fewer proteins and narrowed the list of possible markers and identified D-dopachrome tautomerase (− 1.7, at 3 months) as a possible marker for ethanol-induced early steatohepatitis. The observed differential regulation of proteins have potential to serve as biomarker signature for the detection of steatosis and its progression to steatohepatitis once validated in plasma/serum. -- Graphical abstract: The figure shows the Hierarchial cluster analysis of differentially expressed protein spots obtained after ethanol feeding for 1 (1–3

  4. Anhydrobiosis vs. aging: comparative genomics of protein repair L-isoaspartyl methyltransferases in the sleeping chironomid. .

    Science.gov (United States)

    Gusev, Oleg; Kikawada, Takahiro; Shagimardanova, Elena; Suetsugu, Yoshitaka; Ayupov, Rustam

    Origin of anhydrobiosis in the larvae of the sleeping chironomid Polypedilum vanderplanki represents unique example of set of evolutionary events in a single species, resulted in acquiring new ability allowing survival in extremely changeable environment. Complex comparative analysis of the genome of P. vanderplanki resulted in discovery of a set of features, including existence of the set of unique clusters of genes contributing in desiccation resistance. Surprisingly, in several cases, the genes mainly contributing to the formation of the molecular shield in the larvae are sleeping chironomid-specific and have no homology with genes from other insects, including P. nubifer - a chironomid from the same genus. Protein L-isoaspartyl methyltransferase (PIMT) acts on proteins that have been non-enzymatically damaged due to age, and partially restores aspartic residues, extending life of the polypeptides. PIMT a highly conserved enzyme present in nearly all eukaryotes, and microorganisms mostly in a single copy (or in a few isoforms in certain plants and some bacteria). While conducting a comparative analysis of the genomes of two chironomid midge species different in their ability to stand complete water loss, we have noticed that structure and number of PIMT-coding genes in the desiccation resistant (anhydrobiotic) midge (Polypedilum vanderplanki, Pv) is different from those of the common desiccation-sensitive midge (Polypedilum nubifer, Pn) and the rest of insects. Both species have a clear orthologous PIMT shared by all insects. At the same time, in contrast to Pn which has only one PIMT gene (PnPimt-1), the Pv genome contains 12 additional genes paralogous to Pimt1 (PvPimt-2-12) presumably coding functional PIMT proteins, which are arranged in a single cluster. Remarkably, PvPimt-1 location in the Pv is different from the rest of Pimt-like genes. PvPimt-1 gene is ubiquitously expressed during the life cycle, but expression of the PvPimt2-12 is limited to the eggs

  5. An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases.

    Science.gov (United States)

    Dorgan, Kathleen M; Wooderchak, Whitney L; Wynn, Donraphael P; Karschner, Erin L; Alfaro, Joshua F; Cui, Yinqiu; Zhou, Zhaohui Sunny; Hevel, Joan M

    2006-03-15

    Modification of small molecules and proteins by methyltransferases affects a wide range of biological processes. Here, we report an enzyme-coupled continuous spectrophotometric assay to quantitatively characterize S-adenosyl-L-methionine (AdoMet/SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy/SAH), the transmethylation product of AdoMet-dependent methyltransferases, is hydrolyzed to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase (SAHN/MTAN, EC 3.2.2.9). Subsequently, adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase (EC 3.5.4.2). This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Coupling enzymes are recombinant and easily purified. The utility of this assay was shown using recombinant rat protein arginine N-methyltransferase 1 (PRMT1, EC 2.1.1.125), which catalyzes the mono- and dimethylation of guanidino nitrogens of arginine residues in select proteins. Using this assay, the kinetic parameters of PRMT1 with three synthetic peptides were determined. An advantage of this assay is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Finally, this method may be used to assay other enzymes that produce AdoHcy, 5'-methylthioadenosine, or compounds that can be cleaved by AdoHcy nucleosidase. PMID:16460659

  6. Conservation and Functional Importance of Carbon-Oxygen Hydrogen Bonding in AdoMet-Dependent Methyltransferases

    Energy Technology Data Exchange (ETDEWEB)

    Horowitz, Scott; Dirk, Lynnette M.A.; Yesselman, Joseph D.; Nimtz, Jennifer S.; Adhikari, Upendra; Mehl, Ryan A.; Scheiner, Steve; Houtz, Robert L.; Al-Hashimi, Hashim M.; Trievel, Raymond C. [Oregon State U.; (Michigan); (Utah SU); (HHMI); (Kentucky)

    2013-09-06

    S-Adenosylmethionine (AdoMet)-based methylation is integral to metabolism and signaling. AdoMet-dependent methyltransferases belong to multiple distinct classes and share a catalytic mechanism that arose through convergent evolution; however, fundamental determinants underlying this shared methyl transfer mechanism remain undefined. A survey of high-resolution crystal structures reveals that unconventional carbon–oxygen (CH···O) hydrogen bonds coordinate the AdoMet methyl group in different methyltransferases irrespective of their class, active site structure, or cofactor binding conformation. Corroborating these observations, quantum chemistry calculations demonstrate that these charged interactions formed by the AdoMet sulfonium cation are stronger than typical CH···O hydrogen bonds. Biochemical and structural studies using a model lysine methyltransferase and an active site mutant that abolishes CH···O hydrogen bonding to AdoMet illustrate that these interactions are important for high-affinity AdoMet binding and transition-state stabilization. Further, crystallographic and NMR dynamics experiments of the wild-type enzyme demonstrate that the CH···O hydrogen bonds constrain the motion of the AdoMet methyl group, potentially facilitating its alignment during catalysis. Collectively, the experimental findings with the model methyltransferase and structural survey imply that methyl CH···O hydrogen bonding represents a convergent evolutionary feature of AdoMet-dependent methyltransferases, mediating a universal mechanism for methyl transfer.

  7. The histone methyltransferase SUV420H2 and Heterochromatin Proteins HP1 interact but show different dynamic behaviours

    Directory of Open Access Journals (Sweden)

    Souza Patricia P

    2009-06-01

    Full Text Available Abstract Background Histone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions. In particular, trimethylation at lysine 9 of histone H3 (H3K9 and at lysine 20 of histone H4 (H4K20 governed by the histone methyltransferases SUV39H1/2 and SUV420H1/2 respectively, have emerged as a hallmark of pericentric heterochromatin. Controlled chromatin organization is crucial for gene expression regulation and genome stability. Therefore, it is essential to analyze mechanisms responsible for high order chromatin packing and in particular the interplay between enzymes involved in histone modifications, such as histone methyltransferases and proteins that recognize these epigenetic marks. Results To gain insights into the mechanisms of SUV420H2 recruitment at heterochromatin, we applied a tandem affinity purification approach coupled to mass spectrometry. We identified heterochromatin proteins HP1 as main interacting partners. The regions responsible for the binding were mapped to the heterochromatic targeting module of SUV420H2 and HP1 chromoshadow domain. We studied the dynamic properties of SUV420H2 and the HP1 in living cells using fluorescence recovery after photobleaching. Our results showed that HP1 proteins are highly mobile with different dynamics during the cell cycle, whereas SUV420H2 remains strongly bound to pericentric heterochromatin. An 88 amino-acids region of SUV420H2, the heterochromatic targeting module, recapitulates both, HP1 binding and strong association to heterochromatin. Conclusion FRAP experiments reveal that in contrast to HP1, SUV420H2 is strongly associated to pericentric heterochromatin. Then, the fraction of SUV420H2 captured and characterized by TAP/MS is a soluble fraction which may be in a stable association with HP1. Consequently, SUV420H2 may be recruited to heterochromatin in association with HP1, and stably maintained at its heterochromatin sites in an HP1

  8. Involvement of histone methyltransferase GLP in HIV-1 latency through catalysis of H3K9 dimethylation

    International Nuclear Information System (INIS)

    Understanding the mechanism of HIV-1 latency is crucial to eradication of the viral reservoir in HIV-1-infected individuals. However, the role of histone methyltransferase (HMT) G9a-like protein (GLP) in HIV-1 latency is still unclear. In the present work, we established four clonal cell lines containing HIV-1 vector. We found that the integration sites of most clonal cell lines favored active gene regions. However, we also observed hypomethylation of CpG of HIV 5′LTR in all four clonal cell lines. Additionally, 5′-deoxy-5′-methylthioadenosine (MTA), a broad-spectrum histone methyltransferase inhibitor, was used to examine the role of histone methylation in HIV-1 latency. MTA was found to decrease the level of H3K9 dimethylation, causing reactivation of latent HIV-1 in C11 cells. GLP knockdown by small interfering RNA clearly induced HIV-1 LTR expression. Results suggest that GLP may play a significant role in the maintenance of HIV-1 latency by catalyzing dimethylation of H3K9. - Highlights: ► We have established an in vitro model of HIV-1 latency. ► The integration sites of most clonal cell lines favor in active gene regions. ► Hypomethylation occurs in CpG islands of HIV 5′LTR in all four clonal cell lines. ► MTA can reactivate latent HIV-1 by decreasing the level of H3K9 me2 in C11 cells. ► HMT GLP may play a significant role in the maintenance of HIV-1 latency

  9. Involvement of histone methyltransferase GLP in HIV-1 latency through catalysis of H3K9 dimethylation

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Donglin; Qu, Xiying; Li, Lin; Zhou, Xin; Liu, Sijie; Lin, Shiguan; Wang, Pengfei; Liu, Shaohui; Kong, Chuijin; Wang, Xiaohui; Liu, Lin; Zhu, Huanzhang, E-mail: hzzhu@fudan.edu.cn

    2013-06-05

    Understanding the mechanism of HIV-1 latency is crucial to eradication of the viral reservoir in HIV-1-infected individuals. However, the role of histone methyltransferase (HMT) G9a-like protein (GLP) in HIV-1 latency is still unclear. In the present work, we established four clonal cell lines containing HIV-1 vector. We found that the integration sites of most clonal cell lines favored active gene regions. However, we also observed hypomethylation of CpG of HIV 5′LTR in all four clonal cell lines. Additionally, 5′-deoxy-5′-methylthioadenosine (MTA), a broad-spectrum histone methyltransferase inhibitor, was used to examine the role of histone methylation in HIV-1 latency. MTA was found to decrease the level of H3K9 dimethylation, causing reactivation of latent HIV-1 in C11 cells. GLP knockdown by small interfering RNA clearly induced HIV-1 LTR expression. Results suggest that GLP may play a significant role in the maintenance of HIV-1 latency by catalyzing dimethylation of H3K9. - Highlights: ► We have established an in vitro model of HIV-1 latency. ► The integration sites of most clonal cell lines favor in active gene regions. ► Hypomethylation occurs in CpG islands of HIV 5′LTR in all four clonal cell lines. ► MTA can reactivate latent HIV-1 by decreasing the level of H3K9 me2 in C11 cells. ► HMT GLP may play a significant role in the maintenance of HIV-1 latency.

  10. Methyltransferases mediate cell memory of a genotoxic insult.

    Science.gov (United States)

    Rugo, R E; Mutamba, J T; Mohan, K N; Yee, T; Chaillet, J R; Greenberger, J S; Engelward, B P

    2011-02-10

    Characterization of the direct effects of DNA-damaging agents shows how DNA lesions lead to specific mutations. Yet, serum from Hiroshima survivors, Chernobyl liquidators and radiotherapy patients can induce a clastogenic effect on naive cells, showing indirect induction of genomic instability that persists years after exposure. Such indirect effects are not restricted to ionizing radiation, as chemical genotoxins also induce heritable and transmissible genomic instability phenotypes. Although such indirect induction of genomic instability is well described, the underlying mechanism has remained enigmatic. Here, we show that mouse embryonic stem cells exposed to γ-radiation bear the effects of the insult for weeks. Specifically, conditioned media from the progeny of exposed cells can induce DNA damage and homologous recombination in naive cells. Notably, cells exposed to conditioned media also elicit a genome-destabilizing effect on their neighbouring cells, thus demonstrating transmission of genomic instability. Moreover, we show that the underlying basis for the memory of an insult is completely dependent on two of the major DNA cytosine methyltransferases, Dnmt1 and Dnmt3a. Targeted disruption of these genes in exposed cells completely eliminates transmission of genomic instability. Furthermore, transient inactivation of Dnmt1, using a tet-suppressible allele, clears the memory of the insult, thus protecting neighbouring cells from indirect induction of genomic instability. We have thus demonstrated that a single exposure can lead to long-term, genome-destabilizing effects that spread from cell to cell, and we provide a specific molecular mechanism for these persistent bystander effects. Collectively, our results impact the current understanding of risks from toxin exposures and suggest modes of intervention for suppressing genomic instability in people exposed to carcinogenic genotoxins. PMID:21057543

  11. Catechol-O-methyltransferase, dopamine, and sleep-wake regulation.

    Science.gov (United States)

    Dauvilliers, Yves; Tafti, Mehdi; Landolt, Hans Peter

    2015-08-01

    Sleep and sleep disorders are complex and highly variable phenotypes regulated by many genes and environment. The catechol-O-methyltransferase (COMT) gene is an interesting candidate, being one of the major mammalian enzymes involved in the catabolism of catecholamines. The activity of COMT enzyme is genetically polymorphic due to a guanine-to-adenine transition at codon 158, resulting in a valine (Val) to methionine (Met) substitution. Individuals homozygous for the Val allele show higher COMT activity, and lower dopaminergic signaling in prefrontal cortex (PFC) than subjects homozygous for the Met allele. Since COMT has a crucial role in metabolising dopamine, it was suggested that the common functional polymorphism in the COMT gene impacts on cognitive function related to PFC, sleep-wake regulation, and potentially on sleep pathologies. The COMT Val158Met polymorphism may predict inter-individual differences in brain electroencephalography (EEG) alpha oscillations and recovery processes resulting from partial sleep loss in healthy individuals. The Val158Met polymorphism also exerts a sexual dimorphism and has a strong effect on objective daytime sleepiness in patients with narcolepsy-cataplexy. Since the COMT enzyme inactivates catecholamines, it was hypothesized that the response to stimulant drugs differs between COMT genotypes. Modafinil maintained executive functioning performance and vigilant attention throughout sleep deprivation in subjects with Val/Val genotype, but less in those with Met/Met genotype. Also, homozygous Met/Met patients with narcolepsy responded to lower doses of modafinil compared to Val/Val carriers. We review here the critical role of the common functional COMT gene polymorphism, COMT enzyme activity, and the prefrontal dopamine levels in the regulation of sleep and wakefulness in normal subjects, in narcolepsy and other sleep-related disorders, and its impact on the response to psychostimulants. PMID:25466290

  12. Demonstration of thiopurine methyltransferase activity in the erythrocytes of cats.

    Science.gov (United States)

    Foster, A P; Shaw, S E; Duley, J A; Shobowale-Bakre, E M; Harbour, D A

    2000-01-01

    Azathioprine is a purine analogue used as an immunosuppressive and immunomodulator agent in various mammals, including cats. Several adverse reactions have been reported and have limited the use of the drug in the cat. Adverse reactions to azathioprine in humans have been correlated with reduced activity of thiopurine methyltransferase (TPMT) in erythrocytes. The purpose of this preliminary study was to determine if cats have TPMT activity in their erythrocytes and to compare the values obtained with the normal range for humans and the normal range for dogs in a preliminary report. Activity of the enzyme was measured in blood samples drawn from 41 cats. Blood also was taken from 5 dogs. The mean erythrocyte TPMT activity in the cats was 2.4 +/- 0.4 nmol (range, 1.2-3.9 nmol) per hour per milliliter of red blood cells (U/mL RBC) or 2-8 nmol per hour per gram of hemoglobin (U/g Hb). This range was far lower than the normal human range (8-15 U/mL RBC; 16-33 U/g Hb) and was of monopolar distribution. This observation apparently precludes any diagnostic purpose in assaying erythrocyte TPMT in this species. Erythrocyte TPMT activity in the 5 dogs ranged from 5.5 to 13.1 U/mL RBC (11-27 U/g Hb), which was comparable with normal and carrier ranges for humans, but proof of TPMT genetic polymorphism in either species will require genotyping studies. PMID:11012121

  13. Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins

    Science.gov (United States)

    Gonsalvez, Graydon B.; Tian, Liping; Ospina, Jason K.; Boisvert, François-Michel; Lamond, Angus I.; Matera, A. Gregory

    2007-01-01

    Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells. PMID:17709427

  14. Study on methylation and expressions of p16INK4a,Runx3 and O-6-methylguanine-DNA methyltransferase genes in gastdc carcinogenesis%胃癌形成过程中p16IN4a、Runx3和O-6-甲基鸟嘌呤-DNA甲基转移酶基因甲基化及其表达研究

    Institute of Scientific and Technical Information of China (English)

    张斌; 曹俊; 刘文佳; 陈敏; 邹晓平

    2009-01-01

    目的 研究胃癌形成过程中p16INK4a、Runx3和O-6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因启动子区的高甲基化状态,同时检测MGMT的蛋白表达情况.探讨抑癌基因启动子区高甲基化与胃癌发生的关系.方法 选择经透明帽法进行首次黏膜病变切除者43例,其中异型增生27例,早期胃癌16例.选择胃镜活检证实为慢性萎缩性胃炎伴肠上皮化生者14例.另取20例正常胃黏膜活检组织作为对照.采用甲基化特异聚合酶链反应(MSP)检测每例组织中p16INK4a、Runx3和MGMT基因启动子区的甲基化状态,对所有甲基化p16INK4a产物进行测序,免疫组化检测MGMT蛋白表达情况.结果 肠上皮化生、异型增生和早期胃癌中p16INK4a基因甲基化率依次为14.3%(2/14)、22.2%(6/27)和37.5%(6/16);Runx3基因甲基化率依次为14.3%(2/14)、48.1%(13/27)和50.0%(8/16);MGMT基因甲基化率依次为7.1%(1/14)、48.1%(13/27)和50.0%(8/16).20名正常对照均未检出基因甲基化,与异型增生和早期胃癌相比差异有统计学意义(P<0.05).Runx3和MGMT两种基因在异型增生和早期胃癌中的甲基化率显著高于肠上皮化生组(P<0.05).各组病变中三种基因甲基化联合分析发现,异型增生和早期胃癌中甲基化的基因种类高于肠上皮化生组.差异有统计学意义(P<0.01).基因甲基化与息者年龄、性别、幽门螺杆菌感染以及病变部位无相关性,但p16INK4a和MGMT基因甲基化与血清癌胚抗原水平升高显著相关(P值分别为0.003和0.039).MGMT基因启动子区高甲基化与其蛋白失表达密切相关(χ2=12.821,P=0.001).结论 抑癌基因启动子区高甲基化是基因失活的主要机制,可能是胃癌发生的早期分子事件.p16INK4a、Runx3和MGMT基因启动子区高甲基化在胃癌形成过程中起着重要的作用.%Objective To investigate the promoter methylation of p16INK4a,Runx3 and O-6-methylguanine-DNA methyltransferase genes

  15. Isolation and functional characterization of N-methyltransferases that catalyze betaine synthesis from glycine in a halotolerant photosynthetic organism Aphanothece halophytica.

    Science.gov (United States)

    Waditee, Rungaroon; Tanaka, Yoshito; Aoki, Kenji; Hibino, Takashi; Jikuya, Hiroshi; Takano, Jun; Takabe, Tetsuko; Takabe, Teruhiro

    2003-02-14

    Glycine betaine (N,N,N-trimethylglycine) is an important osmoprotectant and is synthesized in response to abiotic stresses. Although almost all known biosynthetic pathways of betaine are two-step oxidation of choline, here we isolated two N-methyltransferase genes from a halotolerant cyanobacterium Aphanothece halophytica. One of gene products (ORF1) catalyzed the methylation reactions of glycine and sarcosine with S-adenosylmethionine acting as the methyl donor. The other one (ORF2) specifically catalyzed the methylation of dimethylglycine to betaine. Both enzymes are active as monomers. Betaine, a final product, did not show the feed back inhibition for the methyltransferases even in the presence of 2 m. A reaction product, S-adenosyl homocysteine, inhibited the methylation reactions with relatively low affinities. The co-expressing of two enzymes in Escherichia coli increased the betaine level and enhanced the growth rates. Immunoblot analysis revealed that the accumulation levels of both enzymes in A. halophytica cells increased with increasing the salinity. These results indicate that A. halophytica cells synthesize betaine from glycine by a three-step methylation. The changes of amino acids Arg-169 to Lys or Glu in ORF1 and Pro-171 to Gln and/or Met-172 to Arg in ORF2 significantly decreased V(max) and increased K(m) for methyl acceptors (glycine, sarcosine, and dimethylglycine) but modestly affected K(m) for S-adenosylmethionine, indicating the importance of these amino acids for the binding of methyl acceptors. Physiological and functional properties of methyltransferases were discussed. PMID:12466265

  16. SpnH from Saccharopolyspora spinosa encodes a rhamnosyl 4'-O-methyltransferase for biosynthesis of the insecticidal macrolide, spinosyn A.

    Science.gov (United States)

    Huang, Ke-xue; Zahn, James; Han, Lei

    2008-12-01

    Deoxysugar, 2', 3', 4'-tri-O-methylrhamnose is an essential structural component of spinosyn A and D, which are the active ingredients of the commercial insect control agent, Spinosad. The spnH gene, which was previously assigned as a rhamnose O-methyltransferase based on gene sequence homology, was cloned from the wild-type Saccharopolyspora spinosa and from a spinosyn K-producing mutant that was defective in the 4'-O-methylation of 2', 3'-tri-O-methylrhamnose. DNA sequencing confirmed a mutation resulting in an amino acid substitution of G-165 to A-165 in the rhamnosyl 4'-O-methyltransferase of the mutant strain, and the subsequent sequence analysis showed that the mutation occurred in a highly conserved region of the translated amino acid sequence. Both spnH and the gene defective in 4'-O-methylation activity (spnH165A) were expressed heterologously in E. coli and were then purified to homogeneity using a His-tag affinity column. Substrate bioconversion studies showed that the enzyme encoded by spnH, but not spnH165A, could utilize spinosyn K as a substrate. When the wild-type spnH gene was transformed into the spinosyn K-producing mutant, spinosyn A production was restored. These results establish that the enzyme encoded by the spnH gene in wild-type S. spinosa is a rhamnosyl 4'-O-methyltransferase that is responsible for the final rhamnosyl methylation step in the biosynthesis of spinosyn A. PMID:18704529

  17. The RNA-methyltransferase Misu (NSun2 poises epidermal stem cells to differentiate.

    Directory of Open Access Journals (Sweden)

    Sandra Blanco

    2011-12-01

    Full Text Available Homeostasis of most adult tissues is maintained by balancing stem cell self-renewal and differentiation, but whether post-transcriptional mechanisms can regulate this process is unknown. Here, we identify that an RNA methyltransferase (Misu/Nsun2 is required to balance stem cell self-renewal and differentiation in skin. In the epidermis, this methyltransferase is found in a defined sub-population of hair follicle stem cells poised to undergo lineage commitment, and its depletion results in enhanced quiescence and aberrant stem cell differentiation. Our results reveal that post-transcriptional RNA methylation can play a previously unappreciated role in controlling stem cell fate.

  18. H3K9 methyltransferase G9a and the related molecule GLP

    OpenAIRE

    Shinkai, Yoichi; Tachibana, Makoto

    2011-01-01

    The discovery of Suv39h1, the first SET domain-containing histone lysine methyltransferase (HKMT), was reported in 2000. Since then, research on histone methylation has progressed rapidly. Among the identified HKMTs in mammals, G9a and GLP are the primary enzymes for mono- and dimethylation at Lys 9 of histone H3 (H3K9me1 and H3K9me2), and exist predominantly as a G9a–GLP heteromeric complex that appears to be a functional H3K9 methyltransferase in vivo. Recently, many important studies have ...

  19. Negative in vitro selection identifies the rRNA recognition motif for ErmE methyltransferase

    DEFF Research Database (Denmark)

    Nielsen, A K; Douthwaite, S; Vester, B

    1999-01-01

    the adjacent single-stranded region around A2058. An RNA transcript of 72 nt that displays this motif functions as an efficient substrate for the ErmE methyltransferase. Pools of degenerate RNAs were formed by doping 34-nt positions that extend over and beyond the putative Erm recognition motif within...... contained substitutions at single sites, and these are confined to 12 nucleotide positions. These nucleotides, corresponding to A2051-A2060, C2611, and A2614 in 23S rRNA, presumably comprise the RNA recognition motif for ErmE methyltransferase. The structure formed by these nucleotides is highly conserved...

  20. The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

    Energy Technology Data Exchange (ETDEWEB)

    Suriguga,; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun, E-mail: yizc@buaa.edu.cn

    2013-12-15

    Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. - Highlights: • Catechol enhanced hemin-induced hemoglobin accumulation. • COMT-catalyzed methylation acted as detoxication of catechol. • COMT involved in catechol-enhanced erythroid differentiation.

  1. The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

    International Nuclear Information System (INIS)

    Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. - Highlights: • Catechol enhanced hemin-induced hemoglobin accumulation. • COMT-catalyzed methylation acted as detoxication of catechol. • COMT involved in catechol-enhanced erythroid differentiation

  2. Radiometric assay for phenylethanolamine N-methyltransferase and catechol O-methyltransferase in a single tissue sample: application to rat hypothalamic nuclei, pineal gland, and heart

    International Nuclear Information System (INIS)

    A simple and highly sensitive method for simultaneous assay of phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) is described. These enzymes are determined in a single tissue homogenate using S-[methyl-3H] adenosyl-L-methionine as methyl donor and sequentially incubating with the substrates phenylethanolamine and epinephrine. The radioactive products of the enzymatic reactions, N-methylphenylethanolamine and metanephrine, are extracted and then separated by thin-layer chromatography. The identity of the reaction products has been established chromatographically and the conditions for both enzymatic reactions in the assay procedure have been defined. Measurement of PNMT activity in the rat pineal gland or in minute fragments of other tissues (e.g., brain nuclei) has not been possible using previously described methods. Activities of PNMT and COMT in the rat pineal gland, various hypothalamic nuclei, and the auricular and ventricular myocardia are herein reported

  3. The critical role of protein arginine methyltransferase prmt8 in zebrafish embryonic and neural development is non-redundant with its paralogue prmt1.

    Directory of Open Access Journals (Sweden)

    Yu-ling Lin

    Full Text Available Protein arginine methyltransferase (PRMT 1 is the most conserved and widely distributed PRMT in eukaryotes. PRMT8 is a vertebrate-restricted paralogue of PRMT1 with an extra N-terminal sequence and brain-specific expression. We use zebrafish (Danio rerio as a vertebrate model to study PRMT8 function and putative redundancy with PRMT1. The transcripts of zebrafish prmt8 were specifically expressed in adult zebrafish brain and ubiquitously expressed from zygotic to early segmentation stage before the neuronal development. Whole-mount in situ hybridization revealed ubiquitous prmt8 expression pattern during early embryonic stages, similar to that of prmt1. Knockdown of prmt8 with antisense morpholino oligonucleotide phenocopied prmt1-knockdown, with convergence/extension defects at gastrulation. Other abnormalities observed later include short body axis, curled tails, small and malformed brain and eyes. Catalytically inactive prmt8 failed to complement the morphants, indicating the importance of methyltransferase activity. Full-length prmt8 but not prmt1 cRNA can rescue the phenotypic changes. Nevertheless, cRNA encoding Prmt1 fused with the N-terminus of Prmt8 can rescue the prmt8 morphants. In contrast, N-terminus- deleted but not full-length prmt8 cRNA can rescue the prmt1 morphants as efficiently as prmt1 cRNA. Abnormal brain morphologies illustrated with brain markers and loss of fluorescent neurons in a transgenic fish upon prmt8 knockdown confirm the critical roles of prmt8 in neural development. In summery, our study is the first report showing the expression and function of prmt8 in early zebrafish embryogenesis. Our results indicate that prmt8 may play important roles non-overlapping with prmt1 in embryonic and neural development depending on its specific N-terminus.

  4. MMSET is highly expressed and associated with aggressiveness in neuroblastoma

    DEFF Research Database (Denmark)

    Hudlebusch, Heidi Rye; Skotte, Julie; Santoni-Rugiu, Eric;

    2011-01-01

    MMSET (WHSC1/NSD2) is a SET-domain-containing histone lysine methyltransferase, whose expression is deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation associated with poor prognosis. Recent studies have demonstrated that MMSET mRNA levels are increased in other...

  5. Mouse myofibers lacking the SMYD1 methyltransferase are susceptible to atrophy, internalization of nuclei and myofibrillar disarray.

    Science.gov (United States)

    Stewart, M David; Lopez, Suhujey; Nagandla, Harika; Soibam, Benjamin; Benham, Ashley; Nguyen, Jasmine; Valenzuela, Nicolas; Wu, Harry J; Burns, Alan R; Rasmussen, Tara L; Tucker, Haley O; Schwartz, Robert J

    2016-03-01

    The Smyd1 gene encodes a lysine methyltransferase specifically expressed in striated muscle. Because Smyd1-null mouse embryos die from heart malformation prior to formation of skeletal muscle, we developed a Smyd1 conditional-knockout allele to determine the consequence of SMYD1 loss in mammalian skeletal muscle. Ablation of SMYD1 specifically in skeletal myocytes after myofiber differentiation using Myf6(cre) produced a non-degenerative myopathy. Mutant mice exhibited weakness, myofiber hypotrophy, prevalence of oxidative myofibers, reduction in triad numbers, regional myofibrillar disorganization/breakdown and a high percentage of myofibers with centralized nuclei. Notably, we found broad upregulation of muscle development genes in the absence of regenerating or degenerating myofibers. These data suggest that the afflicted fibers are in a continual state of repair in an attempt to restore damaged myofibrils. Disease severity was greater for males than females. Despite equivalent expression in all fiber types, loss of SMYD1 primarily affected fast-twitch muscle, illustrating fiber-type-specific functions for SMYD1. This work illustrates a crucial role for SMYD1 in skeletal muscle physiology and myofibril integrity. PMID:26935107

  6. O6-methylguanine-DNA methyltransferase (MGMT) Promoter methylation is a rare event in soft tissue sarcoma

    International Nuclear Information System (INIS)

    Gene silencing of O6-methylguanine–DNA methyltransferase (MGMT) by promoter methylation improves the outcome of glioblastoma patients after combined therapy of alkylating chemotherapeutic agents and radiation. The purpose of this study was to assess the frequency of MGMT promoter methylation in soft tissue sarcoma to identify patients eligible for alkylating agent chemotherapy such as temozolomide. Paraffin tumor blocks of 75 patients with representative STS subtypes were evaluated. The methylation status of the MGMT promoter was assessed by methylation-specific polymerase-chain-reaction analysis (PCR). Furthermore, immunohistochemistry was applied to verify expression of MGMT. MGMT gene silencing was assumed if MGMT promoter methylation was present and the fraction of tumor cells expressing MGMT was 20% or less. Methylation specific PCR detected methylated MGMT promoter in 10/75 cases. Immunohistochemical staining of nuclear MGMT was negative in 15/75 cases. 6/75 tumor samples showed MGMT promoter methylation and negative immunohistochemical nuclear staining of MGMT. In none of the tested STS subtypes we found a fraction of tumors with MGMT silencing exceeding 22%. MGMT gene silencing is a rare event in soft tissue sarcoma and cannot be recommended as a selection criterion for the therapy of STS patients with alkylating agents such as temozolomide

  7. O6-methylguanine-DNA methyltransferase (MGMT Promoter methylation is a rare event in soft tissue sarcoma

    Directory of Open Access Journals (Sweden)

    Jakob Jens

    2012-10-01

    Full Text Available Abstract Background Gene silencing of O6-methylguanine–DNA methyltransferase (MGMT by promoter methylation improves the outcome of glioblastoma patients after combined therapy of alkylating chemotherapeutic agents and radiation. The purpose of this study was to assess the frequency of MGMT promoter methylation in soft tissue sarcoma to identify patients eligible for alkylating agent chemotherapy such as temozolomide. Findings Paraffin tumor blocks of 75 patients with representative STS subtypes were evaluated. The methylation status of the MGMT promoter was assessed by methylation-specific polymerase-chain-reaction analysis (PCR. Furthermore, immunohistochemistry was applied to verify expression of MGMT. MGMT gene silencing was assumed if MGMT promoter methylation was present and the fraction of tumor cells expressing MGMT was 20% or less. Methylation specific PCR detected methylated MGMT promoter in 10/75 cases. Immunohistochemical staining of nuclear MGMT was negative in 15/75 cases. 6/75 tumor samples showed MGMT promoter methylation and negative immunohistochemical nuclear staining of MGMT. In none of the tested STS subtypes we found a fraction of tumors with MGMT silencing exceeding 22%. Conclusion MGMT gene silencing is a rare event in soft tissue sarcoma and cannot be recommended as a selection criterion for the therapy of STS patients with alkylating agents such as temozolomide.

  8. Wolbachia prophage DNA adenine methyltransferase genes in different Drosophila-Wolbachia associations

    DEFF Research Database (Denmark)

    Saridaki, Aggeliki; Sapountzis, Panagiotis; Harris, Harriet L;

    2011-01-01

    . The importance of DNA methylation in cell fate and biology calls for in depth studying of putative methylation-related genes. We present a molecular and phylogenetic analysis of a putative DNA adenine methyltransferase encoded by a prophage in the Wolbachia genome. Two slightly different copies of the gene, met1...

  9. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity

    DEFF Research Database (Denmark)

    Pasini, Diego; Bracken, Adrian P; Jensen, Michael R;

    2004-01-01

    SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show tha...

  10. Methyltransferase Erm(37) Slips on rRNA to Confer Atypical Resistance in Mycobacterium tuberculosis

    Czech Academy of Sciences Publication Activity Database

    Madsen, Ch. T.; Jakobsen, L.; Buriánková, Karolína; Doucet-Populaire, F.; Perdonet, J. L.; Douthwaite, S.

    2005-01-01

    Roč. 280, č. 47 (2005), s. 38942-38947. ISSN 0021-9258 R&D Projects: GA ČR GA310/03/0292 Institutional research plan: CEZ:AV0Z50200510 Keywords : methyltransferase erm * mycobacterium tuberculosis * rRNA Subject RIV: EE - Microbiology, Virology Impact factor: 5.854, year: 2005

  11. Association of Catechol-O-Methyltransferase (COMT) Polymorphism and Academic Achievement in a Chinese Cohort

    Science.gov (United States)

    Yeh, Ting-Kuang; Chang, Chun-Yen; Hu, Chung-Yi; Yeh, Ting-Chi; Lin, Ming-Yeh

    2009-01-01

    Catechol-O-methyltransferase (COMT) is a methylation enzyme that catalyzes the degradation pathway and inactivation of dopamine. It is accepted widely as being involved in the modulation of dopaminergic physiology and prefrontal cortex (PFC) function. The COMT Val158Met polymorphism is associated with variation in COMT activity. COMT 158Met allele…

  12. Catechol-O-methyltransferase gene methylation and substance use in adolescents: The TRAILS study

    NARCIS (Netherlands)

    L.J. van der Knaap (Lisette); J.M. Schäfer (Johanna); I.H.A. Franken (Ingmar); F.C. Verhulst (Frank); F.V.A. van Oort (Floor); H. Riese (Harriëtte)

    2014-01-01

    textabstractSubstance use often starts in adolescence and poses a major problem for society and individual health. The dopamine system plays a role in substance use, and catechol-O-methyltransferase (COMT) is an important enzyme that degrades dopamine. The Val108/158Met polymorphism modul

  13. Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing

    DEFF Research Database (Denmark)

    Relling, M V; Gardner, E E; Sandborn, W J;

    2011-01-01

    Thiopurine methyltransferase (TPMT) activity exhibits monogenic co-dominant inheritance, with ethnic differences in the frequency of occurrence of variant alleles. With conventional thiopurine doses, homozygous TPMT-deficient patients (~1 in 178 to 1 in 3,736 individuals with two nonfunctional TP...

  14. SABATH Methyltransferases from White Spruce (Picea glauca [Moench] Voss): Gene Cloning, Functional Characterization and Structural Analysis

    Science.gov (United States)

    Known members of the plant SABATH family of methyltransferases have important biological functions by methylating hormones, signaling molecules and other metabolites. While all previously characterized SABATH genes were isolated from angiosperms, in this article, we report on the isolation and funct...

  15. Recognition elements in rRNA for the tylosin resistance methyltransferase RlmA(II)

    DEFF Research Database (Denmark)

    Lebars, Isabelle; Husson, Clotilde; Yoshizawa, Satoko; Douthwaite, Stephen; Fourmy, Dominique

    2007-01-01

    antibiotics. We have previously solved the solution structure of hairpin 35 in the conformation that is recognized by the RlmA(II) methyltransferase from Streptococcus pneumoniae. It was shown that while essential recognition elements are located in hairpin 35, the interactions between RlmA(II) and hairpin 35...

  16. DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents.

    NARCIS (Netherlands)

    B.J. Glassner (Brian); G. Weeda (Geert); J.M. Allan (James); J.L.M. Broekhof (Jose'); N.H.E. Carls (Nick); I. Donker (Ingrid); B.P. Engelward (Bevin); R.J. Hampson (Richard); R. Hersmus (Remko); M.J. Hickman (Mark); R.B. Roth (Richard); H.B. Warren (Henry); M.M. Wu (Mavis); J.H.J. Hoeijmakers (Jan); L.D. Samson (Leona)

    1999-01-01

    textabstractWe have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA methyltransf

  17. Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

    Science.gov (United States)

    Lignin is a significant recalcitrant in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired...

  18. Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability

    DEFF Research Database (Denmark)

    Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; Del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O'Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

    2014-01-01

    and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for...

  19. Local chromatin microenvironment determines DNMT activity : from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase

    NARCIS (Netherlands)

    van der Wijst, Monique G. P.; Venkiteswaran, Muralidhar; Chen, Hui; Xu, Guo-Liang; Plosch, Torsten; Rots, Marianne G.

    2015-01-01

    Insights on active DNA demethylation disproved the original assumption that DNA methylation is a stable epigenetic modification. Interestingly, mammalian DNA methyltransferases 3A and 3B (DNMT-3A and -3B) have also been reported to induce active DNA demethylation, in addition to their well-known fun

  20. Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing

    DEFF Research Database (Denmark)

    Relling, M V; Gardner, E E; Sandborn, W J; Schmiegelow, K; Pui, C-H; Yee, S W; Stein, Paul C.; Carrillo, Maria Berrocal; Evans, W E; Klein, T E

    2011-01-01

    Thiopurine methyltransferase (TPMT) activity exhibits monogenic co-dominant inheritance, with ethnic differences in the frequency of occurrence of variant alleles. With conventional thiopurine doses, homozygous TPMT-deficient patients (~1 in 178 to 1 in 3,736 individuals with two nonfunctional TP...... recommendations (updates at http://www.pharmgkb.org) for azathioprine, mercaptopurine (MP), and thioguanine based on TPMT genotype....

  1. Tyrosine 87 is vital for the activity of human protein arginine methyltransferase 3 (PRMT3)

    Czech Academy of Sciences Publication Activity Database

    Handrková, H.; Petrák, J.; Halada, Petr; Pospíšilová, D.; Čmejla, R.

    2011-01-01

    Roč. 1814, č. 2 (2011), s. 277-282. ISSN 1570-9639 R&D Projects: GA MŠk LC07017 Institutional research plan: CEZ:AV0Z50200510 Keywords : DIAMOND-BLACKFAN ANEMIA * SUBSTRATE-SPECIFICITY * N-METHYLTRANSFERASE Subject RIV: CE - Biochemistry Impact factor: 3.635, year: 2011

  2. A mouse speciation gene encodes a meiotic histone H3 methyltransferase

    Czech Academy of Sciences Publication Activity Database

    Mihola, Ondřej; Trachtulec, Zdeněk; Vlček, Čestmír; Schimenti, J.C.; Forejt, Jiří

    2009-01-01

    Roč. 323, č. 5912 (2009), s. 373-375. ISSN 0036-8075 Institutional research plan: CEZ:AV0Z50520514 Keywords : hybrid sterility * histone H3K4 methyltransferase * Prdm9 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 29.747, year: 2009

  3. An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum.

    Directory of Open Access Journals (Sweden)

    Adela S Oliva Chávez

    Full Text Available Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA, is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6 at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4, but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8, the S-adenosine homocystein-bound (PDB_ID:4OA5, the SAH-Mn2+ bound (PDB_ID:4PCA, and SAM- Mn2+ bound (PDB_ID:4PCL X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary

  4. An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum.

    Science.gov (United States)

    Oliva Chávez, Adela S; Fairman, James W; Felsheim, Roderick F; Nelson, Curtis M; Herron, Michael J; Higgins, LeeAnn; Burkhardt, Nicole Y; Oliver, Jonathan D; Markowski, Todd W; Kurtti, Timothy J; Edwards, Thomas E; Munderloh, Ulrike G

    2015-01-01

    Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn2+ bound (PDB_ID:4PCA), and SAM- Mn2+ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations

  5. Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli

    International Nuclear Information System (INIS)

    The 23S rRNA methyltransferase RlmJ from E. coli has been cloned, expressed, purified and crystallized. X-ray diffraction data to 1.85 Å resolution have been collected from the apo RlmJ crystals. Methyltransferase RlmJ uses the cofactor S-adenosylmethionine to methylate the exocyclic nitrogen N6 of nucleotide A2030 in 23S rRNA during ribosome assembly in Escherichia coli. RlmJ with a C-terminal hexahistidine tag was overexpressed in E. coli and purified as a monomer using Ni2+-affinity and size-exclusion chromatography. The recombinant RlmJ was crystallized using the sitting-drop vapour-diffusion method and a full data set was collected to 1.85 Å resolution from a single apo crystal. The crystals belonged to space group P21, with unit-cell parameters a = 46.9, b = 77.8, c = 82.5 Å, β = 104°. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20 Å3 Da−1

  6. Developing new chemical tools for DNA methyltransferase 1 (DNMT 1): a small-molecule activity-based probe and novel tetrazole-containing inhibitors.

    Science.gov (United States)

    Zhu, Biwei; Ge, Jingyan; Yao, Shao Q

    2015-06-15

    DNA methylation is an important epigenetic modification catalyzed by DNA methyltransferases (DNMTs). Abnormal expression of endogenous DNMTs in human causes alterations in the genome methylation patterns which subsequently lead to the development of cancers. Thus detection of endogenous DNMT activities and efficient inhibition of DNMTs have important therapeutic significance. In this work, a small molecule activity-based probe (ABP) of DNA methyltransferase 1 (DNMT1), T1, was developed. The probe was a clickable analog of tryptophan and was able to covalently label endogenous DNMT1 and inhibit its enzymatic activity more effectively than previously known DNMT1 inhibitors (RG108 and its maleimide analog 1149). In addition, we also discovered a new type of small molecule DNMT inhibitors based on tetrazole-containing compounds which were analogs of 1149. Among these compounds, which we called Gn, one of them (G6) possessed reasonable inhibitory activity against DNMT1 in both in vitro enzymatic assays and cell growth proliferation experiments. Both T1 and G6 showed effective labeling of endogenous DNMT1 from mammalian cells by using in vitro competitive pull-down and live-cell bioimaging experiments. PMID:25801160

  7. Hepatitis B virus can be inhibited by DNA methyltransferase 3a via specific zinc-finger-induced methylation of the X promoter.

    Science.gov (United States)

    Xirong, L; Rui, L; Xiaoli, Y; Qiuyan, H; Bikui, T; Sibo, Z; Naishuo, Z

    2014-02-01

    In this work we explored whether DNA methyltransferase 3a (Dnmt3a) targeted to the HBV X promoter (XP) causes epigenetic suppression of hepatitis B virus (HBV). The C-terminus of Dnmt3a (Dnmt3aC) was fused to a six-zinc-finger peptide specific to XP to form a fused DNA methyltransferase (XPDnmt3aC). The binding and methyl-modifying specificity of XPDnmt3aC were verified with an electrophoretic mobility shift assay and methylation-specific PCR, respectively. XP activity and HBV expression were clearly downregulated in HepG2 cells transfected with plasmid pXPDnmt3aC. The injection of XPDnmt3aC into HBV transgenic (TgHBV) mice also showed significant inhibition, leading to low serum HBV surface protein (HBsAg) levels and a reduced viral load. Thus, XPDnmt3aC specifically silenced HBV via site-selective DNA methylation delivered by zinc-finger peptides. This study establishes the foundation of an epigenetic way of controlling HBV-related diseases. PMID:24794726

  8. Identification of Cyproheptadine as an Inhibitor of SET Domain Containing Lysine Methyltransferase 7/9 (Set7/9) That Regulates Estrogen-Dependent Transcription.

    Science.gov (United States)

    Takemoto, Yasushi; Ito, Akihiro; Niwa, Hideaki; Okamura, Mutsumi; Fujiwara, Takashi; Hirano, Tomoya; Handa, Noriko; Umehara, Takashi; Sonoda, Takeshi; Ogawa, Kenji; Tariq, Mohammad; Nishino, Norikazu; Dan, Shingo; Kagechika, Hiroyuki; Yamori, Takao; Yokoyama, Shigeyuki; Yoshida, Minoru

    2016-04-28

    SET domain containing lysine methyltransferase 7/9 (Set7/9), a histone lysine methyltransferase (HMT), also methylates non-histone proteins including estrogen receptor (ER) α. ERα methylation by Set7/9 stabilizes ERα and activates its transcriptional activities, which are involved in the carcinogenesis of breast cancer. We identified cyproheptadine, a clinically approved antiallergy drug, as a Set7/9 inhibitor in a high-throughput screen using a fluorogenic substrate-based HMT assay. Kinetic and X-ray crystallographic analyses revealed that cyproheptadine binds in the substrate-binding pocket of Set7/9 and inhibits its enzymatic activity by competing with the methyl group acceptor. Treatment of human breast cancer cells (MCF7 cells) with cyproheptadine decreased the expression and transcriptional activity of ERα, thereby inhibiting estrogen-dependent cell growth. Our findings suggest that cyproheptadine can be repurposed for breast cancer treatment or used as a starting point for the discovery of an anti-hormone breast cancer drug through lead optimization. PMID:27088648

  9. A new nuclear function of the Entamoeba histolytica glycolytic enzyme enolase: the metabolic regulation of cytosine-5 methyltransferase 2 (Dnmt2 activity.

    Directory of Open Access Journals (Sweden)

    Ayala Tovy

    2010-02-01

    Full Text Available Cytosine-5 methyltransferases of the Dnmt2 family function as DNA and tRNA methyltransferases. Insight into the role and biological significance of Dnmt2 is greatly hampered by a lack of knowledge about its protein interactions. In this report, we address the subject of protein interaction by identifying enolase through a yeast two-hybrid screen as a Dnmt2-binding protein. Enolase, which is known to catalyze the conversion of 2-phosphoglycerate (2-PG to phosphoenolpyruvate (PEP, was shown to have both a cytoplasmatic and a nuclear localization in the parasite Entamoeba histolytica. We discovered that enolase acts as a Dnmt2 inhibitor. This unexpected inhibitory activity was antagonized by 2-PG, which suggests that glucose metabolism controls the non-glycolytic function of enolase. Interestingly, glucose starvation drives enolase to accumulate within the nucleus, which in turn leads to the formation of additional enolase-E.histolytica DNMT2 homolog (Ehmeth complex, and to a significant reduction of the tRNA(Asp methylation in the parasite. The crucial role of enolase as a Dnmt2 inhibitor was also demonstrated in E.histolytica expressing a nuclear localization signal (NLS-fused-enolase. These results establish enolase as the first Dnmt2 interacting protein, and highlight an unexpected role of a glycolytic enzyme in the modulation of Dnmt2 activity.

  10. Type III methyltransferase M.NgoAX from Neisseria gonorrhoeae FA1090 regulates biofilm formation and human cell invasion

    Directory of Open Access Journals (Sweden)

    Agnieszka eKwiatek

    2015-12-01

    Full Text Available Neisseria gonorrhoeae is the etiological factor of the sexually transmitted gonorrhea disease that may lead, under specific conditions, to systemic infections. The gonococcal genome encodes many Restriction Modification (RM systems, which main biological role is to defend the pathogen from potentially harmful foreign DNA. However, RM systems seem also to be involved in several other functions. In this study, we examined the effect of inactivation the N. gonorrhoeae FA1090 ngo0545 gene encoding M.NgoAX methyltransferase on the global gene expression, biofilm formation, interactions with human epithelial host cells and overall bacterial growth. Expression microarrays showed at least a two-fold deregulation of a total of 121 genes in the NgoAX knock-out mutant compared to the wt strain under standard grow conditions. As determined by the assay with crystal violet, the NgoAX knock-out strain formed a slightly larger biofilm biomass per cell than the wt strain (OD570/600 = 13.8  2.24 and 9.35  2.06, respectively. SCLM observations showed that the biofilm formed by the gonococcal ngo0545 gene mutant is more relaxed and dispersed than the one formed by the wt strain. Thickness of the biofilm formed by both strains was 48.3 (14.9 µm for the mutant and 28.6 (4.0 µm for the wt. This more relaxed feature of the biofilm in respect to adhesion and bacterial interactions seems advantageous for pathogenesis of the NgoAX-deficient gonococci at the stage of human epithelial cell invasion. Indeed, the overall adhesion of mutant bacterial cells to human cells was lower than adhesion of the wt gonococci (adhesion index = 0.672 ( 0.2 and 2.15 ( 1.53, respectively; yet, a higher number of mutant than wt bacteria were found inside the Hec-1-B epithelial cells (invasion index = 3.38 ( 0.93  105 for mutant and 4.67 ( 3.09  104 for the wt strain. These results indicate that NgoAX-deficient cells have lower ability to attach to human cells

  11. Physiology and Posttranscriptional Regulation of Methanol:Coenzyme M Methyltransferase Isozymes in Methanosarcina acetivorans C2A ▿ §

    OpenAIRE

    Opulencia, Rina B.; Bose, Arpita; Metcalf, William W.

    2009-01-01

    Methanosarcina species possess three operons (mtaCB1, mtaCB2, and mtaCB3) encoding methanol-specific methyltransferase 1 (MT1) isozymes and two genes (mtaA1 and mtaA2) with the potential to encode a methanol-specific methyltransferase 2 (MT2). Previous genetic studies showed that these genes are differentially regulated and encode enzymes with distinct levels of methyltransferase activity. Here, the effects of promoter strength on growth and on the rate of methane production were examined by ...

  12. Systematic analysis of O-methyltransferase gene family and identification of potential members involved in the formation of O-methylated flavonoids in Citrus.

    Science.gov (United States)

    Liu, Xiaogang; Luo, Yan; Wu, Hongkun; Xi, Wanpeng; Yu, Jie; Zhang, Qiuyun; Zhou, Zhiqin

    2016-01-10

    The O-methylation of various secondary metabolites is mainly catalyzed by S-adenosyl-l-methionine (SAM)-dependent O-methyltransferase (OMT) proteins that are encoded by the O-methyltransferase gene family. Citrus fruits are a rich source of O-methylated flavonoids that have a broad spectrum of biological activities, including anti-inflammatory, anticarcinogenic, and antiatherogenic properties. However, little is known about this gene family and its members that are involved in the O-methylation of flavonoids and their regulation in Citrus. In this study, 58 OMT genes were identified from the entire Citrus sinensis genome and compared with those from 3 other representative dicot plants. A comprehensive analysis was performed, including functional/substrate predictions, identification of chromosomal locations, phylogenetic relationships, gene structures, and conserved motifs. Distribution mapping revealed that the 58 OMT genes were unevenly distributed on the 9 citrus chromosomes. Phylogenetic analysis of 164 OMT proteins from C.sinensis, Arabidopsis thaliana, Populus trichocarpa, and Vitis vinifera showed that these proteins were categorized into group I (COMT subfamily) and group II (CCoAOMT subfamily), which were further divided into 10 and 2 subgroups, respectively. Finally, digital gene expression and quantitative real-time polymerase chain reaction analyses revealed that citrus OMT genes had distinct temporal and spatial expression patterns in different tissues and developmental stages. Interestingly, 18 and 11 of the 27 genes predicted to be involved in O-methylation of flavonoids had higher expression in the peel and pulp during fruit development, respectively. The citrus OMT gene family identified in this study might help in the selection of appropriate candidate genes and facilitate functional studies in Citrus. PMID:26407870

  13. GRIP1-associated SET-domain methyltransferase in glucocorticoid receptor target gene expression

    OpenAIRE

    Chinenov, Yurii; Sacta, Maria A.; Cruz, Anna R.; Rogatsky, Inez

    2008-01-01

    Transcriptional regulators such as the glucocorticoid receptor (GR) recruit multiple cofactors to activate or repress transcription. Although most cofactors are intrinsically bifunctional, little is known about the molecular mechanisms dictating the specific polarity of regulation. Furthermore, chromatin modifications thought to be confined to silent loci appear in actively transcribed genes suggesting that similar enzymatic activities may mediate constitutive and transient chromatin states. ...

  14. Arsenic Methylation in Arabidopsis thaliana Expressing an Algal Arsenite Methyltransferase Gene Increases Arsenic Phytotoxicity.

    Science.gov (United States)

    Tang, Zhong; Lv, Yanling; Chen, Fei; Zhang, Wenwen; Rosen, Barry P; Zhao, Fang-Jie

    2016-04-01

    Arsenic (As) contamination in soil can lead to elevated transfer of As to the food chain. One potential mitigation strategy is to genetically engineer plants to enable them to transform inorganic As to methylated and volatile As species. In this study, we genetically engineered two ecotypes of Arabidopsis thaliana with the arsenite (As(III)) S-adenosylmethyltransferase (arsM) gene from the eukaryotic alga Chlamydomonas reinhardtii. The transgenic A. thaliana plants gained a strong ability to methylate As, converting most of the inorganic As into dimethylarsenate [DMA(V)] in the shoots. Small amounts of volatile As were detected from the transgenic plants. However, the transgenic plants became more sensitive to As(III) in the medium, suggesting that DMA(V) is more phytotoxic than inorganic As. The study demonstrates a negative consequence of engineered As methylation in plants and points to a need for arsM genes with a strong ability to methylate As to volatile species. PMID:26998776

  15. YccW is the m5C methyltransferase specific for 23S rRNA nucleotide 1962

    DEFF Research Database (Denmark)

    Purta, Elzbieta; O'Connor, Michelle; Bujnicki, Janusz M;

    2008-01-01

    . However, YccW does not methylate assembled 50S subunits, and this is somewhat surprising as the published crystal structures show nucleotide C1962 to be fully accessible at the subunit interface. YccW-directed methylation at nucleotide C1962 is conserved in bacteria, and loss of this methylation in E....... coli marginally reduces its growth rate. YccW had previously eluded identification because it displays only limited sequence similarity to the m(5)C methyltransferases RsmB and RsmF and is in fact more similar to known m(5)U (5-methyluridine) RNA methyltransferases. In keeping with the previously...... proposed nomenclature system for bacterial rRNA methyltransferases, yccW is now designated as the rRNA large subunit methyltransferase gene rlmI....

  16. N-6-Adenine-Specific DNA Methyltransferase 1 (N6AMT1) Polymorphisms and Arsenic Methylation in Andean Women

    OpenAIRE

    Harari, Florencia; Engström, Karin; Concha, Gabriela; Colque, Graciela; Vahter, Marie; Broberg, Karin

    2013-01-01

    BACKGROUND: In humans, inorganic arsenic is metabolized to methylated metabolites mainly by arsenic (+3 oxidation state) methyltransferase (AS3MT). AS3MT polymorphisms are associated with arsenic metabolism efficiency. Recently, a putative N-6-adenine-specific DNA methyltransferase 1 (N6AMT1) was found to methylate arsenic in vitro. OBJECTIVE: We evaluated the role of N6AMT1 polymorphisms in arsenic methylation efficiency in humans. METHODS: We assessed arsenic methylation efficiency in 188 w...

  17. The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia

    OpenAIRE

    Pappano, William N.; Guo, Jun; He, Yupeng; Ferguson, Debra; Jagadeeswaran, Sujatha; Osterling, Donald J.; Gao, Wenqing; Spence, Julie K.; Pliushchev, Marina; Sweis, Ramzi F.; Buchanan, Fritz G.; Michaelides, Michael R.; Shoemaker, Alexander R.; Tse, Chris; Chiang, Gary G.

    2015-01-01

    Histone methyltransferases are epigenetic regulators that modify key lysine and arginine residues on histones and are believed to play an important role in cancer development and maintenance. These epigenetic modifications are potentially reversible and as a result this class of enzymes has drawn great interest as potential therapeutic targets of small molecule inhibitors. Previous studies have suggested that the histone lysine methyltransferase G9a (EHMT2) is required to perpetuate malignant...

  18. Investigating the potential role of genetic and epigenetic variation of DNA methyltransferase genes in hyperplastic polyposis syndrome.

    Directory of Open Access Journals (Sweden)

    Musa Drini

    Full Text Available BACKGROUND: Hyperplastic Polyposis Syndrome (HPS is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC. At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP, potentially linked to aberrant DNA methyltransferase (DNMT activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT, and negative regulators of Wnt signaling (such as WIF1. In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS. METHODS: We utilized high resolution melting (HRM analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters. RESULTS: No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene was over-represented in cases with HPS (p<0.01 compared with population controls. The DNMT1, DNMT3A and DNMT3B promoters were unmethylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells with methylation level negatively correlated to expression level in normal colonic tissue. DNMT3L promoter hypomethylation was more often found in polyps harbouring KRAS mutations (p = 0.0053. BRAF mutations were common (11 out of 21 polyps, whilst KRAS mutations were identified in 4 of 21 polyps. CONCLUSIONS: Genetic or epigenetic alterations in DNMT genes do not appear to be associated with HPS, but further investigation of genetic variation at rs62106244 is justified given the high frequency of the minor allele in this case series.

  19. Protein repair L-isoaspartyl methyltransferase 1 (PIMT1) in rice improves seed longevity by preserving embryo vigor and viability.

    Science.gov (United States)

    Wei, Yidong; Xu, Huibin; Diao, Lirong; Zhu, Yongsheng; Xie, Hongguang; Cai, Qiuhua; Wu, Fangxi; Wang, Zonghua; Zhang, Jianfu; Xie, Huaan

    2015-11-01

    Damaged proteins containing abnormal isoaspartyl (isoAsp) accumulate as seeds age and the abnormality is thought to undermine seed vigor. Protein-L-isoaspartyl methyltransferase (PIMT) is involved in isoAsp-containing protein repair. Two PIMT genes from rice (Oryza sativa L.), designated as OsPIMT1 and OsPIMT2, were isolated and investigated for their roles. The results indicated that OsPIMT2 was mainly present in green tissues, but OsPIMT1 largely accumulated in embryos. Confocal visualization of the transient expression of OsPIMTs showed that OsPIMT2 was localized in the chloroplast and nucleus, whereas OsPIMT1 was predominately found in the cytosol. Artificial aging results highlighted the sensitivity of the seeds of OsPIMT1 mutant line when subjected to accelerated aging. Overexpression of OsPIMT1 in transgenic seeds reduced the accumulation of isoAsp-containing protein in embryos, and increased embryo viability. The germination percentage of transgenic seeds overexpressing OsPIMT1 increased 9-15% compared to the WT seeds after 21-day of artificial aging, whereas seeds from the OsPIMT1 RNAi lines overaccumulated isoAsp in embryos and experienced rapid loss of seed germinability. Taken together, these data strongly indicated that OsPIMT1-related seed longevity improvement is probably due to the repair of detrimental isoAsp-containing proteins that over accumulate in embryos when subjected to accelerated aging. PMID:26438231

  20. The DNA methyltransferase inhibitor zebularine induces mitochondria-mediated apoptosis in gastric cancer cells in vitro and in vivo

    International Nuclear Information System (INIS)

    Highlights: ► Zebularine inhibited cell growth of gastric cancer in a time- and dose-dependent manner. ► Chromatin condensation and nuclear fragmentation were induced. ► Zebularine promoted apoptosis via mitochondrial pathways. ► Tumorigenicity was inhibited by zebularine. -- Abstract: DNA methyltransferase (DNMT) inhibitor zebularine has been reported to potentiate the anti-tumor effect by reactivating the expression of tumor suppressor genes and apoptosis-related genes in various malignant cells. However, the apoptotic signaling pathway in gastric cancer cells induced by zebularine is not well understood. In the study, the effects of zebularine on the growth and apoptosis of gastric cancer cells were investigated by MTT assay, Hoechst assay, Western blot analysis, flow cytometric analysis of annexin V-FITC/PI staining, and TUNEL assay. Zebularine was an effective inhibitor of human gastric cancer cells proliferation in vitro and in vivo. The effects were dose dependent. A zebularine concentration of 50 μM accounted for the inhibition of cell proliferation of 67% at 48 h. The treatment with zebularine upregulated Bax, and decreased Bcl-2 protein. Caspase-3 was activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, zebularine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay in xenograft tumor mouse model. These results demonstrated that zebularine induced apoptosis in gastric cancer cells via mitochondrial pathways, and zebularine might become a therapeutic approach for the treatment of gastric cancer.

  1. Molecular Cloning and Functional Characterization of a Novel Isoflavone 3′-O-methyltransferase from Pueraria lobata

    Science.gov (United States)

    Li, Jia; Li, Changfu; Gou, Junbo; Zhang, Yansheng

    2016-01-01

    Pueraria lobata roots accumulate 3′-, 4′- and 7-O-methylated isoflavones and many of these methylated compounds exhibit various pharmacological activities. Either the 4′- or 7-O-methylation activity has been investigated at molecular levels in several legume species. However, the gene encoding the isoflavone 3′-O-methyltransferase (OMT) has not yet been isolated from any plant species. In this study, we reported the first cDNA encoding the isoflavone 3′-OMT from P. lobata (designated PlOMT4). Heterologous expressions in yeast and Escherichia coli cells showed that the gene product exhibits an enzyme activity to methylate the 3′-hydroxy group of the isoflavone substrate. The transcript abundance of PlOMT4 matches well with its enzymatic product in different organs of P. lobata and in the plant roots in response to methyl jasmonate elicitation. Integration of the biochemical with metabolic and transcript data supported the proposed function of PlOMT4. The identification of PlOMT4 would not only help to understand the isoflavonoid metabolism in P. lobata but also potentially provide an enzyme catalyst for methylating existing drug candidates to improve their hydrophobicity.

  2. Association study between the rs165599 catechol-O-methyltransferase genetic polymorphism and schizophrenia in a Brazilian sample

    Directory of Open Access Journals (Sweden)

    Quirino Cordeiro

    2012-12-01

    Full Text Available Schizophrenia is a severe psychiatric disorder with frequent recurrent psychotic relapses and progressive functional impairment. It results from a poorly understood gene-environment interaction. The gene encoding catechol-O-methyltransferase (COMT is a likely candidate for schizophrenia. Its rs165599 (A/G polymorphism has been shown to be associated with alteration of COMT gene expression. Therefore, the present study aimed to investigate a possible association between schizophrenia and this polymorphism. The distribution of the alleles and genotypes of this polymorphism was investigated in a Brazilian sample of 245 patients and 834 controls. The genotypic frequencies were in Hardy-Weinberg equilibrium and no statistically significant differences were found between cases and controls when analyzed according to gender or schizophrenia subtypes. There was also no difference in homozygosis between cases and controls. Thus, in the sample studied, there was no evidence of any association between schizophrenia and rs165599 (A/G polymorphism in the non-coding region 3' of the COMT gene.

  3. Posttranslational Regulation of O(6)-Methylguanine-DNA Methyltransferase (MGMT) and New Opportunities for Treatment of Brain Cancers.

    Science.gov (United States)

    Srivenugopal, Kalkunte S; Rawat, Amit; Niture, Suryakant K; Paranjpe, Ameya; Velu, Chinavenmani; Venugopal, Sanjay N; Madala, Hanumantha Rao; Basak, Debasish; Punganuru, Surendra R

    2016-01-01

    O(6)-Methylguanine-DNA-methyltransferase (MGMT) is an antimutagenic DNA repair protein highly expressed in human brain tumors. Because MGMT repairs the mutagenic, carcinogenic and cytotoxic O(6)-alkylguanine adducts, including those generated by the clinically used anticancer alkylating agents, it has emerged as a central and rational target for overcoming tumor resistance to alkylating agents. Although the pseudosubstrates for MGMT [O(6)-benzylguanine, O(6)-(4- bromothenyl)guanine] have gained attention as powerful and clinically-relevant inhibitors, bone marrow suppression due to excessive alkylation damage has diminished this strategy. Our laboratory has been working on various posttranslational modifications of MGMT that affect its protein stability, DNA repair activity and response to oxidative stress. While these modifications greatly impact the physiological regulation of MGMT, they also highlight the opportunities for inactivating DNA repair and new drug discovery in this specific area. This review briefly describes the newer aspects of MGMT posttranslational regulation by ubiquitination, sumoylation and glutathionylation and reveals how the reactivity of the active site Cys145 can be exploited for potent inhibition and depletion of MGMT by thiol-reacting drugs such as the disulfiram and various dithiocarbamate derivatives. The possible repurposing of these nontoxic and safe drugs for improved therapy of pediatric and adult brain tumors is discussed. PMID:26202203

  4. Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/γ-globin looping.

    Science.gov (United States)

    Krivega, Ivan; Byrnes, Colleen; de Vasconcellos, Jaira F; Lee, Y Terry; Kaushal, Megha; Dean, Ann; Miller, Jeffery L

    2015-07-30

    Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and β-thalassemia. Transcription of β-globin genes is regulated by the distant locus control region (LCR), which is brought into direct gene contact by the LDB1/GATA-1/TAL1/LMO2-containing complex. Inhibition of G9a H3K9 methyltransferase by the chemical compound UNC0638 activates fetal and represses adult β-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on β-globin gene expression using ex vivo differentiation of CD34(+) erythroid progenitor cells from peripheral blood of healthy adult donors. UNC0638 inhibition of G9a caused dosed accumulation of HbF up to 30% of total hemoglobin in differentiated cells. Elevation of HbF was associated with significant activation of fetal γ-globin and repression of adult β-globin transcription. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus and gain of LDB1 complex occupancy at the γ-globin promoters as well as de novo formation of LCR/γ-globin contacts. Our findings demonstrate that G9a establishes epigenetic conditions preventing activation of γ-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of β-hemoglobinopathies. PMID:25979948

  5. Inhibition of H3K9 methyltransferase G9a induces autophagy and apoptosis in oral squamous cell carcinoma

    International Nuclear Information System (INIS)

    Objective: To explore whether inhibition of H3K9 Methyltransferase G9a could exert an antitumoral effect in oral squamous cell carcinoma (OSCC). Materials and methods: First we checked G9a expression in two OSCC cell lines Tca8113 and KB. Next we used a special G9a inhibitor BIX01294 (BIX) to explore the effect of inhibition of G9a on OSCC in vitro. Cell growth was tested by typlan blue staining, MTT assay and Brdu immunofluorescence staining. Cell autophagy was examined by monodansylcadaverine (MDC) staining, LC3-II immunofluorescence staining and LC3-II western blot assay. Cell apoptosis was checked by FITC Annexin-V and PI labeling, tunnel staining and caspase 3 western blot assay. Finally, the effect of inhibition of G9a on clonogenesis and tumorigenesis capacity of OSCC was analyzed by soft agar growth and xenograft model. Results: Here we showed that G9a was expressed in both Tca8113 and KB cells. Inhibition of G9a using BIX significantly reduced cell growth and proliferation in Tca8113 and KB. Inhibition of G9a induced cell autophagy with conversion of LC3-I to LC3-II and cell apoptosis with the expression of cleaved caspase 3. We also found that inhibition of G9a reduced colony formation in soft agar and repressed tumor growth in mouse xenograph model. Conclusion: Our results suggested that G9a might be a potential epigenetic target for OSCC treatment. - Highlights: • Inhibition of G9a reduced cell growth and proliferation in OSCC cells. • Inhibition of G9a induces autophagy and apoptosis in OSCC cells. • Inhibition of G9a repressed tumor growth in mouse xenograph model

  6. Inhibition of H3K9 methyltransferase G9a induces autophagy and apoptosis in oral squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Aishu; Qiu, Yu [Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing Medical University, Chongqing, 401147 (China); Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing, 401147 (China); Cui, Hongjuan [State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716 (China); Fu, Gang, E-mail: fg.ras@hotmail.com [Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing Medical University, Chongqing, 401147 (China); Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing, 401147 (China)

    2015-03-27

    Objective: To explore whether inhibition of H3K9 Methyltransferase G9a could exert an antitumoral effect in oral squamous cell carcinoma (OSCC). Materials and methods: First we checked G9a expression in two OSCC cell lines Tca8113 and KB. Next we used a special G9a inhibitor BIX01294 (BIX) to explore the effect of inhibition of G9a on OSCC in vitro. Cell growth was tested by typlan blue staining, MTT assay and Brdu immunofluorescence staining. Cell autophagy was examined by monodansylcadaverine (MDC) staining, LC3-II immunofluorescence staining and LC3-II western blot assay. Cell apoptosis was checked by FITC Annexin-V and PI labeling, tunnel staining and caspase 3 western blot assay. Finally, the effect of inhibition of G9a on clonogenesis and tumorigenesis capacity of OSCC was analyzed by soft agar growth and xenograft model. Results: Here we showed that G9a was expressed in both Tca8113 and KB cells. Inhibition of G9a using BIX significantly reduced cell growth and proliferation in Tca8113 and KB. Inhibition of G9a induced cell autophagy with conversion of LC3-I to LC3-II and cell apoptosis with the expression of cleaved caspase 3. We also found that inhibition of G9a reduced colony formation in soft agar and repressed tumor growth in mouse xenograph model. Conclusion: Our results suggested that G9a might be a potential epigenetic target for OSCC treatment. - Highlights: • Inhibition of G9a reduced cell growth and proliferation in OSCC cells. • Inhibition of G9a induces autophagy and apoptosis in OSCC cells. • Inhibition of G9a repressed tumor growth in mouse xenograph model.

  7. Structural, Biochemical, and Phylogenetic Analyses Suggest That Indole-3-Acetic Acid Methyltransferase Is an Evolutionarily Ancient Member of the SABATH Family

    Energy Technology Data Exchange (ETDEWEB)

    Zhao,N.; Ferrer, J.; Ross, J.; Guan, J.; Yang, Y.; Pichersky, E.; Noel, J.; Chen, F.

    2008-01-01

    The plant SABATH protein family encompasses a group of related small-molecule methyltransferases (MTs) that catalyze the S-adenosyl-L-methionine-dependent methylation of natural chemicals encompassing widely divergent structures. Indole-3-acetic acid (IAA) methyltransferase (IAMT) is a member of the SABATH family that modulates IAA homeostasis in plant tissues through methylation of IAA's free carboxyl group. The crystal structure of Arabidopsis (Arabidopsis thaliana) IAMT (AtIAMT1) was determined and refined to 2.75 Angstroms resolution. The overall tertiary and quaternary structures closely resemble the two-domain bilobed monomer and the dimeric arrangement, respectively, previously observed for the related salicylic acid carboxyl methyltransferase from Clarkia breweri (CbSAMT). To further our understanding of the biological function and evolution of SABATHs, especially of IAMT, we analyzed the SABATH gene family in the rice (Oryza sativa) genome. Forty-one OsSABATH genes were identified. Expression analysis showed that more than one-half of the OsSABATH genes were transcribed in one or multiple organs. The OsSABATH gene most similar to AtIAMT1 is OsSABATH4. Escherichia coli-expressed OsSABATH4 protein displayed the highest level of catalytic activity toward IAA and was therefore named OsIAMT1. OsIAMT1 exhibited kinetic properties similar to AtIAMT1 and poplar IAMT (PtIAMT1). Structural modeling of OsIAMT1 and PtIAMT1 using the experimentally determined structure of AtIAMT1 reported here as a template revealed conserved structural features of IAMTs within the active-site cavity that are divergent from functionally distinct members of the SABATH family, such as CbSAMT. Phylogenetic analysis revealed that IAMTs from Arabidopsis, rice, and poplar (Populus spp.) form a monophyletic group. Thus, structural, biochemical, and phylogenetic evidence supports the hypothesis that IAMT is an evolutionarily ancient member of the SABATH family likely to play a critical

  8. Increase of O6-methylguanine-DNA-methyltransferase and N3-methyladenine glycosylase RNA transcripts in rat hepatoma cells treated with DNA-damaging agents

    International Nuclear Information System (INIS)

    A variety of DNA-damaging agents increase the O6-methylguanine-DNA-methyltransferase (transferase) and the N3-methyladenine (3-meAde)-DNA-glycosylase activities in a rat hepatoma cell line (H4 cells). Using two cDNA expressing either the rat 3-meAde-DNA-glycosylase or the transferase, the level of mRNA transcripts was measured by hybridization in H4 cells treated with three different inducing agents, gamma-rays, cis-dichlorodiammine platinum II or N-methyl-9-hydroxy ellipticinium. The two mRNA increased 24 hours after the cell treatments but this enhanced transcription was a transient phenomenon, as it was no longer observed after 96 hours. No significant DNA amplification was detectable in the treated cells

  9. Hypomethylation of ERVs in the sperm of mice haploinsufficient for the histone methyltransferase Setdb1 correlates with a paternal effect on phenotype.

    Science.gov (United States)

    Daxinger, Lucia; Oey, Harald; Isbel, Luke; Whitelaw, Nadia C; Youngson, Neil A; Spurling, Alex; Vonk, Kelly K D; Whitelaw, Emma

    2016-01-01

    The number of reports of paternal epigenetic influences on the phenotype of offspring in rodents is increasing but the molecular events involved remain unclear. Here, we show that haploinsufficiency for the histone 3 lysine 9 methyltransferase Setdb1 in the sire can influence the coat colour phenotype of wild type offspring. This effect occurs when the allele that directly drives coat colour is inherited from the dam, inferring that the effect involves an "in trans" step. The implication of this finding is that epigenetic state of the sperm can alter the expression of genes inherited on the maternally derived chromosomes. Whole genome bisulphite sequencing revealed that Setdb1 mutant mice show DNA hypomethylation at specific classes of transposable elements in the sperm. Our results identify Setdb1 as a paternal effect gene in the mouse and suggest that epigenetic inheritance may be more likely in individuals with altered levels of epigenetic modifiers. PMID:27112447

  10. Discovery of sphingosine 1-O-methyltransferase in rat kidney and liver homogenates

    Institute of Scientific and Technical Information of China (English)

    Santosh J SACKET; Dong-soon IM

    2008-01-01

    Aim:To characterize sphingosine methyltransferase in rat tissues.Methods:By using S-adenosyl-L-(methyl-3H) methionine,enzymatic activity was measured in the rat liver and kidney homogenates.Results:The optimum pH and reaction time for the enzyme assay were pH 7.8 and 1 h.ZnCl2 inhibited the activity,but not MgCl2,CaCl2,CoCl2,or NiCl2.In the kidney homogenate,enzymatic activity was detectable in the cytosol and all membrane fractions from the plasma membrane and other organelles; however,in the liver homogenate,enzymatic activity was detectable in all membrane fractions,but not in the cytosol.We also tested the enzymatic activity with structurally-modified sphingosine derivatives.Conclusion:We found sphingosine l-O-methyltransferase activity in the rat liver and kidney homogenates.

  11. Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

    DEFF Research Database (Denmark)

    Vranken, Charlotte; Deen, Jochem; Dirix, Lieve;

    2014-01-01

    We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore...... to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay...... the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes...

  12. Crystallization and preliminary X-ray diffraction studies of a catechol-O-methyltransferase/inhibitor complex

    International Nuclear Information System (INIS)

    Catechol-O-methyltransferase has been co-crystallized with a novel inhibitor, which has potential therapeutic application in the Parkinson’s disease therapy. Inhibitors of the enzyme catechol-O-methyltransferase (COMT) are used as co-adjuvants in the therapy of Parkinson’s disease. A recombinant form of the soluble cytosolic COMT from rat has been co-crystallized with a new potent inhibitor, BIA 8-176 [(3,4-dihydroxy-2-nitrophenyl)phenylmethanone], by the vapour-diffusion method using PEG 6K as precipitant. Crystals diffract to 1.6 Å resolution on a synchrotron-radiation source and belong to the monoclinic space group P21, with unit-cell parameters a = 52.77, b = 79.63, c = 61.54 Å, β = 91.14°

  13. Crystallization and preliminary X-ray diffraction studies of a catechol-O-methyltransferase/inhibitor complex

    Energy Technology Data Exchange (ETDEWEB)

    Rodrigues, M. L. [Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. República, Apt. 127, 2781-901 Oeiras (Portugal); Bonifácio, M. J.; Soares-da-Silva, P. [Department of Research and Development, BIAL, 4785 S. Mamede do Coronado (Portugal); Carrondo, M. A.; Archer, M., E-mail: archer@itqb.unl.pt [Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. República, Apt. 127, 2781-901 Oeiras (Portugal)

    2005-01-01

    Catechol-O-methyltransferase has been co-crystallized with a novel inhibitor, which has potential therapeutic application in the Parkinson’s disease therapy. Inhibitors of the enzyme catechol-O-methyltransferase (COMT) are used as co-adjuvants in the therapy of Parkinson’s disease. A recombinant form of the soluble cytosolic COMT from rat has been co-crystallized with a new potent inhibitor, BIA 8-176 [(3,4-dihydroxy-2-nitrophenyl)phenylmethanone], by the vapour-diffusion method using PEG 6K as precipitant. Crystals diffract to 1.6 Å resolution on a synchrotron-radiation source and belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 52.77, b = 79.63, c = 61.54 Å, β = 91.14°.

  14. Structure and possible mechanism of the CcbJ methyltransferase from Streptomyces caelestis

    Czech Academy of Sciences Publication Activity Database

    Bauer, J.; Ondrovičová, G.; Najmanová, Lucie; Pevala, V.; Kameník, Zdeněk; Koštan, J.; Janata, Jiří; Kutejová, Eva

    2014-01-01

    Roč. 70, APR 2014 (2014), s. 943-957. ISSN 0907-4449 R&D Projects: GA MŠk(CZ) EE2.3.30.0003; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : CATECHOL-O-METHYLTRANSFERASE * SN2-LIKE TRANSITION-STATE * CRYSTAL-STRUCTURES Subject RIV: CE - Biochemistry Impact factor: 7.232, year: 2013

  15. Identification, Characterization, and Ontogenic Study of a Catechol O-methyltransferase from Zebrafish

    OpenAIRE

    Alazizi, Adnan; Liu, Ming-Yih; Williams, Frederick E.; Kurogi, Katsuhisa; Sakakibara, Yoichi; Suiko, Masahito; Liu, Ming-Cheh

    2010-01-01

    To establish the zebrafish as a model for investigating the methylation pathway of drug metabolism, we embarked on the molecular cloning of the zebrafish catechol O-methyltransferase (COMT). By searching the GenBank database, a zebrafish nucleotide sequence encoding a putative COMT was identified. Based on the sequence information, we designed and synthesized oligonucleotides corresponding to its 5’- and 3’-coding regions of this zebrafish COMT. Using the first-strand cDNA reverse-transcribed...

  16. Survival and tumorigenesis in O6-methylguanine DNA methyltransferase-deficient mice following cyclophosphamide exposure

    OpenAIRE

    Nagasubramanian, Ramamoorthy; Hansen, Ryan J.; Delaney, Shannon M.; Cherian, Mathew M.; Samson, Leona D.; Kogan, Scott C.; Dolan, M. Eileen

    2008-01-01

    O6-methylguanine DNA methyltransferase (MGMT) deficiency is associated with an increased susceptibility to alkylating agent toxicity. To understand the contribution of MGMT in protecting against cyclophosphamide (CP)-induced toxicity, mutagenesis and tumorigenesis, we compared the biological effects of this agent in transgenic Mgmt knockout and wild-type mice. In addition, neurofibromin (Nf1)+/− background was used to increase the likelihood of CP-induced tumorigenesis. Cohorts of Mgmt-profic...

  17. Somatic mutations in glioblastoma are associated with methylguanine-DNA methyltransferase methylation

    OpenAIRE

    McDonald, Kerrie L.; Tabone, Tania; Nowak, Anna K; Erber, Wendy N.

    2015-01-01

    The high level of methylguanine-DNA methyltransferase (MGMT) in glioblastoma is responsible for resistance to alkylating agents, such as temozolomide (TMZ). In glioblastomas with a methylated MGMT promoter, MGMT deficiency is presumed, resulting in an enhanced effect of TMZ. The aim of the present study was to investigate whether genomic alterations work synergistically with MGMT methylation status and contribute to the response to treatment and overall prognosis in glioblastoma. The current ...

  18. HISTONE H3 LYSINE 9 METHYLTRANSFERASE G9a IS A TRANSCRIPTIONAL COACTIVATOR FOR NUCLEAR RECEPTORS*

    OpenAIRE

    David Y. Lee; Northrop, Jeffrey P.; Kuo, Min-Hao; Stallcup, Michael R.

    2006-01-01

    Methylation of Lys-9 of histone H3 has been associated with repression of transcription. G9a is a histone H3 Lys-9 methyltransferase localized in euchromatin and acts as a corepressor for specific transcription factors. Here we demonstrate that G9a also functions as a coactivator for nuclear receptors, cooperating synergistically with nuclear receptor coactivators GRIP1, CARM1, and p300 in transient transfection assays. This synergy depends strongly on the arginine-specific protein methyltran...

  19. The methyltransferase G9a regulates HoxA9-dependent transcription in AML

    OpenAIRE

    Lehnertz, Bernhard; Pabst, Caroline; Su, Le; Miller, Michelle; Liu, Feng; Yi, Lin; Zhang, Regan; Krosl, Jana; Yung, Eric; Kirschner, Jeanette; Rosten, Patty; Underhill, T. Michael; Jin, Jian; Hébert, Josée; Sauvageau, Guy

    2014-01-01

    Lehnertz et al. identify the histone methyltransferase G9a as a selective regulator of fast proliferating myeloid progenitors with no discernible function in hematopoietic stem cells. Loss of G9a significantly delays disease progression and reduces leukemia stem cell frequency in mouse models of acute myeloid leukemia. G9a interacts with the leukemogenic transcription factor HoxA9 and regulates HoxA9-dependent transcription. These results highlight G9a inhibition as a means to counteract the ...

  20. CCAAT displacement protein/cut homolog recruits G9a histone lysine methyltransferase to repress transcription

    OpenAIRE

    Nishio, Hitomi; Walsh, Martin J.

    2004-01-01

    CCAAT displacement protein/cut homolog (CDP/cut) is a highly conserved homeodomain protein that contains three cut repeat sequences. CDP/cut is a transcriptional factor for many diverse cellular and viral genes that are involved in most cellular processes, including differentiation, development, and proliferation. Here, we report that CDP/cut interacts with a histone lysine methyltransferase (HKMT), G9a, in vivo and in vitro. The deletion of the cut repeats within CDP/cut abrogates the intera...

  1. Negative Regulation of JAK2 by H3K9 Methyltransferase G9a in Leukemia

    OpenAIRE

    Son, Hye-Ju; Kim, Ji-Young; Hahn, Yoonsoo; Seo, Sang-Beom

    2012-01-01

    Histone methylation at specific lysine residues is a crucial regulatory process in transcriptional regulation. Using chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis, we found that the H3K9-me2 target gene JAK2 was an important factor during differentiation of the HL-60 promyelocytic leukemia cell line by all-trans-retinoic acid (ATRA) treatment. Here, we report that the H3K9 methyltransferase G9a negatively regulated JAK2 transcription in histone methyltransferas...

  2. Three-dimensional culture sensitizes epithelial ovarian cancer cells to EZH2 methyltransferase inhibition

    Science.gov (United States)

    Amatangelo, Michael D.; Garipov, Azat; Li, Hua; Conejo-Garcia, Jose R.; Speicher, David W.; Zhang, Rugang

    2013-01-01

    Inhibitors of EZH2 methyltransferase activity have been demonstrated to selectively suppress the growth of diffused large B cell lymphoma (DLBCL) cells with gain-of-function mutations in EZH2, while exhibiting very limited effects on the growth of DLBCL cells with wild-type EZH2. Given that EZH2 is often overexpressed but not mutated in solid tumors, it is important to investigate the determinants of sensitivity of solid tumor cells to EZH2 inhibitors. In the current study, we show that three-dimensional (3D) culture of epithelial ovarian cancer (EOC) cells that overexpress EZH2 sensitizes these cells to EZH2 methyltransferase inhibition. Treatment of EOC cells with GSK343, a specific inhibitor of EZH2 methyltransferase, decreases the level of H3K27Me3, the product of EZH2’s enzymatic activity. However, GSK343 exhibited limited effects on the growth of EOC cells in conventional two-dimensional (2D) culture. In contrast, GSK343 significantly suppressed the growth of EOC cells cultured in 3D matrigel extracellular matrix (ECM), which more closely mimics the tumor microenvironment in vivo. Notably, GSK343 induces apoptosis of EOC cells in 3D but not 2D culture. In addition, GSK343 significantly inhibited the invasion of EOC cells. In summary, we show that the 3D ECM sensitizes EOC cells to EZH2 methyltransferase inhibition, which suppresses cell growth, induces apoptosis and inhibits invasion. Our findings imply that in EZH2 wild-type solid tumors, the ECM tumor microenvironment plays an important role in determining sensitivity to EZH2 inhibition and suggest that targeting the ECM represents a novel strategy for enhancing EZH2 inhibitor efficacy. PMID:23759589

  3. Kupplung der DNA-Methyltransferase M.SssI mit Triplehelix-bildenden Oligodesoxynucleotiden

    OpenAIRE

    Monami, Amélie Joséphine

    2007-01-01

    DNA modifying enzymes, like DNA methyltransferases (DNA MTases) and restriction endonucleases (REases), could in principle be used to map or manipulate genomes. However, these enzymes are inappropriate for that purpose because of their generally short recognition sequences. An increase of their sequence specificity would therefore be desirable. One strategy to produce megaspecific DNA modifying enzymes is to couple them with triple helix forming oligodeoxynucleotide (TFO). These additional DN...

  4. Catecholamine-o-methyltransferase polymorphisms are associated with postoperative pain intensity.

    LENUS (Irish Health Repository)

    Lee, Peter J

    2011-02-01

    single nucleotide polymorphisms (SNPs) in the genes for catecholamine-O-methyltransferase (COMT), μ-opioid receptor and GTP cyclohydrolase (GCH1) have been linked to acute and chronic pain states. COMT polymorphisms are associated with experimental pain sensitivity and a chronic pain state. No such association has been identified perioperatively. We carried out a prospective observational clinical trial to examine associations between these parameters and the development of postoperative pain in patients undergoing third molar (M3) extraction.

  5. Identification and Characterization of a Highly Conserved Crenarchaeal Protein Lysine Methyltransferase with Broad Substrate Specificity

    OpenAIRE

    Chu, Yindi; Zhang, Zhenfeng; Wang, Qian; Luo, Yuanming; Huang, Li

    2012-01-01

    Protein lysine methylation occurs extensively in the Crenarchaeota, a major kingdom in the Archaea. However, the enzymes responsible for this type of posttranslational modification have not been found. Here we report the identification and characterization of the first crenarchaeal protein lysine methyltransferase, designated aKMT, from the hyperthermophilic crenarchaeon Sulfolobus islandicus. The enzyme was capable of transferring methyl groups to selected lysine residues in a substrate prot...

  6. Genetic, physiological and biochemical characterization of multiple methanol methyltransferase isozymes in Methanosarcina acetivorans C2A.

    Science.gov (United States)

    Pritchett, Matthew A; Metcalf, William W

    2005-06-01

    Biochemical evidence suggests that methanol catabolism in Methanosarcina species requires the concerted effort of methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MtaB), a corrinoid-containing methyl-accepting protein (MtaC) and Co-methyl-5-hydroxybenzimidazolylcobamide:2-mercapto-ethanesulphonic acid methyltransferase (MtaA). Here we show that Methanosarcina acetivorans possesses three operons encoding putative methanol-specific MtaB and corrinoid proteins: mtaCB1, mtaCB2 and mtaCB3. Deletion mutants lacking the three operons, in all possible combinations, were constructed and characterized. Strains deleted for any two of the operons grew on methanol, whereas strains lacking all three did not. Therefore, each operon encodes a bona fide methanol-utilizing MtaB/corrinoid protein pair. Most of the mutants were similar to the wild-type strain, with the exception of the DeltamtaCB1 DeltamtaCB2 double mutant, which grew more slowly and had reduced cell yields on methanol medium. However, all mutants displayed significantly longer lag times when switching from growth on trimethylamine to growth on methanol. This indicates that all three operons are required for wild-type growth on methanol and suggests that each operon has a distinct role in the metabolism of this substrate. The combined methanol:CoM methyltransferase activity of strains carrying only mtaCB1 was twofold higher than strains carrying only mtaCB2 and fourfold higher than strains carrying only mtaCB3. Interestingly, the presence of the mtaCB2 and mtaCB3 operons, in addition to the mtaCB1 operon, did not increase the overall methyltransferase activity, suggesting that these strains may be limited by MtaA availability. All deletion mutants were unaffected with respect to growth on trimethylamine and acetate corroborating biochemical evidence indicating that each methanogenic substrate has specific methyltransfer enzymes. PMID:15882413

  7. Is catechol-o-methyltransferase gene polymorphism a risk factor in the development of premenstrual syndrome?

    OpenAIRE

    Deveci, Esma Ozturk; Incebiyik, Adnan; Selek, Salih; Camuzcuoglu, Aysun; Hilali, Nese Gul; Camuzcuoglu, Hakan; Erdal, Mehmet Emin; Vural, Mehmet

    2014-01-01

    Objective The objective of this study was to investigate whether there was a correlation between catechol-o-methyltransferase (COMT) gene polymorphism, which is believed to play a role in the etiology of psychotic disorders, and premenstrual syndrome (PMS). Methods Fifty-three women with regular menstrual cycles, aged between 18 and 46 years and diagnosed with PMS according to the American Congress of Obstetrics and Gynecology criteria were included in this study as the study group, and 53 he...

  8. Sensorimotor gating of schizophrenia patients depends on catechol O-methyltransferase Val158Met polymorphism

    OpenAIRE

    Quednow, B B; Wagner, M.; Mössner, R; Maier, W; Kühn, K U

    2010-01-01

    It has been recently shown that Catechol O-methyltransferase (COMT) Val(158)Met polymorphism strongly influences prepulse inhibition (PPI) of the acoustic startle response (ASR) in healthy human volunteers. Given that schizophrenia patients exhibit impairment in PPI and that COMT is a putative susceptibility gene for schizophrenia, we investigated the impact of the COMT Val(158)Met polymorphisms on PPI in schizophrenic inpatients. We analyzed COMT Val(158)Met polymorphisms and assessed startl...

  9. Polymorphism of the catechol-O-methyltransferase gene in Han Chinese patients with psoriasis vulgaris

    OpenAIRE

    Lin Gao; Hong Li; Kai Li; Zhu Shen; Ling Liu; Chunying Li; Zhengdong Zhang; Yufeng Liu

    2009-01-01

    Psoriasis vulgaris is defined by a series of linked cellular changes in the skin: hyperplasia of epidermal keratinocytes, vascular hyperplasia and ectasia, and infiltration of T lymphocytes, neutrophils and other types of leukocytes in the affected skin. Catechol-O-methyltransferase ( COMT ) 158 polymorphism can reduce the activity of the COMT enzyme that may trigger defective differentiation of keratinocytes in psoriasis. Immunocytes can degrade and inactivate catecholamines via monamine oxi...

  10. Catechol-O-methyltransferase (COMT) influences the connectivity of the prefrontal cortex at rest

    OpenAIRE

    Tunbridge, Elizabeth M.; Farrell, Sarah M.; Harrison, Paul J; Mackay, Clare E

    2013-01-01

    Catechol-O-methyltransferase (COMT) modulates dopamine in the prefrontal cortex (PFC) and influences PFC dopamine-dependent cognitive task performance. A human COMT polymorphism (Val158Met) alters enzyme activity and is associated with both the activation and functional connectivity of the PFC during task performance, particularly working memory. Here, we used functional magnetic resonance imaging and a data-driven, independent components analysis (ICA) approach to compare resting state funct...

  11. Polymorphism of the catechol-O-methyltransferase gene in Han Chinese patients with psoriasis vulgaris

    OpenAIRE

    Gao, Lin; Li, Hong; Li, Kai; Shen, Zhu; Liu, Ling; Li, Chunying; Zhang, Zhengdong; Liu, Yufeng

    2009-01-01

    Psoriasis vulgaris is defined by a series of linked cellular changes in the skin: hyperplasia of epidermal keratinocytes, vascular hyperplasia and ectasia, and infiltration of T lymphocytes, neutrophils and other types of leukocytes in the affected skin. Catechol-O-methyltransferase (COMT) 158 polymorphism can reduce the activity of the COMT enzyme that may trigger defective differentiation of keratinocytes in psoriasis. Immunocytes can degrade and inactivate catecholamines via monamine oxida...

  12. Towards more specific O6-Methylguanine-DNA methyltransferase (MGMT) inactivators

    OpenAIRE

    CORDEIRO MACHADO, ALESSANDRA; MC MURRY, THOMAS; Rozas, Isabel

    2011-01-01

    PUBLISHED Searching for a novel family of inactivators of the human DNA repair protein O6?methylguanine?DNA methyltransferase (MGMT) which is known to bind to the DNA minor groove, we have computationally modelled and synthesised two series of 2?amino?6?aryloxy?5?nitropyrimidines with morpholino or aminodiaryl substituents (potential minor groove binders) at the 4?position. Synthesis of these compounds was achieved by successive substitution of each of the two Cl atoms of 2?amino?4,6?dichl...

  13. O6-methylguanine-DNA methyltransferase (MGMT): can function explain a suicidal mechanism?

    OpenAIRE

    Gouws, Chrisna; Pretorius, Petrus Jacobus

    2011-01-01

    Why does O6-methylguanine-DNA methyltransferase (MGMT), an indispensable DNA repair enzyme, have a mechanism which seems to run counter to its importance? This enzyme is key to the removal of detrimental alkyl adducts from guanine bases. Although the mechanism is well known, an unusual feature surrounds its mode of action, which is its so-called suicidal endpoint. In addition, induction of MGMT is highly variable and its kinetics is atypical. These features raise some questions on the seeming...

  14. Inhibition of methyltransferases accelerates degradation of cFLIP and sensitizes B-cell lymphoma cells to TRAIL-induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Frank K Braun

    Full Text Available Non-Hodgkin lymphomas (NHLs are characterized by specific abnormalities that alter cell cycle regulation, DNA damage response, and apoptotic signaling. It is believed that cancer cells are particularly sensitive to cell death induced by tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL. However, many cancer cells show blocked TRAIL signaling due to up-regulated expression of anti-apoptotic factors, such as cFLIP. This hurdle to TRAIL's tumor cytotoxicity might be overcome by combining TRAIL-based therapy with drugs that reverse blockages of its apoptotic signaling. In this study, we investigated the impact of a pan-methyltransferase inhibitor (3-deazaneplanocin A, or DZNep on TRAIL-induced apoptosis in aggressive B-cell NHLs: mantle cell, Burkitt, and diffuse large B-cell lymphomas. We characterized TRAIL apoptosis regulation and caspase activation in several NHL-derived cell lines pre-treated with DZNep. We found that DZNep increased cancer cell sensitivity to TRAIL signaling by promoting caspase-8 processing through accelerated cFLIP degradation. No change in cFLIP mRNA level indicated independence of promoter methylation alterations in methyltransferase activity induced by DZNep profoundly affected cFLIP mRNA stability and protein stability. This appears to be in part through increased levels of cFLIP-targeting microRNAs (miR-512-3p and miR-346. However, additional microRNAs and cFLIP-regulating mechanisms appear to be involved in DZNep-mediated enhanced response to extrinsic apoptotic stimuli. The capacity of DZNep to target cFLIP expression on multiple levels underscores DZNep's potential in TRAIL-based therapies for B-cell NHLs.

  15. Arsenic (+3 oxidation state) methyltransferase and the inorganic arsenic methylation phenotype

    International Nuclear Information System (INIS)

    Inorganic arsenic is enzymatically methylated; hence, its ingestion results in exposure to the parent compound and various methylated arsenicals. Both experimental and epidemiological evidences suggest that some of the adverse health effects associated with chronic exposure to inorganic arsenic may be mediated by these methylated metabolites. If i As methylation is an activation process, then the phenotype for inorganic arsenic methylation may determine risk associated with exposure to this metalloid. We examined inorganic arsenic methylation phenotypes and arsenic (+3 oxidation state) methyltransferase genotypes in four species: three that methylate inorganic arsenic (human (Homo sapiens), rat (Rattus norwegicus), and mouse (Mus musculus)) and one that does not methylate inorganic arsenic (chimpanzee, Pan troglodytes). The predicted protein products from arsenic (+3 oxidation state) methyltransferase are similar in size for rat (369 amino acid residues), mouse (376 residues), and human (375 residues). By comparison, a 275-nucleotide deletion beginning at nucleotide 612 in the chimpanzee gene sequence causes a frameshift that leads to a nonsense mutation for a premature stop codon after amino acid 205. The null phenotype for inorganic arsenic methylation in the chimpanzee is likely due to the deletion in the gene for arsenic (+3 oxidation state) methyltransferase that yields an inactive truncated protein. This lineage-specific loss of function caused by the deletion event must have occurred in the Pan lineage after Homo-Pan divergence about 5 million years ago

  16. Chromatin Targeting of de Novo DNA Methyltransferases by the PWWP Domain

    Institute of Scientific and Technical Information of China (English)

    Ying-ZiGe; Min-TiePu; HumairaGowher; Hai-PingWu; Jian-PingDing; AlbertJeltsch; Guo-LiangXu

    2005-01-01

    DNA methylation patterns of mammalian genomes are generated in gametogenesis and early embryonic development. Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the process. Both en-zymes contain a long N-terminal regulatory region linked to a conserved C-terminal domain responsible forthe catalytic activity. Although a PWWP domain in the N-terminal region has been shown to bind DNA in vitro, it is unclear how the DNA methyltransferases access their substrate in chromatin in vivo. We show here that the two proteins are associated with chromatin including mitotic chromosomes in mammalian cells, and the PWWP domain is essential for the chromatin targeting of the enzymes. The functional significance of PWWPmediated chromatin targeting is suggested by the fact that a missense mutation in this domain of human DNMT3B causes immunodeficiency, centromeric heterochromatin instability, facial anomalies (ICF) syndrome, which is characterized by loss of methylation insatellite DNA, pericentromeric instability, and immunodeficiency. We demonstrate that the mutant protein completely loses its chromatin targeting capacity. Our data establish the PWWP domain as a novel chromatin/chromosome-targeting module and suggest that the PWWP-mediated chromatin association is essential for the function of the de novo methyltransferases during development.

  17. Rapid restriction enzyme free detection of DNA methyltransferase activity based on DNA-templated silver nanoclusters.

    Science.gov (United States)

    Kermani, Hanie Ahmadzade; Hosseini, Morteza; Dadmehr, Mehdi; Ganjali, Mohammad Reza

    2016-06-01

    DNA methylation has significant roles in gene regulation. DNA methyltransferase (MTase) enzyme characterizes DNA methylation and also induces an aberrant methylation pattern that is related to many diseases, especially cancers. Thus, it is required to develop a method to detect the DNA MTase activity. In this study, we developed a new sensitive and reliable method for methyltransferase activity assay by employing DNA-templated silver nanoclusters (DNA/Ag NCs) without using restriction enzymes. The Ag NCs have been utilized for the determination of M.SssI MTase activity and its inhibition. We designed an oligonucleotide probe which contained an inserted six-cytosine loop as Ag NCs formation template. The changes in fluorescence intensity were monitored to quantify the M.SssI activity. The fluorescence spectra showed a linear decrease in the range of 0.4 to 20 U/ml with a detection limit of 0.1 U/ml, which was significant compared with previous reports. The proposed method was applied successfully for demonstrating the Gentamicin effect as MTase inhibitor. The proposed method showed convenient reproducibility and sensitivity indicating its potential for the determination of methyltransferase activity. PMID:27052776

  18. Purification of homologous protein carboxyl methyltransferase isozymes from human and bovine erythrocytes

    International Nuclear Information System (INIS)

    The authors have purified the two major isozymes of the L-isoaspartyl/D-aspartyl protein methyltransferase from both human and bovine erythrocytes. These four enzymes all have polypeptide molecular weights of approximately 26,500 and appear to be monomers in solution. Each of these enzymes cross-reacts with antibodies directed against protein carboxyl methyltransferase I from bovine brain. Their structures also appear to be similar when analyzed by dodecyl sulfate gel electrophoresis for the large fragments produced by digestion with Staphylococcus aureus protease V8 or when analyzed by high-performance liquid chromatography (HPLC) for tryptic peptides. The structural relatedness of these enzymes was confirmed by sequence analysis of a total of 433 residues in 32 tryptic fragments of the human erythrocyte isozymes I and II and of the bovine erythrocyte isozyme II. They found sequence identity or probable identity in 111 out of 112 residues when they compared the human isozymes I and II and identities in 127 out of 134 residues when the human and bovine isozymes II were compared. These results suggest that the erythrocyte isozymes from both organisms may have nearly identical structures and confirm the similarities in the function of these methyltransferases that have been previously demonstrated

  19. Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

    Energy Technology Data Exchange (ETDEWEB)

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy); Lamballeire, Xavier de; Brisbare, Nadege [Unité des Virus Emergents, Faculté de Médecine, 27 Boulevard Jean Moulin, 13005 Marseille (France); Dalle, Karen; Lantez, Violaine; Egloff, Marie-Pierre; Coutard, Bruno; Canard, Bruno [Laboratoire Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS ESIL, Case 932, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France); Gould, Ernest; Forrester, Naomi [CEH Oxford, Mansfield Road, Oxford OX1 3SR (United Kingdom); Bolognesi, Martino, E-mail: martino.bolognesi@unimi.it [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy)

    2006-08-01

    Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup.

  20. MtrA of the sodium ion pumping methyltransferase binds cobalamin in a unique mode.

    Science.gov (United States)

    Wagner, Tristan; Ermler, Ulrich; Shima, Seigo

    2016-01-01

    In the three domains of life, vitamin B12 (cobalamin) is primarily used in methyltransferase and isomerase reactions. The methyltransferase complex MtrA-H of methanogenic archaea has a key function in energy conservation by catalysing the methyl transfer from methyl-tetrahydromethanopterin to coenzyme M and its coupling with sodium-ion translocation. The cobalamin-binding subunit MtrA is not homologous to any known B12-binding proteins and is proposed as the motor of the sodium-ion pump. Here, we present crystal structures of the soluble domain of the membrane-associated MtrA from Methanocaldococcus jannaschii and the cytoplasmic MtrA homologue/cobalamin complex from Methanothermus fervidus. The MtrA fold corresponds to the Rossmann-type α/β fold, which is also found in many cobalamin-containing proteins. Surprisingly, the cobalamin-binding site of MtrA differed greatly from all the other cobalamin-binding sites. Nevertheless, the hydrogen-bond linkage at the lower axial-ligand site of cobalt was equivalently constructed to that found in other methyltransferases and mutases. A distinct polypeptide segment fixed through the hydrogen-bond linkage in the relaxed Co(III) state might be involved in propagating the energy released upon corrinoid demethylation to the sodium-translocation site by a conformational change. PMID:27324530

  1. Epigenetic Determinants of Erythropoiesis: Role of the Histone Methyltransferase SetD8 in Promoting Erythroid Cell Maturation and Survival.

    Science.gov (United States)

    DeVilbiss, Andrew W; Sanalkumar, Rajendran; Hall, Bryan D R; Katsumura, Koichi R; de Andrade, Isabela Fraga; Bresnick, Emery H

    2015-06-01

    Erythropoiesis, in which committed progenitor cells generate millions of erythrocytes daily, involves dramatic changes in the chromatin structure and transcriptome of erythroblasts, prior to their enucleation. While the involvement of the master-regulatory transcription factors GATA binding protein 1 (GATA-1) and GATA-2 in this process is established, the mechanistic contributions of many chromatin-modifying/remodeling enzymes in red cell biology remain enigmatic. We demonstrated that SetD8, a histone methyltransferase that catalyzes monomethylation of histone H4 at lysine 20 (H4K20me1), is a context-dependent GATA-1 corepressor in erythroid cells. To determine whether SetD8 controls erythroid maturation and/or function, we used a small hairpin RNA (shRNA)-based loss-of-function strategy in a primary murine erythroblast culture system. In this system, SetD8 promoted erythroblast maturation and survival, and this did not involve upregulation of the established regulator of erythroblast survival Bcl-x(L). SetD8 catalyzed H4K20me1 at a critical Gata2 cis element and restricted occupancy by an enhancer of Gata2 transcription, Scl/TAL1, thereby repressing Gata2 transcription. Elevating GATA-2 levels in erythroid precursors yielded a maturation block comparable to that induced by SetD8 downregulation. As lowering GATA-2 expression in the context of SetD8 knockdown did not rescue erythroid maturation, we propose that SetD8 regulation of erythroid maturation involves multiple target genes. These results establish SetD8 as a determinant of erythroid cell maturation and provide a framework for understanding how a broadly expressed histone-modifying enzyme mediates cell-type-specific GATA factor function. PMID:25855754

  2. The catechol-O-methyltransferase inhibitory potential of Z-vallesiachotamine by in silicoand in vitro approaches

    Directory of Open Access Journals (Sweden)

    Carolina dos Santos Passos

    2015-08-01

    Full Text Available AbstractZ-Vallesiachotamine is a monoterpene indole alkaloid that has a β-N-acrylate group in its structure. This class of compounds has already been described in different Psychotriaspecies. Our research group observed that E/Z-vallesiachotamine exhibits a multifunctional feature, being able to inhibit targets related to neurodegeneration, such as monoamine oxidase A, sirtuins 1 and 2, and butyrylcholinesterase enzymes. Aiming at better characterizing the multifunctional profile of this compound, its effect on cathecol-O-methyltransferase activity was investigated. The cathecol-O-methyltransferase activity was evaluated in vitro by a fluorescence-based method, using S-(5′-adenosyl-l-methionine as methyl donor and aesculetin as substrate. The assay optimization was performed varying the concentrations of methyl donor (S-(5′-adenosyl-l-methionine and enzyme. It was observed that the highest concentrations of both factors (2.25 U of the enzyme and 100 µM of S-(5′-adenosyl-l-methionine afforded the more reproducible results. The in vitro assay demonstrated that Z-vallesiachotamine was able to inhibit the cathecol-O-methyltransferase activity with an IC50 close to 200 µM. Molecular docking studies indicated that Z-vallesiachotamine can bind the catechol pocket of catechol-O-methyltransferase enzyme. The present work demonstrated for the first time the inhibitory properties of Z-vallesiachotamine on cathecol-O-methyltransferase enzyme, affording additional evidence regarding its multifunctional effects in targets related to neurodegenerative diseases.

  3. The ASH1 HOMOLOG 2 (ASHH2 histone H3 methyltransferase is required for ovule and anther development in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Paul E Grini

    Full Text Available BACKGROUND: SET-domain proteins are histone lysine (K methyltransferases (HMTase implicated in defining transcriptionally permissive or repressive chromatin. The Arabidopsis ASH1 HOMOLOG 2 (ASHH2 protein (also called SDG8, EFS and CCR1 has been suggested to methylate H3K4 and/or H3K36 and is similar to Drosophila ASH1, a positive maintainer of gene expression, and yeast Set2, a H3K36 HMTase. Mutation of the ASHH2 gene has pleiotropic developmental effects. Here we focus on the role of ASHH2 in plant reproduction. METHODOLOGY/PRINCIPAL FINDINGS: A slightly reduced transmission of the ashh2 allele in reciprocal crosses implied involvement in gametogenesis or gamete function. However, the main requirement of ASHH2 is sporophytic. On the female side, close to 80% of mature ovules lack embryo sac. On the male side, anthers frequently develop without pollen sacs or with specific defects in the tapetum layer, resulting in reduction in the number of functional pollen per anther by up to approximately 90%. In consistence with the phenotypic findings, an ASHH2 promoter-reporter gene was expressed at the site of megaspore mother cell formation as well as tapetum layers and pollen. ashh2 mutations also result in homeotic changes in floral organ identity. Transcriptional profiling identified more than 300 up-regulated and 600 down-regulated genes in ashh2 mutant inflorescences, whereof the latter included genes involved in determination of floral organ identity, embryo sac and anther/pollen development. This was confirmed by real-time PCR. In the chromatin of such genes (AP1, AtDMC1 and MYB99 we observed a reduction of H3K36 trimethylation (me3, but not H3K4me3 or H3K36me2. CONCLUSIONS/SIGNIFICANCE: The severe distortion of reproductive organ development in ashh2 mutants, argues that ASHH2 is required for the correct expression of genes essential to reproductive development. The reduction in the ashh2 mutant of H3K36me3 on down-regulated genes relevant to

  4. Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

    DEFF Research Database (Denmark)

    Karminska, K. H.; Purta, E.; Hansen, L .H.; Bujnicki, J. M.; Vester, B.; Long, Katherine

    2010-01-01

    The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase...

  5. 2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase.

    Directory of Open Access Journals (Sweden)

    Hongping Dong

    Full Text Available RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2'-O methyltransferase activities that are required for the formation of 5' type I cap (m(7GpppAm of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4 specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2'-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N⁶-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2'-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2'-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2'-O-methyladenosine. The 2'-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2'-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2'-O methylation of internal adenosine of

  6. Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

    Science.gov (United States)

    Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude

    2016-04-01

    Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity. PMID:26785049

  7. Human erythrocyte phenol O-methyltransferase: Radiochemical microassay and biochemical properties

    International Nuclear Information System (INIS)

    A radiochemical microassay for the determination of phenol O-methyltransferase (PMT) activity in human red blood cell membranes has been developed. Acetaminophen was used as the substrate. The apparent Michaelis-Menten Ksub(M) value for acetaminophen was 21.2 X 10-3M. The apparent Ksub(M) value for S-adenosyl-L-methionine, a co-substrate for the reaction, was 4.8 X 10-6 M, and the pH optimum of the reaction was approximately 9.0 with four different buffer systems. Phenol was also tested as a substrate and had an apparent Ksub(M) value of 2.0 X 10-3 M. Human erythrocyte (RBC) membrane PMT activity did not have the biochemical characteristics of catechol O-methyltransferase, another RBC membrane methyltransferase enzyme activity. Blood samples obtained from 212 randomly selected adult white subjects had a mean activity of 134.5 +41.5 pmol of p-acetanisidide formed per mg protein per hour (mean +- S.D.). Activities varied from 44 to 282 units. There were no differences in the mean activities of samples from men and women. Experiments in which mixtures of 'low' and 'high' activity RBC membrane preparations were assayed for PMT provided no evidence that the variations in enzyme activity were due to the presence of endogenous PMT activators or inhibitors. RBC membrane PMT activity in blood from 9 patients with renal failure, a pathological state in which there are elevated circulating levels of phenols, was found to be significantly decreased with average activity of 76.2+-9.7 (mean +- S.E.M., P <0.001). (Auth.)

  8. Development of fed-batch profiles for efficient biosynthesis of catechol-O-methyltransferase

    OpenAIRE

    G.M. Espírito Santo; A.Q. Pedro; D. Oppolzer; Bonifácio, M J; Queiroz, J. A.; Silva, F.; L.A. Passarinha

    2014-01-01

    Catechol-O-methyltransferase (COMT, EC 2.1.1.6) plays a crucial role in dopamine metabolism which has intimately linked this enzyme to some neurodegenerative diseases, such as Parkinson's disease. In recent years, in the attempt of developing new therapeutic strategies for Parkinson's disease, there has been a growing interest in the search for effective COMT inhibitors. In order to do so, large amounts of COMT in an active form are needed, and the best way to achieve this is by up-scaling it...

  9. Structural Basis for Binding of RNA and Cofactor by a KsgA Methyltransferase

    OpenAIRE

    Tu, Chao; Tropea, Joseph E.; Austin, Brian P; Court, Donald L.; Waugh, David S.; Ji, Xinhua

    2009-01-01

    Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. Here, we report structures of Aquifex aeolicus KsgA, in its ligand-free form, in complex with RNA and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), providing the first pieces of structural information on KsgA-RNA and KsgA-SAH interactions. Moreover, the stru...

  10. Histone methyltransferase Suv39h1 represses MyoD-stimulated myogenic differentiation

    OpenAIRE

    Mal, Asoke K.

    2006-01-01

    Suv39h1 is a histone H3 lysine-9 (H3-K9) specific methyltransferase (HMT) that is associated with gene silencing through chromatin modification. The transition from proliferation into differentiation of muscle cell is accompanied by transcriptional activation of previously silent muscle genes. I report Suv39h1 interaction with myogenic regulator MyoD in proliferating muscle cells and its HMT activity, which is associated with MyoD, diminishes as differentiation proceeds. The Suv39h1–MyoD comp...

  11. The DmtA methyltransferase contributes to Aspergillus flavus conidiation, sclerotial production, aflatoxin biosynthesis and virulence

    OpenAIRE

    Kunlong Yang; Linlin Liang; Fanlei Ran; Yinghang Liu; Zhenguo Li; Huahui Lan; Peili Gao; Zhenhong Zhuang; Feng Zhang; Xinyi Nie; Shimuye Kalayu Yirga; Shihua Wang

    2016-01-01

    DNA methylation is essential for epigenetic regulation of gene transcription and development in many animals, plants and fungi. We investigated whether DNA methylation plays a role in the development and secondary metabolism of Aspergillus flavus, identified the DmtA methyltransferase from A. flavus, and produced a dmtA knock-out mutant by replacing the dmtA coding sequence with the pyrG selectable marker. The A. flavus dmtA null mutant lines produced white fluffy mycelium in liquid medium, a...

  12. Histone methyltransferase G9a contributes to H3K27 methylation in vivo

    Institute of Scientific and Technical Information of China (English)

    Hui Wu; Bing Zhu; Xiuzhen Chen; Jun Xiong; Yingfeng Li; Hong Li; Xiaojun Ding; Sheng Liu; She Chen; Shaorong Gao

    2011-01-01

    @@ Dear Editor, Histone modifications play a vital role in the conformation and function of their associated chromatin templates[1].Histone H3K27 methylation mediated by the PRC2 complex is critical for transcriptional regulation,Polycomb silencing,Drosophila segmentation,mammalian X inactivation and cancer[1].Interestingly,H3K27me1(H3 mono-methylated at residue K27)levels in vivo remain unaffected after PRC2 disruption[2,3],which is an indication for the existence of other contributing histone methyltransferase(s)to H3K27me1.

  13. EmtA, a rRNA methyltransferase conferring high-level evernimicin resistance

    DEFF Research Database (Denmark)

    Mann, P. A.; Xiong, L.; Mankin, A. S.;

    2001-01-01

    Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high-level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355...... exhibited a dramatic reduction in evernimicin binding, shown by both cell-free translation assays and direct-binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin...

  14. Juvenile hormone acid methyltransferase: A key regulatory enzyme for insect metamorphosis

    OpenAIRE

    Shinoda, Tetsuro; Itoyama, Kyo

    2003-01-01

    Juvenile hormone (JH) acid methyltransferase (JHAMT) is an enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis pathway in insects. By fluorescent mRNA differential display, we have cloned a cDNA encoding JHAMT from the corpora allata (CA) of the silkworm, Bombyx mori (BmJHAMT). The BmJHAMT cDNA encodes an ORF of 278 aa with a calculated molecular mass of 32,544 Da. The predicted amino acid sequence contains a conserved S-adenosyl-l-me...

  15. Crystal structure of phosphoethanolamine methyltransferase from Plasmodium falciparum in complex with amodiaquine

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Soon Goo; Alpert, Tara D.; Jez, Joseph M. (WU)

    2012-07-17

    Phosphoethanolamine N-methyltransferase (PMT) is essential for phospholipid biogenesis in the malarial parasite Plasmodium falciparum. PfPMT catalyzes the triple methylation of phosphoethanolamine to produce phosphocholine, which is then used for phosphatidylcholine synthesis. Here we describe the 2.0 {angstrom} resolution X-ray crystal structure of PfPMT in complex with amodiaquine. To better characterize inhibition of PfPMT by amodiaquine, we determined the IC{sub 50} values of a series of aminoquinolines using a direct radiochemical assay. Both structural and functional analyses provide a possible approach for the development of new small molecule inhibitors of PfPMT.

  16. O6-Methylguanine-DNA methyltransferase deficiency in developing brain: Implications for brain tumorigenesis

    OpenAIRE

    Bobola, Michael S.; Blank, A.; Berger, Mitchel S.; Silber, John R

    2007-01-01

    The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a cardinal defense against the mutagenic and carcinogenic effects of alkylating agents. We have reported evidence that absence of detectable MGMT activity (MGMT− phenotype) in human brain is a predisposing factor for primary brain tumors that affects ca. 12% of individuals [J.R. Silber, et. al. Proc. Natl. Acad. Sci. USA 93 (1996) 6941–6946]. We report here that MGMT− phenotype in the brain of children and adults, and the...

  17. DNA Methyltransferase Gene dDnmt2 and Longevity of Drosophila

    Institute of Scientific and Technical Information of China (English)

    Meng-JauLin; Lin-YaTang; M.NarsaReddy; C.K.JamesShen

    2005-01-01

    The DNA methylation program of the fruit fly Drosophila melanogaster is carried out by the single DNA methyltransferase gene dDnmt2, the function of which is unknown before. We present evidence that intactness of the gene is required for maintenance of the normal life span of the fruit flies. In contrast, overexpression of dDnmt2 could extend Drosophila life span. The study links the Drosophila DNA methylation program with the small heatshock proteins and longevity/aging and has interesting implication on the eukaryotic DNA methyl-ation programs in general.

  18. Discovery of an in vivo Chemical Probe of the Lysine Methyltransferases G9a and GLP

    OpenAIRE

    Liu, Feng; Barsyte-Lovejoy, Dalia; Li, Fengling; Xiong, Yan; Korboukh, Victoria; Huang, Xi-Ping; Allali-Hassani, Abdellah; Janzen, William P.; Roth, Bryan L.; Frye, Stephen V.; Arrowsmith, Cheryl H.; Brown, Peter J.; Vedadi, Masoud; Jin, Jian

    2013-01-01

    Among epigenetic “writers”, “readers”, and “erasers”, the lysine methyltransferases G9a and GLP, which catalyze mono- and dimethylation of histone H3 lysine 9 (H3K9me2) and non-histone proteins, have been implicated in a variety of human diseases. A “toolkit” of well-characterized chemical probes will allow biological and disease hypotheses concerning these proteins to be tested in cell-based and animal models with high confidence. We previously discovered potent and selective G9a/GLP inhibit...

  19. Protein lysine methyltransferase G9a acts on non-histone targets

    OpenAIRE

    Rathert, Philipp; Dhayalan, Arunkumar; Murakami, Marie; Zhang, Xing; Tamas, Raluca; Jurkowska, Renata; Komatsu, Yasuhiko; Shinkai, Yoichi; Cheng, Xiaodong; Jeltsch, Albert

    2008-01-01

    By methylation of peptide arrays, we determined the specificity profile of the protein methyltransferase G9a. We show that it mostly recognizes an Arg-Lys sequence and that its activity is inhibited by methylation of the arginine residue. Using the specificity profile, we identified new non-histone protein targets of G9a, including CDYL1, WIZ, ACINUS and G9a (automethylation), as well as peptides derived from CSB. We demonstrate potential downstream signaling pathways for methylation of non-h...

  20. A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells

    OpenAIRE

    Vedadi, Masoud; Barsyte-Lovejoy, Dalia; Liu, Feng; Rival-Gervier, Sylvie; Allali-Hassani, Abdellah; Labrie, Viviane; Wigle, Tim J.; DiMaggio, Peter A.; Wasney, Gregory A.; Siarheyeva, Alena; Dong, Aiping; Tempel, Wolfram; Wang, Sun-Chong; Chen, Xin; Chau, Irene

    2011-01-01

    Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for sm...

  1. Quantitative proteomics reveals direct and indirect alterations in the histone code following methyltransferase knockdown

    OpenAIRE

    Plazas-Mayorca, Mariana D.; Bloom, Joshua S.; Zeissler, Ulrike; LeRoy, Gary; Young, Nicolas L.; DiMaggio, Peter A.; Krugylak, Leonid; Schneider, Robert; Garcia, Benjamin A

    2010-01-01

    Histones are highly conserved proteins that organize cellular DNA. These proteins, especially their N-terminal domains, are adorned with many post-translational modifications (PTMs) such as lysine methylation, which are associated with active or repressed transcriptional states. The lysine methyltransferase G9a and its interaction partner Glp1 can mono- or dimethylate histone H3 on lysine (H3K9me1 or me2); possible cross-talk between these modifications and other PTMs on the same or other his...

  2. A fluorescence resonance energy transfer-based method for histone methyltransferases

    DEFF Research Database (Denmark)

    Devkota, Kanchan; Lohse, Brian; Nyby Jakobsen, Camilla;

    2015-01-01

    A simple dye–quencher fluorescence resonance energy transfer (FRET)-based assay for methyltransferases was developed and used to determine kinetic parameters and inhibitory activity at EHMT1 and EHMT2. Peptides mimicking the truncated histone H3 tail were functionalized in each end with a dye and a...... quencher, respectively. When lysine-9 residues in the peptides were methylated, they were protected from cleavage by endoproteinase–EndoLysC, whereas unmethylated peptides were cleaved, resulting in an increase in fluorescent intensity....

  3. Regioselectivity of catechol O-methyltransferase confers enhancement of catalytic activity

    Science.gov (United States)

    Tsao, Douglas; Liu, Shubin; Dokholyan, Nikolay V.

    2011-04-01

    Catechol O-methyltransferase (COMT) metabolizes catechol moieties by methylating a single hydroxyl group at the meta- or para- hydroxyl position. Hydrophobic amino acids near the active site of COMT influence the regioselectivity of this reaction. Our sequence analysis highlights their importance by showing that these residues are highly conserved throughout evolution. Reaction barriers calculated in the gas phase reveal a lower barrier during methylation at the meta- position, suggesting that the observed meta-regioselectivity of COMT can be attributed to the substrate itself, and that COMT has evolved residues to orient the substrate in a manner that increases the rate of catalysis.

  4. Unexpected Distinct Roles of the Related Histone H3 Lysine 9 Methyltransferases G9a and G9a-Like Protein in Myoblasts.

    Science.gov (United States)

    Battisti, Valentine; Pontis, Julien; Boyarchuk, Ekaterina; Fritsch, Lauriane; Robin, Philippe; Ait-Si-Ali, Slimane; Joliot, Véronique

    2016-06-01

    Lysine methyltransferases G9a and GLP (G9a-like protein) are highly homologous and form functional heterodimeric complexes that establish mono- and dimethylation on histone H3 lysine 9 (H3K9me1, H3K9me2) in euchromatin. Here, we describe unexpected distinct roles for G9a and GLP in skeletal muscle terminal differentiation. Indeed, gain- or loss-of-function assays in myoblasts showed, in agreement with previous reports, that G9a inhibits terminal differentiation. While GLP plays a more intricate role in muscle differentiation,in one hand, both GLP gain and loss of function inhibit late steps of differentiation; on the other hand, in contrast to G9a, GLP overexpression promotes abnormal precocious expression of muscle differentiation-specific genes already in proliferating myoblasts. In agreement, transcriptomic analysis indicates that G9a and GLP regulate different sets of genes. Thus, GLP, but not G9a, inhibits proteasome subunit-encoding genes expression, resulting in an inhibition of the proteasome activities. Subsequently, GLP, but not G9a, overexpression stabilizes MyoD that is likely to be responsible for muscle markers expression in proliferating myoblasts. PMID:27056598

  5. The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development

    OpenAIRE

    Stabell, Marianne; Eskeland, Ragnhild; Bjørkmo, Mona; Larsson, Jan; Aalen, Reidunn B.; Imhof, Axel; Lambertsson, Andrew

    2006-01-01

    Mammalian G9a is a histone H3 Lys-9 (H3–K9) methyltransferase localized in euchromatin and acts as a co-regulator for specific transcription factors. G9a is required for proper development in mammals as g9a−/g9a− mice show growth retardation and early lethality. Here we describe the cloning, the biochemical and genetical analyses of the Drosophila homolog dG9a. We show that dG9a shares the structural organization of mammalian G9a, and that it is a multi-catalytic histone methyltransferase wit...

  6. Functional analysis of the N- and C-terminus of mammalian G9a histone H3 methyltransferase

    OpenAIRE

    Estève, Pierre-Olivier; Patnaik, Debasis; Chin, Hang Gyeong; Benner, Jack; Teitell, Michael A.; Pradhan, Sriharsa

    2005-01-01

    Methylation of lysine 9 (K9) in the N-terminus tail of histone H3 (H3) in chromatin is associated with transcriptionally silenced genes and is mediated by histone methyltransferases. Murine G9a is a 1263 amino acid H3-K9 methyltransferase that possesses characteristic SET domain and ANK repeats. In this paper, we have used a series of green fluorescent protein-tagged deletion constructs to identify two nuclear localization signals (NLS), the first NLS embedded between amino acids 24 and 109 a...

  7. Set7/9, a methyltransferase, regulates the thermogenic program during brown adipocyte differentiation through the modulation of p53 acetylation.

    Science.gov (United States)

    Son, Min Jeong; Kim, Won Kon; Park, Anna; Oh, Kyoung-Jin; Kim, Jeong-Hoon; Han, Baek Soo; Kim, Il Chul; Chi, Seung-Wook; Park, Sung Goo; Lee, Sang Chul; Bae, Kwang-Hee

    2016-08-15

    Brown adipose tissue, which is mainly composed of brown adipocytes, plays a key role in the regulation of energy balance via dissipation of extra energy as heat, and consequently counteracts obesity and its associated-disorders. Therefore, brown adipocyte differentiation should be tightly controlled at the multiple regulation steps. Among these, the regulation at the level of post-translational modifications (PTMs) is largely unknown. Here, we investigated the changes in the expression level of the enzymes involved in protein lysine methylation during brown adipocyte differentiation by using quantitative real-time PCR (qPCR) array analysis. Several enzymes showing differential expression patterns were identified. In particular, the expression level of methyltransferase Set7/9 was dramatically repressed during brown adipocyte differentiation. Although there was no significant change in lipid accumulation, ectopic expression of Set7/9 led to enhanced expression of several key thermogenic genes, such as uncoupling protein-1 (UCP-1), Cidea, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and PR domain containing 16 (PRDM16). In contrast, knockdown of endogenous Set7/9 led to significantly reduced expression of these thermogenic genes. Furthermore, suppressed mitochondrial DNA content and decreased oxygen consumption rate were also detected upon Set7/9 knockdown. We found that p53 acetylation was regulated by Set7/9-dependent interaction with Sirt1. Based on these results, we suggest that Set7/9 acts as a fine regulator of the thermogenic program during brown adipocyte differentiation by regulation of p53 acetylation. Thus, Set7/9 could be used as a valuable target for regulating thermogenic capacity and consequently to overcome obesity and its related metabolic diseases. PMID:27132805

  8. MGMT expression: insights into its regulation. 1. Epigenetic factors

    OpenAIRE

    Iatsyshyna A. P.

    2013-01-01

    O6-methylguanine-DNA methyltransferase (MGMT) is the DNA repair enzyme responsible for removing of alkylation adducts from the O6-guanine in DNA. Despite MGMT prevents mutations and cell death, this enzyme can provide resistance of cancer cells to alkylating agents of chemotherapy. The high intra- and inter-individual variations in the human MGMT expression level have been observed indicating to a complicated regulation of this gene. This review is focused on the study of epigenetic factors w...

  9. MGMT expression: insights into its regulation. 2. Single nucleotide polymorphisms

    OpenAIRE

    Iatsyshyna A. P.; Pidpala O. V.; Lukash L. L.

    2013-01-01

    High intra- and interindividual variations in the expression levels of the human O6-methylguanine-DNA methyltransferase (MGMT) gene have been observed. This DNA repair enzyme can be a cause of resistance of cancer cells to alkylating chemotherapy. It has been studied the association of single nucleotide polymorphisms (SNPs) of MGMT with the risk for different types of cancer, progression-free survival in patients with cancer treated with alkylating chemotherapy, as well as an effect of SNPs o...

  10. Hippocampal dysfunction in the Euchromatin histone methyltransferase 1 heterozygous knockout mouse model for Kleefstra syndrome.

    Science.gov (United States)

    Balemans, Monique C M; Kasri, Nael Nadif; Kopanitsa, Maksym V; Afinowi, Nurudeen O; Ramakers, Ger; Peters, Theo A; Beynon, Andy J; Janssen, Sanne M; van Summeren, Rik C J; Eeftens, Jorine M; Eikelenboom, Nathalie; Benevento, Marco; Tachibana, Makoto; Shinkai, Yoichi; Kleefstra, Tjitske; van Bokhoven, Hans; Van der Zee, Catharina E E M

    2013-03-01

    Euchromatin histone methyltransferase 1 (EHMT1) is a highly conserved protein that catalyzes mono- and dimethylation of histone H3 lysine 9, thereby epigenetically regulating transcription. Kleefstra syndrome (KS), is caused by haploinsufficiency of the EHMT1 gene, and is an example of an emerging group of intellectual disability (ID) disorders caused by genes encoding epigenetic regulators of neuronal gene activity. Little is known about the mechanisms underlying this disorder, prompting us to study the Euchromatin histone methyltransferase 1 heterozygous knockout (Ehmt1(+/-)) mice as a model for KS. In agreement with the cognitive disturbances observed in patients with KS, we detected deficits in fear extinction learning and both novel and spatial object recognition in Ehmt1(+/-) mice. These learning and memory deficits were associated with a significant reduction in dendritic arborization and the number of mature spines in hippocampal CA1 pyramidal neurons of Ehmt1(+/-) mice. In-depth analysis of the electrophysiological properties of CA3-CA1 synapses revealed no differences in basal synaptic transmission or theta-burst induced long-term potentiation (LTP). However, paired-pulse facilitation (PPF) was significantly increased in Ehmt1(+/-) neurons, pointing to a potential deficiency in presynaptic neurotransmitter release. Accordingly, a reduction in the frequency of miniature excitatory post-synaptic currents (mEPSCs) was observed in Ehmt1(+/-) neurons. These data demonstrate that Ehmt1 haploinsufficiency in mice leads to learning deficits and synaptic dysfunction, providing a possible mechanism for the ID phenotype in patients with KS. PMID:23175442

  11. Thiopurine methyltransferase activity in the erythrocytes of adults and children: and HPLC-linked assay.

    Science.gov (United States)

    Micheli, V; Jacomelli, G; Fioravanti, A; Morozzi, G; Marcolongo, R; Pompucci, G

    1997-03-18

    A non-radioactive method that uses reverse-phase high performance liquid chromatography is described for the determination of thiopurine methyltransferase (E.C. 2.1.1.67) activity in human erythrocytes. The method is based on the direct quantitation of 6-methyl-mercaptopurine produced from 6-mercaptopurine by crude erythrocyte lysates. The method is accurate and reliable and suitable for diagnostic use. Activity values in control adults ranged from 5 to 32 pmol/h/mg haemoglobin. The activity in the erythrocytes of adult males was significantly higher compared to females (21 +/- 5 and 15 +/- 8 pmol/h/mg haemoglobin, respectively). The activity measured in the erythrocytes of children (22 +/- 5 pmol/h/mg haemoglobin) did not show any significant difference compared to adults. Thiopurine methyltransferase activity was measured in a female patient with systemic sclerosis who developed severe bone marrow depression after treatment with azathioprine and allopurinol. Activity (6.3 +/- 0.5 pmol/h/mg haemoglobin) was found in the lowest range of controls thus supporting the hypothesis that it could be responsible for increased azathioprine cytotoxicity. PMID:9086303

  12. Bacteriophage adenine methyltransferase: a life cycle regulator? Modelled using Vibrio harveyi myovirus like.

    Science.gov (United States)

    Bochow, S; Elliman, J; Owens, L

    2012-11-01

    The adenine methyltransferase (DAM) gene methylates GATC sequences that have been demonstrated in various bacteria to be a powerful gene regulator functioning as an epigenetic switch, particularly with virulence gene regulation. However, overproduction of DAM can lead to mutations, giving rise to variability that may be important for adaptation to environmental change. While most bacterial hosts carry a DAM gene, not all bacteriophage carry this gene. Currently, there is no literature regarding the role DAM plays in life cycle regulation of bacteriophage. Vibrio campbellii strain 642 carries the bacteriophage Vibrio harveyi myovirus like (VHML) that has been proven to increase virulence. The complete genome sequence of VHML bacteriophage revealed a putative adenine methyltransferase gene. Using VHML, a new model of phage life cycle regulation, where DAM plays a central role between the lysogenic and lytic states, will be hypothesized. In short, DAM methylates the rha antirepressor gene and once methylation is removed, homologous CI repressor protein becomes repressed and non-functional leading to the switching to the lytic cycle. Greater understanding of life cycle regulation at the genetic level can, in the future, lead to the genesis of chimeric bacteriophage with greater control over their life cycle for their safe use as probiotics within the aquaculture industry. PMID:22681538

  13. Improved radioenzymatic assay for plasma norepinephrine using purified phenylethanolamine n-methyltransferase

    International Nuclear Information System (INIS)

    Radioenzymatic assays have been developed for catecholamines using either catechol O-methyltransferase (COMT) or phenylethanolamine N-methyltransferase (PNMT). Assays using PNMT are specific for norepinephrine (NE) and require minimal manipulative effort but until now have been less sensitive than the more complex procedures using COMT. The authors report an improved purification scheme for bovine PNMT which has permitted development of an NE assay with dramatically improved sensitivity (0.5 pg), specificity and reproducibility (C.V. < 5%). PNMT was purified by sequential pH 5.0 treatment and dialysis and by column chromatographic procedures using DEAE-Sephacel, Sepharcryl S-200 and Phenyl-Boronate Agarose. Recovery of PNMT through the purification scheme was 50%, while blank recovery was <.001%. NE can be directly quantified in 25 ul of human plasma and an 80 tube assay can be completed within 4 h. The capillary to venous plasma NE gradient was examined in 8 normotensive male subjects. Capillary plasma (NE (211.2 +/- 61.3 pg/ml)) was lower than venous plasma NE (366.6 +/- 92.5 pg/ml) in all subjects (p < 0.005). This difference suggests that capillary (NE) may be a unique indicator of sympathetic nervous system activity in vivo. In conclusion, purification of PNMT has facilitated development of an improved radioenzymatic for NE with significantly improved sensitivity

  14. X-ray crystal structure of N-6 adenine deoxyribose nucleic acid methyltransferase from Streptococcus pneumoniae

    Science.gov (United States)

    Tran, Phidung Hong

    X-ray diffraction by using resonant anomalous scattering has become a popular tool for solving crystal structures in the last ten years with the expanded availability of tunable synchrotron radiation for protein crystallography. Mercury atoms were used for phasing. The crystal structure of N-6 deoxyribose nucleic acid methyltransferase from Streptoccocus pneumoniae (DpnM) was solved by using the Multiple Anomalous Diffraction technique. The crystal structure reveals the formation of mercaptide between the mercury ion and the thiol group on the cysteine amino acid in a hydrophobic environment. The crystal structure contains the bound ligand, S- adenosyl-l-methionine on the surface of the concave opening. The direction of the β-strands on the beta sheets are identical to other solved methyltransferases. The highly conserved motifs, DPPY and the FxGxG, are found to be important in ligand binding and possibly in methyl group transfer. The structure has a concave cleft with an opening on the order of 30 Å that can accommodate a DNA duplex. By molecular modelling coupled to sequence alignment, two other highly conserved residues Arg21 and Gly19 are found to be important in catalysis.

  15. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders.

    Science.gov (United States)

    Ernst, Thomas; Chase, Andrew J; Score, Joannah; Hidalgo-Curtis, Claire E; Bryant, Catherine; Jones, Amy V; Waghorn, Katherine; Zoi, Katerina; Ross, Fiona M; Reiter, Andreas; Hochhaus, Andreas; Drexler, Hans G; Duncombe, Andrew; Cervantes, Francisco; Oscier, David; Boultwood, Jacqueline; Grand, Francis H; Cross, Nicholas C P

    2010-08-01

    Abnormalities of chromosome 7q are common in myeloid malignancies, but no specific target genes have yet been identified. Here, we describe the finding of homozygous EZH2 mutations in 9 of 12 individuals with 7q acquired uniparental disomy. Screening of a total of 614 individuals with myeloid disorders revealed 49 monoallelic or biallelic EZH2 mutations in 42 individuals; the mutations were found most commonly in those with myelodysplastic/myeloproliferative neoplasms (27 out of 219 individuals, or 12%) and in those with myelofibrosis (4 out of 30 individuals, or 13%). EZH2 encodes the catalytic subunit of the polycomb repressive complex 2 (PRC2), a highly conserved histone H3 lysine 27 (H3K27) methyltransferase that influences stem cell renewal by epigenetic repression of genes involved in cell fate decisions. EZH2 has oncogenic activity, and its overexpression has previously been causally linked to differentiation blocks in epithelial tumors. Notably, the mutations we identified resulted in premature chain termination or direct abrogation of histone methyltransferase activity, suggesting that EZH2 acts as a tumor suppressor for myeloid malignancies. PMID:20601953

  16. Nerve Injury Diminishes Opioid Analgesia through Lysine Methyltransferase-mediated Transcriptional Repression of μ-Opioid Receptors in Primary Sensory Neurons.

    Science.gov (United States)

    Zhang, Yuhao; Chen, Shao-Rui; Laumet, Geoffroy; Chen, Hong; Pan, Hui-Lin

    2016-04-15

    The μ-opioid receptor (MOR, encoded by Oprm1) agonists are the mainstay analgesics for treating moderate to severe pain. Nerve injury causes down-regulation of MORs in the dorsal root ganglion (DRG) and diminishes the opioid effect on neuropathic pain. However, the epigenetic mechanisms underlying the diminished MOR expression caused by nerve injury are not clear. G9a (encoded by Ehmt2), a histone 3 at lysine 9 methyltransferase, is a key chromatin regulator responsible for gene silencing. In this study, we determined the role of G9a in diminished MOR expression and opioid analgesic effects in animal models of neuropathic pain. We found that nerve injury in rats induced a long-lasting reduction in the expression level of MORs in the DRG but not in the spinal cord. Nerve injury consistently increased the enrichment of the G9a product histone 3 at lysine 9 dimethylation in the promoter of Oprm1 in the DRG. G9a inhibition or siRNA knockdown fully reversed MOR expression in the injured DRG and potentiated the morphine effect on pain hypersensitivity induced by nerve injury. In mice lacking Ehmt2 in DRG neurons, nerve injury failed to reduce the expression level of MORs and the morphine effect. In addition, G9a inhibition or Ehmt2 knockout in DRG neurons normalized nerve injury-induced reduction in the inhibitory effect of the opioid on synaptic glutamate release from primary afferent nerves. Our findings indicate that G9a contributes critically to transcriptional repression of MORs in primary sensory neurons in neuropathic pain. G9a inhibitors may be used to enhance the opioid analgesic effect in the treatment of chronic neuropathic pain. PMID:26917724

  17. Association of Histone Methyltransferase G9a and Overall Survival After Liver Resection of Patients With Hepatocellular Carcinoma With a Median Observation of 40 Months.

    Science.gov (United States)

    Bai, Kai; Cao, Yi; Huang, Chao; Chen, Jianwei; Zhang, Xiaojin; Jiang, Yi

    2016-01-01

    Approximately 50% of patients with hepatocellular carcinoma (HCC) reside in China. HCC is associated with very high mortality compared with other cancers. Although numerous factors influence the survival of patients with HCC who undergo liver resection, the role of the tumor biomarker histone methyltransferase (G9a) is unknown.We enrolled 350 patients with HCC who underwent liver resection and followed them for 40 months. Patients' clinicopathologic characteristics were acquired from medical records, and overall survival was determined using multiple methods. We conducted an immunohistochemical analysis of study G9a expression in HCC tissues. We used χ test to evaluate the significance of the relationships between G9a and other factors and Cox proportional hazards regression to estimate the hazard ratios and 95% confidence intervals.The levels of alpha-fetoprotein were significantly higher in patients with G9a-positive tumors. TNM stage, elevated alpha-fetoprotein level, and G9a overexpression were associated with worse outcomes.High expression of G9a was associated with worse outcomes, indicating that G9a may serve as a prognostic biomarker for patients with HCC who undergo surgical resection. Because of its role in cell proliferation, G9a represents a potential therapeutic target. PMID:26765460

  18. Severe Hypomyelination and Developmental Defects Are Caused in Mice Lacking Protein Arginine Methyltransferase 1 (PRMT1) in the Central Nervous System.

    Science.gov (United States)

    Hashimoto, Misuzu; Murata, Kazuya; Ishida, Junji; Kanou, Akihiko; Kasuya, Yoshitoshi; Fukamizu, Akiyoshi

    2016-01-29

    Protein arginine methyltransferase 1 (PRMT1) is involved in cell proliferation, DNA damage response, and transcriptional regulation. Although PRMT1 is extensively expressed in the CNS at embryonic and perinatal stages, the physiological role of PRMT1 has been poorly understood. Here, to investigate the primary function of PRMT1 in the CNS, we generated CNS-specific PRMT1 knock-out mice by the Cre-loxP system. These mice exhibited postnatal growth retardation with tremors, and most of them died within 2 weeks after birth. Brain histological analyses revealed prominent cell reduction in the white matter tracts of the mutant mice. Furthermore, ultrastructural analysis demonstrated that myelin sheath was almost completely ablated in the CNS of these animals. In agreement with hypomyelination, we also observed that most major myelin proteins including myelin basic protein (MBP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and myelin-associated glycoprotein (MAG) were dramatically decreased, although neuronal and astrocytic markers were preserved in the brain of CNS-specific PRMT1 knock-out mice. These animals had a reduced number of OLIG2(+) oligodendrocyte lineage cells in the white matter. We found that expressions of transcription factors essential for oligodendrocyte specification and further maturation were significantly suppressed in the brain of the mutant mice. Our findings provide evidence that PRMT1 is required for CNS development, especially for oligodendrocyte maturation processes. PMID:26637354

  19. Burkholderia glumae ToxA Is a Dual-Specificity Methyltransferase That Catalyzes the Last Two Steps of Toxoflavin Biosynthesis.

    Science.gov (United States)

    Fenwick, Michael K; Philmus, Benjamin; Begley, Tadhg P; Ealick, Steven E

    2016-05-17

    Toxoflavin is a major virulence factor of the rice pathogen Burkholderia glumae. The tox operon of B. glumae contains five putative toxoflavin biosynthetic genes toxABCDE. ToxA is a predicted S-adenosylmethionine-dependent methyltransferase, and toxA knockouts of B. glumae are less virulent in plant infection models. In this study, we show that ToxA performs two consecutive methylations to convert the putative azapteridine intermediate, 1,6-didemethyltoxoflavin, to toxoflavin. In addition, we report a series of crystal structures of ToxA complexes that reveals the molecular basis of the dual methyltransferase activity. The results suggest sequential methylations with initial methylation at N6 of 1,6-didemethyltoxoflavin followed by methylation at N1. The two azapteridine orientations that position N6 or N1 for methylation are coplanar with a 140° rotation between them. The structure of ToxA contains a class I methyltransferase fold having an N-terminal extension that either closes over the active site or is largely disordered. The ordered conformation places Tyr7 at a position of a structurally conserved tyrosine site of unknown function in various methyltransferases. Crystal structures of ToxA-Y7F consistently show a closed active site, whereas structures of ToxA-Y7A consistently show an open active site, suggesting that the hydroxyl group of Tyr7 plays a role in opening and closing the active site during the multistep reaction. PMID:27070241

  20. Thirteen new patients with guanidinoacetate methyltransferase deficiency and functional characterization of nineteen novel missense variants in the GAMT gene

    DEFF Research Database (Denmark)

    Mercimek-Mahmutoglu, Saadet; Ndika, Joseph; Kanhai, Warsha;

    2014-01-01

    Guanidinoacetate methyltransferase deficiency (GAMT-D) is an autosomal recessively inherited disorder of creatine biosynthesis. Creatine deficiency on cranial proton magnetic resonance spectroscopy, and elevated guanidinoacetate levels in body fluids are the biomarkers of GAMT-D. In 74 patients 5...

  1. The Cfr rRNA methyltransferase confers resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics

    DEFF Research Database (Denmark)

    Long, K. S.; Poehlsgaard, Jacob; Kehrenberg, C.; Schwarz, S.; Vester, B.

    2006-01-01

    A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin and...

  2. No up-regulation of the phosphatidylethanolamine N-methyltransferase pathway and choline production by sex hormones in cats

    NARCIS (Netherlands)

    Valtolina, Chiara; Vaandrager, Arie B; Favier, Robert P; Robben, Joris H; Tuohetahuntila, Maidina; Kummeling, Anne; Jeusette, Isabelle; Rothuizen, Jan

    2015-01-01

    BACKGROUND: Feline hepatic lipidosis (FHL) is a common cholestatic disease affecting cats of any breed, age and sex. Both choline deficiency and low hepatic phosphatidylethanolamine N-methyltransferase (PEMT) activity are associated with hepatic lipidosis (HL) in humans, mice and rats. The PEMT expr

  3. Implications of fast-time scale dynamics of human DNA/RNA cytosine methyltransferases (DNMTs) for protein function

    Czech Academy of Sciences Publication Activity Database

    Evans, D. A.; Bronowska, Agnieszka Katarzyna

    2010-01-01

    Roč. 125, 3/6 (2010), s. 407-418. ISSN 1432-881X Institutional research plan: CEZ:AV0Z40550506 Keywords : MD simulations * DNA/RNA methyltransferase * enthalpy-entropy compensation Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.903, year: 2010

  4. Flexible double-headed cytosine-linked 2'-deoxycytidine nucleotides. Synthesis, polymerase incorporation to DNA and interaction with DNA methyltransferases

    Czech Academy of Sciences Publication Activity Database

    Kielkowski, Pavel; Cahová, Hana; Pohl, Radek; Hocek, Michal

    2016-01-01

    Roč. 24, č. 6 (2016), s. 1268-1276. ISSN 0968-0896 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : nucleosides * nucleotides * pyrimidines * DNA methyltransferases * DNA polymerases Subject RIV: CC - Organic Chemistry Impact factor: 2.793, year: 2014

  5. An assay for human erythrocyte catechol-O-methyltransferase activity using a catechol estrogen as the substrate

    International Nuclear Information System (INIS)

    A radiometric assay for catechol-O-methyltransferase (COMT) activity in human erythrocytes is described that employs 2-hydroxy[3H]estrone, and nonradiolabeled S-adenosylmethionine (SAM) as the cosubstrates. The ease of separation of the product of the reaction, 2-methoxy[3H)estrone from 2-hydroxy-[3H]estrone makes it possible to achieve low reaction blanks. The assay is very sensitive, and only 200 μl of whole blood are used per determination. The assay is highly reproducible. The interassay variability (coefficient of variation) was 6.5% for 24 assays of COMT activity in red blood cells in blood obtained daily for 24 days from one person. In incubations conducted at 370C for 30 min, the catechol-O-methyltransferase activity was a linear function of enzyme concentration (equivalent to 11 to 180 μl of packed red blood cells). Employing this assay, we evaluated the catalytic conversion of 2-hydroxyestrone to 2-methoxy-estrone by catechol-O-methyltransferase from human red blood cells and found that the apparent Michaelis constant and the apparent maximal rate of reaction were 3 X 10-7 M and 6.7 X 10-9 mol.ml-1 erythrocytes . h-1, respectively. The catechol-O-methyltransferase activity measured in erythrocytes obtained from 100 healthy subjects (men and nonpregnant women) was 8.2 +- 0.17 (mean +- S.E.) nmol 2-methoxyestrone . ml-1 erythrocytes. h-1. (Auth.)

  6. Phylogenetic, molecular, and biochemical characterization of caffeic aicd O-methyltransferase (COMT) gene family in Brachypodium distachyon

    Science.gov (United States)

    Caffeic acid O-methyltransferase (COMT) is one of the important enzymes controlling lignin monomer production in plant cell wall synthesis. Analysis of the genome sequence of new grass model Brachypodium distachyon identified four COMT gene homologues, designated as BdCOMT1, BdCOMT2, BdCOMT3, and ...

  7. Current understanding of the interplay between catechol-O-methyltransferase genetic variants, sleep, brain development and cognitive performance in schizophrenia

    NARCIS (Netherlands)

    Tucci, Valter; Lassi, Glenda; Kas, Martien J

    2012-01-01

    Abnormal sleep is an endophenotype of schizophrenia. Here we provide an overview of the genetic mechanisms that link specific sleep physiological processes to schizophrenia-related cognitive defects. In particular, we will review the possible relationships between catechol-O-methyltransferase (COMT)

  8. Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria

    DEFF Research Database (Denmark)

    Giessing, Anders; Jensen, Søren Skov; Rasmussen, Anette;

    2009-01-01

    The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding to an a...

  9. Polymorphisms in O-methyltransferase genes are associated with stover cell wall digestibility in European maize (Zea mays L.)

    DEFF Research Database (Denmark)

    Brenner, Everton A; Zein, Imad; Chen, Yongsheng;

    2010-01-01

    Background OMT (O-methyltransferase) genes are involved in lignin biosynthesis, which relates to stover cell wall digestibility. Reduced lignin content is an important determinant of both forage quality and ethanol conversion efficiency of maize stover. Results Variation in genomic sequences codi...

  10. An integrated epigenetic and genetic analysis of DNA methyltransferase genes (DNMTs) in tumor resistant and susceptible chicken lines

    Science.gov (United States)

    Both epigenetic alterations and genetic variations play essential roles in tumorigenesis. The epigenetic modification of DNA methylation is catalyzed and maintained by the DNA methyltransferases (DNMT3a, DNMT3b and DNMT1). DNA mutations and DNA methylation profiles of DNMTs themselves and their rela...

  11. Methanol:coenzyme M methyltransferase from Methanosarcina barkeri. Zinc dependence and thermodynamics of the methanol:cob(I)alamin methyltransferase reaction.

    Science.gov (United States)

    Sauer, K; Thauer, R K

    1997-10-01

    In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methyl-coenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated methyltransferases 1 (MT1) and 2 (MT2). Transferase MT1, which is composed of a 50-kDa subunit, MtaB, and a 27-kDa corrinoid-harbouring subunit, MtaC, has been shown recently to catalyze the methylation of free cob(I)alamin with methanol [Sauer, K., Harms, U. & Thauer, R. K. (1997) Eur. J. Biochem. 243, 670-677]. We report here that this reaction is catalyzed by subunit MtaB overproduced in Escherichia coli. MtaB also catalyzed the formation of methanol from methylcobalamin and H2O, the hydrolysis being associated with a free-energy change deltaG(o)' of approximately +7.0 kJ/mol. MtaB was found to contain 1 mol zinc, and its activity to be zinc dependent (pK(Zn2+) = 9.3). The zinc dependence of the MT2 (MtaA)-catalyzed reaction is also described (pK(Zn2+) = 9.6). PMID:9363780

  12. Mutant MMP-9 and HGF gene transfer enhance resolution of CCl4-induced liver fibrosis in rats: role of ASH1 and EZH2 methyltransferases repression.

    Directory of Open Access Journals (Sweden)

    Hussein Atta

    Full Text Available Hepatocyte growth factor (HGF gene transfer inhibits liver fibrosis by regulating aberrant cellular functions, while mutant matrix metalloproteinase-9 (mMMP-9 enhances matrix degradation by neutralizing the elevated tissue inhibitor of metalloproteinase-1 (TIMP-1. It was shown that ASH1 and EZH2 methyltransferases are involved in development of liver fibrosis; however, their role in the resolution phase of liver fibrosis has not been investigated. This study evaluated the role of ASH1 and EZH2 in two mechanistically different therapeutic modalities, HGF and mMMP-9 gene transfer in CCl4 induced rat liver fibrosis. Liver fibrosis was induced in rats with twice a week intraperitoneal injection of CCl4 for 8 weeks. Adenovirus vectors encoding mMMP-9 or HGF genes were injected through tail vein at weeks six and seven and were sacrificed one week after the second injection. A healthy animal group was likewise injected with saline to serve as a negative control. Rats treated with mMMP-9 showed significantly lower fibrosis score, less Sirius red stained collagen area, reduced hydroxyproline and ALT concentration, decreased transforming growth factor beta 1 (TGF-β1 mRNA and lower labeling indices of α smooth muscle actin (α-SMA and proliferating cell nuclear antigen (PCNA stained cells compared with HGF- or saline-treated rats. Furthermore, TIMP-1 protein expression in mMMP-9 group was markedly reduced compared with all fibrotic groups. ASH1 and EZH2 protein expression was significantly elevated in fibrotic liver and significantly decreased in mMMP-9- and HGF-treated compared to saline-treated fibrotic livers with further reduction in the mMMP-9 group.Gene transfer of mMMP-9 and HGF reduced liver fibrosis in rats. ASH1 and EZH2 methyltransferases are significantly reduced in mMMP-9 and HGF treated rats which underlines the central role of these enzymes during fibrogenesis. Future studies should evaluate the role of selective pharmacologic inhibitors

  13. Hypermethylation of CpG island in O6-methylguanine-DNA methyltransferase gene was associated with K-rasG to A mutation in colorectal tumor

    Institute of Scientific and Technical Information of China (English)

    Jian Qi; You-Qing Zhu; Mei-Fang Huang; Dong Yang

    2005-01-01

    AIM: To investigate the functions of promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) gene in colorectal tumorigenesis and progression.METHODS: The promoter hypermethylation of MGMT gene was detected in 27 sporadic colorectal adenomas,62 sporadic colorectal carcinomas and 20 normal colorectal mucosa tissues by methylation-specific PCR. At the same time, the expression of MGMT protein was carried out in the same samples using immunohistochemistry. Mutantallele-specific amplification was used to detect K-rasG to A point mutation in codon 12.RESULTS: None of the normal colorectal mucosa tissues showed methylated bands. Promoter hypermethylation was detected in 40.7% (11 of 27) of adenomas and 43.5% (27 of 62) of carcinomas. MGMT proteins were expressed in nucleus and cytoplasm of normal colorectal mucosa tissues. Loss of MGMT expression was found in 22.2% (6 of 27) of adenomas and 45.2% (28 of 62) of carcinomas. The difference between them was significant (P = 0.041). In the 6 adenomas and 28 carcinomas losing MGMT expression, 5 and 24 cases presented methylation,respectively (P = 0.027, P<0.001). Thirteen of the 19 colorectal tumors with K-rasG to A point mutation in codon 12 had methylated MGMT(P = 0.011). The frequencies of K-rasG to A point mutation were 35.3% (12 of 34) and 12.7% (7 of 55) in tumors losing MGMT expression and with normal expression, respectively.CONCLUSION: Promoter hypermethylation and loss of expression of MGMT gene were common events in colorectal tumorigenesis, and loss of expression of MGMT occurs more frequently in carcinomas than in adenomas in sporadic patients. Hypermethylation of the CpG island of MGMT gene was associated with loss of MGMT expression and K-ras G to A point mutation in colorectal tumor. The frequency of K-ras G to A point mutation was increased in tumors losing MGMT expression. It suggests that epigenetic inactivation of MGMT plays an important role in colorectal neoplasia.

  14. The role of gene body cytosine modifications in MGMT expression and sensitivity to temozolomide

    OpenAIRE

    Moen, Erika L.; Stark, Amy L.; Zhang, Wei; Dolan, M. Eileen; Godley, Lucy A.

    2014-01-01

    The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is known to play a role in sensitivity to temozolomide. Promoter hypermethylation of MGMT is commonly used to predict low expression levels of MGMT in gliomas, despite observed discordance between promoter methylation and protein levels. Here, we investigated the functional role of gene body cytosine modification in regulating levels of MGMT gene expression and sensitivity to temozolomide. In 91 human glioblastoma samples, w...

  15. Identification of regions correlating MGMT promoter methylation and gene expression in glioblastomas

    OpenAIRE

    Everhard, Sibille; Tost, Jörg; Abdalaoui, Hafida El; Crinière, Emmanuelle; Busato, Florence; Marie, Yannick; Gut, Ivo G.; Sanson, Marc; Mokhtari, Karima; Laigle-Donadey, Florence; Hoang-Xuan, Khê; Delattre, Jean-Yves; Thillet, Joëlle

    2009-01-01

    The O6-methylguanine-DNA methyltransferase gene (MGMT) is methylated in several cancers, including gliomas. However, the functional role of cysteine-phosphate-guanine (CpG) island (CGI) methylation in MGMT silencing is still controversial. The aim of this study was to investigate whether MGMT CGI methylation correlates inversely with RNA expression of MGMT in glioblastomas and to determine the CpG region whose methylation best reflects the level of expression. The methylation level of CpG sit...

  16. Prognostic impact of MGMT promoter methylation and MGMT and CD133 expression in colorectal adenocarcinoma

    OpenAIRE

    Oliver, Jaime Antonio; Ortiz Quesada, Ra??l; Melguizo Alonso, Consolaci??n; ??lvarez, Pablo Juan; G??mez-Mill??n, Jaime; Prados, Jos??

    2014-01-01

    Background: New biomarkers are needed for the prognosis of advanced colorectal cancer, which remains incurable by conventional treatments. O6-methylguanine DNA methyltransferase (MGMT) methylation and protein expression have been related to colorectal cancer treatment failure and tumor progression. Moreover, the presence in these tumors of cancer stem cells, which are characterized by CD133 expression, has been associated with chemoresistance, radioresistance, metastasis, and local recurr...

  17. O6-methylguanine-DNA methyltransferase (MGMT): can function explain a suicidal mechanism?

    Science.gov (United States)

    Gouws, Chrisna; Pretorius, Pieter J

    2011-11-01

    Why does O(6)-methylguanine-DNA methyltransferase (MGMT), an indispensable DNA repair enzyme, have a mechanism which seems to run counter to its importance? This enzyme is key to the removal of detrimental alkyl adducts from guanine bases. Although the mechanism is well known, an unusual feature surrounds its mode of action, which is its so-called suicidal endpoint. In addition, induction of MGMT is highly variable and its kinetics is atypical. These features raise some questions on the seemingly paradoxical mechanism. In this manuscript we point out that, although there is ample literature regarding the "how" of the MGMT enzyme, we found a lack of information on "why" this specific mechanism is in place. We then ask whether we know all there is to know about MGMT, or if perhaps there is a further as yet unknown function for MGMT, or if the suicidal mechanism may play some kind of protective role in the cell. PMID:21864987

  18. Structural Basis for Binding of RNA and Cofactor by a KsgA Methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Tu, Chao; Tropea, Joseph E.; Austin, Brian P.; Court, Donald L.; Waugh, David S.; Ji, Xinhua; (NCI)

    2009-03-27

    Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. Here we report structures of Aquifex aeolicus KsgA, in its ligand-free form, in complex with RNA, and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), revealing critical structural information on KsgA-RNA and KsgA-SAH interactions. Moreover, the structures show how conformational changes that occur upon RNA binding create the cofactor-binding site. There are nine conserved functional motifs (motifs IVIII and X) in KsgA. Prior to RNA binding, motifs I and VIII are flexible, each exhibiting two distinct conformations. Upon RNA binding, the two motifs become stabilized in one of these conformations, which is compatible with the binding of SAH. Motif X, which is also stabilized upon RNA binding, is directly involved in the binding of SAH.

  19. Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases

    DEFF Research Database (Denmark)

    Ntokou, Eleni; Hansen, Lykke Haastrup; Kongsted, Jacob;

    2015-01-01

    -ray structure of RlmN. We used a trinucleotide as target sequence and assessed its positioning at the active site for methylation. The calculations are in accordance with different poses of the trinucleotide in the two enzymes indicating major evolutionary changes to shift the C2/C8 specificities. To explore......Cfr and RlmN methyltransferases both modify adenine 2503 in 23S rRNA (Escherichia coli numbering). RlmN methylates position C2 of adenine while Cfr methylates position C8, and to a lesser extent C2, conferring antibiotic resistance to peptidyl transferase inhibitors. Cfr and RlmN show high sequence...... interchangeability between Cfr and RlmN we constructed various combinations of their genes. The function of the mixed genes was investigated by RNA primer extension analysis to reveal methylation at 23S rRNA position A2503 and by MIC analysis to reveal antibiotic resistance. The catalytic site is expected to be...

  20. Adding a Lysine Mimic in the Design of Potent Inhibitors of Histone Lysine Methyltransferases

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Yanqi; Ganesh, Thota; Horton, John R.; Spannhoff, Astrid; Liu, Jin; Sun, Aiming; Zhang, Xing; Bedford, Mark T.; Shinkai, Yoichi; Snyder, James P.; Cheng, Xiaodong (Emory); (Kyoto); (Texas)

    2010-07-19

    Dynamic histone lysine methylation involves the activities of modifying enzymes (writers), enzymes removing modifications (erasers), and readers of the histone code. One common feature of these activities is the recognition of lysines in methylated and unmethylated states, whether they are substrates, reaction products, or binding partners. We applied the concept of adding a lysine mimic to an established inhibitor (BIX-01294) of histone H3 lysine 9 methyltransferases G9a and G9a-like protein by including a 5-aminopentyloxy moiety, which is inserted into the target lysine-binding channel and becomes methylated by G9a-like protein, albeit slowly. The compound enhances its potency in vitro and reduces cell toxicity in vivo. We suggest that adding a lysine or methyl-lysine mimic should be considered in the design of small-molecule inhibitors for other methyl-lysine writers, erasers, and readers.

  1. Molecular basis for oncohistone H3 recognition by SETD2 methyltransferase.

    Science.gov (United States)

    Yang, Shuang; Zheng, Xiangdong; Lu, Chao; Li, Guo-Min; Allis, C David; Li, Haitao

    2016-07-15

    High-frequency point mutations of genes encoding histones have been identified recently as novel drivers in a number of tumors. Specifically, the H3K36M/I mutations were shown to be oncogenic in chondroblastomas and undifferentiated sarcomas by inhibiting H3K36 methyltransferases, including SETD2. Here we report the crystal structures of the SETD2 catalytic domain bound to H3K36M or H3K36I peptides with SAH (S-adenosylhomocysteine). In the complex structure, the catalytic domain adopts an open conformation, with the K36M/I peptide snuggly positioned in a newly formed substrate channel. Our structural and biochemical data reveal the molecular basis underying oncohistone recognition by and inhibition of SETD2. PMID:27474439

  2. Multi-site-specific 16S rRNA methyltransferase RsmF from Thermus thermophilus

    DEFF Research Database (Denmark)

    Demirci, Hasan; Larsen, Line H G; Hansen, Trine;

    2010-01-01

    Cells devote a significant effort toward the production of multiple modified nucleotides in rRNAs, which fine tune the ribosome function. Here, we report that two methyltransferases, RsmB and RsmF, are responsible for all four 5-methylcytidine (m(5)C) modifications in 16S rRNA of Thermus...... thermophilus. Like Escherichia coli RsmB, T. thermophilus RsmB produces m(5)C967. In contrast to E. coli RsmF, which introduces a single m(5)C1407 modification, T. thermophilus RsmF modifies three positions, generating m(5)C1400 and m(5)C1404 in addition to m(5)C1407. These three residues are clustered near...

  3. Nucleosome Binding Alters the Substrate Bonding Environment of Histone H3 Lysine 36 Methyltransferase NSD2.

    Science.gov (United States)

    Poulin, Myles B; Schneck, Jessica L; Matico, Rosalie E; Hou, Wangfang; McDevitt, Patrick J; Holbert, Marc; Schramm, Vern L

    2016-06-01

    Nuclear receptor-binding SET domain protein 2 (NSD2) is a histone H3 lysine 36 (H3K36)-specific methyltransferase enzyme that is overexpressed in a number of cancers, including multiple myeloma. NSD2 binds to S-adenosyl-l-methionine (SAM) and nucleosome substrates to catalyze the transfer of a methyl group from SAM to the ε-amino group of histone H3K36. Equilibrium binding isotope effects and density functional theory calculations indicate that the SAM methyl group is sterically constrained in complex with NSD2, and that this steric constraint is released upon nucleosome binding. Together, these results show that nucleosome binding to NSD2 induces a significant change in the chemical environment of enzyme-bound SAM. PMID:27183271

  4. Characterization of three O-methyltransferases involved in noscapine biosynthesis in opium poppy.

    Science.gov (United States)

    Dang, Thu-Thuy T; Facchini, Peter J

    2012-06-01

    Noscapine is a benzylisoquinoline alkaloid produced in opium poppy (Papaver somniferum) and other members of the Papaveraceae. It has been used as a cough suppressant and more recently was shown to possess anticancer activity. However, the biosynthesis of noscapine in opium poppy has not been established. A proposed pathway leading from (S)-reticuline to noscapine includes (S)-scoulerine, (S)-canadine, and (S)-N-methylcanadine as intermediates. Stem cDNA libraries and latex extracts of eight opium poppy cultivars displaying different alkaloid profiles were subjected to massively parallel pyrosequencing and liquid chromatography-tandem mass spectrometry, respectively. Comparative transcript and metabolite profiling revealed the occurrence of three cDNAs encoding O-methyltransferases designated as SOMT1, SOMT2, and SOMT3 that correlated with the accumulation of noscapine in the eight cultivars. SOMT transcripts were detected in all opium poppy organs but were most abundant in aerial organs, where noscapine primarily accumulates. SOMT2 and SOMT3 showed strict substrate specificity and regiospecificity as 9-O-methyltransferases targeting (S)-scoulerine. In contrast, SOMT1 was able to sequentially 9- and 2-O-methylate (S)-scoulerine, yielding (S)-tetrahydropalmatine. SOMT1 also sequentially 3'- and 7-O-methylated both (S)-norreticuline and (S)-reticuline with relatively high substrate affinity, yielding (S)-tetrahydropapaverine and (S)-laudanosine, respectively. The metabolic functions of SOMT1, SOMT2, and SOMT3 were investigated in planta using virus-induced gene silencing. Reduction of SOMT1 or SOMT2 transcript levels resulted in a significant decrease in noscapine accumulation. Reduced SOMT1 transcript levels also caused a decrease in papaverine accumulation, confirming the selective roles for these enzymes in the biosynthesis of both alkaloids in opium poppy. PMID:22535422

  5. Functional characterisation of three o-methyltransferases involved in the biosynthesis of phenolglycolipids in Mycobacterium tuberculosis.

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    Roxane Simeone

    Full Text Available Phenolic glycolipids are produced by a very limited number of slow-growing mycobacterial species, most of which are pathogen for humans. In Mycobacterium tuberculosis, the etiologic agent of tuberculosis, these molecules play a role in the pathogenicity by modulating the host immune response during infection. The major variant of phenolic glycolipids produced by M. tuberculosis, named PGL-tb, consists of a large lipid core terminated by a glycosylated aromatic nucleus. The carbohydrate part is composed of three sugar residues, two rhamnosyl units and a terminal fucosyl residue, which is per-O-methylated, and seems to be important for pathogenicity. While most of the genes responsible for the synthesis of the lipid core domain and the saccharide appendage of PGL-tb have been characterized, the enzymes involved in the O-methylation of the fucosyl residue of PGL-tb remain unknown. In this study we report the identification and characterization of the methyltransferases required for the O-methylation of the terminal fucosyl residue of PGL-tb. These enzymes are encoded by genes Rv2954c, Rv2955c and Rv2956. Mutants of M. tuberculosis harboring deletion within these genes were constructed. Purification and analysis of the phenolglycolipids produced by these strains, using a combination of mass spectrometry and NMR spectroscopy, revealed that Rv2954c, Rv2955c and Rv2956 encode the methyltransferases that respectively catalysed the O-methylation of the hydroxyl groups located at positions 3, 4 and 2 of the terminal fucosyl residue of PGL-tb. Our data also suggest that methylation at these positions is a sequential process, starting with position 2, followed by positions 4 and 3.

  6. Computational Investigation of the Interplay of Substrate Positioning and Reactivity in Catechol O-Methyltransferase.

    Science.gov (United States)

    Patra, Niladri; Ioannidis, Efthymios I; Kulik, Heather J

    2016-01-01

    Catechol O-methyltransferase (COMT) is a SAM- and Mg2+-dependent methyltransferase that regulates neurotransmitters through methylation. Simulations and experiments have identified divergent catecholamine substrate orientations in the COMT active site: molecular dynamics simulations have favored a monodentate coordination of catecholate substrates to the active site Mg2+, and crystal structures instead preserve bidentate coordination along with short (2.65 Å) methyl donor-acceptor distances. We carry out longer dynamics (up to 350 ns) to quantify interconversion between bidentate and monodentate binding poses. We provide a systematic determination of the relative free energy of the monodentate and bidentate structures in order to identify whether structural differences alter the nature of the methyl transfer mechanism and source of enzymatic rate enhancement. We demonstrate that the bidentate and monodentate binding modes are close in energy but separated by a 7 kcal/mol free energy barrier. Analysis of interactions in the two binding modes reveals that the driving force for monodentate catecholate orientations in classical molecular dynamics simulations is derived from stronger electrostatic stabilization afforded by alternate Mg2+ coordination with strongly charged active site carboxylates. Mixed semi-empirical-classical (SQM/MM) substrate C-O distances (2.7 Å) for the bidentate case are in excellent agreement with COMT X-ray crystal structures, as long as charge transfer between the substrates, Mg2+, and surrounding ligands is permitted. SQM/MM free energy barriers for methyl transfer from bidentate and monodentate catecholate configurations are comparable at around 21-22 kcal/mol, in good agreement with experiment (18-19 kcal/mol). Overall, the work suggests that both binding poses are viable for methyl transfer, and accurate descriptions of charge transfer and electrostatics are needed to provide balanced relative barriers when multiple binding poses are

  7. Suv4-20h histone methyltransferases promote neuroectodermal differentiation by silencing the pluripotency-associated Oct-25 gene.

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    Dario Nicetto

    Full Text Available Post-translational modifications (PTMs of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as a direct target of xSu4-20h enzyme mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4-20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved

  8. Modulation of cell adhesion and migration by the histone methyltransferase subunit mDpy-30 and its interacting proteins.

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    Bin Xia

    Full Text Available We have previously shown that a subset of mDpy-30, an accessory subunit of the nuclear histone H3 lysine 4 methyltransferase (H3K4MT complex, also localizes at the trans-Golgi network (TGN, where its recruitment is mediated by the TGN-localized ARF guanine nucleotide exchange factor (ArfGEF BIG1. Depletion of mDpy-30 inhibits the endosome-to-TGN transport of internalized CIMPR receptors and concurrently promotes their accumulation at the cell protrusion. These observations suggest mDpy-30 may play a novel role at the crossroads of endosomal trafficking, nuclear transcription and adhesion/migration. Here we provide novel mechanistic and functional insight into this association. First, we demonstrate a direct interaction between mDpy-30 and BIG1 and locate the binding region in the N-terminus of BIG1. Second, we provide evidence that the depletion or overexpression of mDpy-30 enhances or inhibits cellular adhesion/migration of glioma cells in vitro, respectively. A similar increase in cell adhesion/migration is observed in cells with reduced levels of BIG1 or other H3K4MT subunits. Third, knockdown of mDpy-30, BIG1, or the RbBP5 H3K4MT subunit increases the targeting of beta1 integrin to cell protrusions, and suppression of H3K4MT activity by depleting mDpy-30 or RbBP5 leads to increased protein and mRNA levels of beta1 integrin. Moreover, stimulation of cell adhesion/migration via mDpy-30 knockdown is abolished after treating cells with a function-blocking antibody to beta1 integrin. Taken together, these data indicate that mDpy-30 and its interacting proteins function as a novel class of cellular adhesion/migration modulators partially by affecting the subcellular distribution of endosomal compartments as well as the expression of key adhesion/migration proteins such as beta1 integrin.

  9. Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

    Science.gov (United States)

    Henderson, Morgan L; Kreuzer, Kenneth N

    2015-01-01

    Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones. PMID:25993347

  10. Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

    Directory of Open Access Journals (Sweden)

    Morgan L Henderson

    Full Text Available Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs, located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs. Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation: RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA. In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

  11. Inhibition of Nonsmall Cell Lung Cancer Cell Migration by Protein Arginine Methyltransferase 1-small Hairpin RNA Through Inhibiting Epithelial-mesenchymal Transition, Extracellular Matrix Degradation, and Src Phosphorylation In Vitro

    Directory of Open Access Journals (Sweden)

    Ting Zhang

    2015-01-01

    Full Text Available Background: Protein arginine methyltransferases 1 (PRMT1 is over-expressed in a variety of cancers, including lung cancer, and is correlated with a poor prognosis of tumor development. This study aimed to investigate the role of PRMT1 in nonsmall cell lung cancer (NSCLC migration in vitro. Methods: In this study, PRMT1 expression in the NSCLC cell line A549 was silenced using lentiviral vector-mediated short hairpin RNAs. Cell migration was measured using both scratch wound healing and transwell cell migration assays. The mRNA expression levels of matrix metalloproteinase 2 (MMP-2 and tissue inhibitor of metalloproteinase 1, 2 (TIMP1, 2 were measured using quantitative real-time reverse transcription-polymerase chain reaction. The expression levels of protein markers for epithelial-mesenchymal transition (EMT (E-cadherin, N-cadherin, focal adhesion kinase (FAK, Src, AKT, and their corresponding phosphorylated states were detected by Western blot. Results: Cell migration was significantly inhibited in the PRMT1 silenced group compared to the control group. The mRNA expression of MMP-2 decreased while TIMP1 and TIMP2 increased significantly. E-cadherin mRNA expression also increased while N-cadherin decreased. Only phosphorylated Src levels decreased in the silenced group while FAK or AKT remained unchanged. Conclusions: PRMT1-small hairpin RNA inhibits the migration abilities of NSCLC A549 cells by inhibiting EMT, extracellular matrix degradation, and Src phosphorylation in vitro.

  12. Large-Scale, Protection-Free Synthesis of Se-Adenosyl-l-selenomethionine Analogues and Their Application as Cofactor Surrogates of Methyltransferases

    OpenAIRE

    Bothwell, Ian R.; Luo, Minkui

    2014-01-01

    S-Adenosyl-l-methionine (SAM) analogues have previously demonstrated their utility as chemical reporters of methyltransferases. Here we describe the facile, large-scale synthesis of Se-alkyl Se-adenosyl-l-selenomethionine (SeAM) analogues and their precursor, Se-adenosyl-l-selenohomocysteine (SeAH). Comparison of SeAM analogues with their equivalent SAM analogues suggests that sulfonium-to-selenonium substitution can enhance their compatibility with certain protein methyltransferases, favorin...

  13. Research Progress of N-methyltransferases Involved in Caffeine Biosynthesis%咖啡碱合成N-甲基转移酶研究进展

    Institute of Scientific and Technical Information of China (English)

    晏嫦妤; 任秋婧; 陈小芳; 李斌; 陈忠正

    2014-01-01

    Caffeine is one of the main quality and functional components in beverage such as tea, coffee, etc. At present, a xanthosine→7-methylxanthosine→7-methylxanthine→theobromine→caffeine pathway is supported as the major route to caffeine, and it is synthesized through three methylation reactions by N- methyltransferase and a nucleosidase reaction by nucleoside hydrolase. N- methyltransferases(NMTs) are important enzymes in caffeine synthesis process. In this paper, the profile of caffeine in plant and the pathway of caffeine synthesis were introduced, and the research progress on the enzymology properties, gene cloning and expression, the relationship of gene function and structure of NMTs involved in caffeine biosynthesis were mainly reviewed. Finally, the research emphasis in the field for the future was discussed and prospected.%咖啡碱是茶叶、咖啡等饮料的主要品质成分及功能成分之一。在植物体内咖啡碱生物合成的核心途径为:黄嘌呤核苷→7-甲基黄嘌呤核苷→7-甲基黄嘌呤→可可碱→咖啡碱,其中包括3步由 N-甲基转移酶催化的转甲基化反应和1步由核糖核苷水解酶催化的脱核苷反应。N-甲基转移酶是参与咖啡碱生物合成的关键酶类。介绍了植物中咖啡碱的基本情况及其生物合成途径,重点综述了咖啡碱合成 N-甲基转移酶的酶学特性、N M Ts 的克隆、基因结构与功能的关系以及基因表达调控研究等方面国内外的研究进展,并对未来该领域的研究重点进行了探讨和展望。

  14. Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells

    Directory of Open Access Journals (Sweden)

    Sharma Shikhar

    2012-01-01

    Full Text Available Abstract Background DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear. Results Here we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation. Conclusions Taken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.

  15. Distribution of the type III DNA methyltransferases modA, modB and modD among Neisseria meningitidis genotypes: implications for gene regulation and virulence.

    Science.gov (United States)

    Tan, Aimee; Hill, Dorothea M C; Harrison, Odile B; Srikhanta, Yogitha N; Jennings, Michael P; Maiden, Martin C J; Seib, Kate L

    2016-01-01

    Neisseria meningitidis is a human-specific bacterium that varies in invasive potential. All meningococci are carried in the nasopharynx, and most genotypes are very infrequently associated with invasive meningococcal disease; however, those belonging to the 'hyperinvasive lineages' are more frequently associated with sepsis or meningitis. Genome content is highly conserved between carriage and disease isolates, and differential gene expression has been proposed as a major determinant of the hyperinvasive phenotype. Three phase variable DNA methyltransferases (ModA, ModB and ModD), which mediate epigenetic regulation of distinct phase variable regulons (phasevarions), have been identified in N. meningitidis. Each mod gene has distinct alleles, defined by their Mod DNA recognition domain, and these target and methylate different DNA sequences, thereby regulating distinct gene sets. Here 211 meningococcal carriage and >1,400 disease isolates were surveyed for the distribution of meningococcal mod alleles. While modA11-12 and modB1-2 were found in most isolates, rarer alleles (e.g., modA15, modB4, modD1-6) were specific to particular genotypes as defined by clonal complex. This suggests that phase variable Mod proteins may be associated with distinct phenotypes and hence invasive potential of N. meningitidis strains. PMID:26867950

  16. Mitochondrial uncoupler carbonyl cyanide M-chlorophenylhydrazone induces the multimer assembly and activity of repair enzyme protein L-isoaspartyl methyltransferase.

    Science.gov (United States)

    Fanélus, Irvens; Desrosiers, Richard R

    2013-07-01

    The protein L-isoaspartyl methyltransferase (PIMT) repairs damaged aspartyl residues in proteins. It is commonly described as a cytosolic protein highly expressed in brain tissues. Here, we report that PIMT is an active monomeric as well as a multimeric protein in mitochondria isolated from neuroblastoma cells. Upon treatments with mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), PIMT monomers level decreased by half while that of PIMT multimers was higher. Gel electrophoresis under reducing conditions of CCCP-induced PIMT multimers led to PIMT monomers accumulation, indicating that multimers resulted from disulfide-linked PIMT monomers. The antioxidant ascorbic acid significantly lowered CCCP-induced formation of PIMT multimers, suggesting that reactive oxygen species contributed to PIMT multimerization. In addition, the elevation of PIMT multimers catalytic activity upon treatments with CCCP was severely inhibited by the reducing agent dithiothreitol. This indicated that PIMT monomers have lower enzymatic activity following CCCP treatments and that activation of PIMT multimers is essentially dependent on the formation of disulfide-linked monomers of PIMT. Furthermore, the perturbation of mitochondrial function by CCCP promoted the accumulation of damaged aspartyl residues in proteins with high molecular weights. Thus, this study demonstrates the formation of active PIMT multimers associated with mitochondria that could play a key role in repairing damaged proteins accumulating during mitochondrial dysfunction. PMID:23319267

  17. Achaete-scute complex homolog-1 promotes DNA repair in the lung carcinogenesis through matrix metalloproteinase-7 and O(6-methylguanine-DNA methyltransferase.

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    Xiao-Yang Wang

    Full Text Available Lung cancer is the leading cause of cancer-related deaths in the world. Achaete-scute complex homolog-1 (Ascl1 is a member of the basic helix-loop-helix (bHLH transcription factor family that has multiple functions in the normal and neoplastic lung such as the regulation of neuroendocrine differentiation, prevention of apoptosis and promotion of tumor-initiating cells. We now show that Ascl1 directly regulates matrix metalloproteinase-7 (MMP-7 and O(6-methylguanine-DNA methyltransferase (MGMT. Loss- and gain-of-function experiments in human bronchial epithelial and lung carcinoma cell lines revealed that Ascl1, MMP-7 and MGMT are able to protect cells from the tobacco-specific nitrosamine NNK-induced DNA damage and the alkylating agent cisplatin-induced apoptosis. We also examined the role of Ascl1 in NNK-induced lung tumorigenesis in vivo. Using transgenic mice which constitutively expressed human Ascl1 in airway lining cells, we found that there was a delay in lung tumorigenesis. We conclude that Ascl1 potentially enhances DNA repair through activation of MMP-7 and MGMT which may impact lung carcinogenesis and chemoresistance. The study has uncovered a novel and unexpected function of Ascl1 which will contribute to better understanding of lung carcinogenesis and the broad implications of transcription factors in tobacco-related carcinogenesis.

  18. Thiopurine S-methyltransferase deficiency: Two nucleotide transitions define the most prevalent mutant allele associated with loss of catalytic activity in caucasians

    Energy Technology Data Exchange (ETDEWEB)

    Tai, Hung-Liang; Krynetski, E.Y.; Yates, C.R. [Univ. of Tennessee, Memphis, TN (United States)] [and others

    1996-04-01

    The autosomal recessive trait of thiopurine S-methyltransferase (TPMT) deficiency is associated with severe hematopoietic toxicity when patients are treated with standard doses of mercaptopurine, azathioprine, or thioguanine. To define the molecular mechanism of this genetic polymorphism, we cloned and characterized the cDNA of a TPMT-deficient patient, which revealed a novel mutant allele (TPMT*3) containing two nucleotide transitions (G{sup 460}{yields}A and A{sup 719}{yields}G) producing amino acid changes at codons 154 (Ala{yields}Thr) and 240 (Tyr{yields}Cys), differing from the rare mutant TPMT allele we previously identified (i.e., TPMT*2 with only G{sup 238}{yields}C). Site-directed mutagenesis and heterologous expression established that either TPMT*3 mutation alone leads to a reduction in catalytic activity (G{sup 460}{yields}A, ninefold reduction; A{sup 179}{yields}G, 1.4-fold reduction), while the presence of both mutations leads to complete loss of activity. Using mutation specific PCR-RFLP analysis, the TPMT*3 allele was detected in genomic DNA from {approx}75% of unrelated white subjects with heterozygous phenotypes, indicating that TPMT*3 is the most prevalent mutant allele associated with TPMT-deficiency in Caucasians. 31 refs., 5 figs., 1 tab.

  19. Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate

    Energy Technology Data Exchange (ETDEWEB)

    Luka, Zigmund; Pakhomova, Svetlana; Loukachevitch, Lioudmila V; Newcomer, Marcia E; Wagner, Conrad [Vanderbilt; (LSU)

    2012-06-27

    Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.

  20. Post-translational protein modifications in type 1 diabetes: a role for the repair enzyme protein-L-isoaspartate (D-aspartate) O-methyltransferase?

    DEFF Research Database (Denmark)

    Wägner, A M; Cloos, P; Bergholdt, R;

    2007-01-01

    15.8+/-2.7 mmol; p<0.01 for high- and low-dose groups vs control group) and residual beta cells were more frequently identified (43% vs 71% vs 86%; p<0.05 for high-dose vs control group) in the treated animals. CONCLUSIONS/INTERPRETATION: The results support a role for post-translational......AIMS/HYPOTHESIS: Post-translational modifications, such as isomerisation of native proteins, may create new antigenic epitopes and play a role in the development of the autoimmune response. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), encoded by the gene PCMT1, is an enzyme that...... expression of Pcmt1, was tested on MIN6 and INS1 cells, to assess its effect on Pcmt1 mRNA and PIMT levels (RT-PCR and western blot) and apoptosis. Forty-five diabetes-prone BioBreeding (BB) Ottawa Karlsburg (OK) rats were randomised to receive 0, 14 or 500 microg/kg (denoted as the control, low-dose and...

  1. 30S Subunit-Dependent Activation of the Sorangium cellulosum So ce56 Aminoglycoside Resistance-Conferring 16S rRNA Methyltransferase Kmr

    Science.gov (United States)

    Savic, Miloje; Sunita, S.; Zelinskaya, Natalia; Desai, Pooja M.; Macmaster, Rachel; Vinal, Kellie

    2015-01-01

    Methylation of bacterial 16S rRNA within the ribosomal decoding center confers exceptionally high resistance to aminoglycoside antibiotics. This resistance mechanism is exploited by aminoglycoside producers for self-protection while functionally equivalent methyltransferases have been acquired by human and animal pathogenic bacteria. Here, we report structural and functional analyses of the Sorangium cellulosum So ce56 aminoglycoside resistance-conferring methyltransferase Kmr. Our results demonstrate that Kmr is a 16S rRNA methyltransferase acting at residue A1408 to confer a canonical aminoglycoside resistance spectrum in Escherichia coli. Kmr possesses a class I methyltransferase core fold but with dramatic differences in the regions which augment this structure to confer substrate specificity in functionally related enzymes. Most strikingly, the region linking core β-strands 6 and 7, which forms part of the S-adenosyl-l-methionine (SAM) binding pocket and contributes to base flipping by the m1A1408 methyltransferase NpmA, is disordered in Kmr, correlating with an exceptionally weak affinity for SAM. Kmr is unexpectedly insensitive to substitutions of residues critical for activity of other 16S rRNA (A1408) methyltransferases and also to the effects of by-product inhibition by S-adenosylhomocysteine (SAH). Collectively, our results indicate that adoption of a catalytically competent Kmr conformation and binding of the obligatory cosubstrate SAM must be induced by interaction with the 30S subunit substrate. PMID:25733511

  2. (Accumulation of methyl-deficient rat liver messenger ribonucleic acid on ethionine administration). Progress report. [Methyltransferase activity in Ehrlich ascites tumor cells and effects of phorbol ester on methyltransferase activity

    Energy Technology Data Exchange (ETDEWEB)

    Borek, E.

    1980-01-01

    Enzyme fractions were isolated from Ehrlich ascites cells which introduced methyl groups into methyl deficient rat liver mRNA and unmethylated vaccinia mRNA. The methyl groups were incorporated at the 5' end into cap 1 structures by the viral enzyme, whereas both cap 0 and cap 1 structures were formed by the Ehrlich ascites cell enzymes. Preliminary results indicate the presence of adenine N/sup 6/-methyltransferase activity in Ehrlich ascites cells. These results indicate that mRNA deficient in 5'-cap methylation and in internal methylation of adenine accumulated in rats on exposure to ethionine. The methyl-deficient mRNA isolated from the liver of ethionine-fed rats differed in its translational properties from mRNA isolated from control animals. Preliminary experiments indicate that single topical application of 17n moles of TPA to mouse skin altered tRNA methyltransferases. The extent of methylation was increased over 2-fold in mouse skin treated with TPA for 48 hours. These changes have been observed as early as 12 hours following TPA treatment. In contrast, the application of initiating dose of DMBA had no effect on these enzymes. It should be emphasized that the changes in tRNA methyltransferases produced by TPA are not merely an increase of the concentration of the enzyme, rather that they represent alterations of specificity of a battery of enzymes. In turn the change in enzyme specificity can produce alterations in the structure of tRNA. (ERB)

  3. The ankyrin repeats of G9a and GLP histone methyltransferases are mono- and dimethyllysine binding modules

    Energy Technology Data Exchange (ETDEWEB)

    Collins, Robert E.; Northrop, Jeffrey P.; Horton, John R.; Lee, David Y.; Zhang, Xing; Stallcup, Michael R.; Cheng, Xiaodong (USC); (Emory)

    2008-03-19

    Histone modifications have important roles in transcriptional control, mitosis and heterochromatin formation. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by mono- and dimethylating histone H3 at Lys9 (H3K9). Here we demonstrate that the ankyrin repeat domains of G9a and GLP bind with strong preference to N-terminal H3 peptides containing mono- or dimethyl K9. X-ray crystallography revealed the basis for recognition of the methylated lysine by a partial hydrophobic cage with three tryptophans and one acidic residue. Substitution of key residues in the cage eliminated the H3 tail interaction. Hence, G9a and GLP contain a new type of methyllysine binding module (the ankyrin repeat domains) and are the first examples of protein (histone) methyltransferases harboring in a single polypeptide the activities that generate and read the same epigenetic mark.

  4. A female adnexal tumor of probable Wolffian origin showing positive O-6-methylguanine-DNA methyltransferase methylation

    Science.gov (United States)

    Kwon, Min Jung; Yun, Min Jeong

    2016-01-01

    Female adnexal tumor of probable Wolffian origin (FATWO) is a rare disease entity that arises from the mesonephric duct system. FATWO is different than other gynecological cancers in terms of embryology. Here, we describe the case of a 52-year-old woman with malignant FATWO. The patient underwent explorative laparotomy and surgical staging after a frozen section revealed malignancy. Detailed examination of the pathologic findings were consistent with FATWO. Counseling and further testing were provided to the patient to assess the risk of germline mutation and epigenetic change. An O-6-methylguanine-DNA methyltransferase gene methylation test was positive, and all other tests were normal. This is the first study to report a case of O-6-methylguanine-DNA methyltransferase methylation with FATWO in Korea. PMID:27462603

  5. Role for DNA Methylation in the Regulation of miR-200c and miR-141 Expression in Normal and Cancer Cells

    Czech Academy of Sciences Publication Activity Database

    Vrba, Lukáš; Jensen, T.J.; Garbe, J.; Heimark, R.L.; Cress, A.E.; Dickinson, S.; Stampfer, M.R.; Futscher, B. W.

    2010-01-01

    Roč. 5, č. 1 (2010), e8697. E-ISSN 1932-6203 Institutional research plan: CEZ:AV0Z50510513 Keywords : GENE-EXPRESSION * HISTONE METHYLTRANSFERASE * E-CADHERIN Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.411, year: 2010

  6. Structure of the Q237W mutant of HhaI DNA methyltransferase: an insight into protein-protein interactions

    OpenAIRE

    Dong, Aiping; Zhou, Lan; Zhang, Xing; Stickel, Shawn; Roberts, Richard J.; Cheng, Xiaodong

    2004-01-01

    We have determined the structure of a mutant (Q237W) of HhaI DNA methyltransferase, complexed with the methyl-donor product AdoHcy. The Q237W mutant proteins were crystallized in the monoclinic space group C2 with two molecules in the crystallographic asymmetric unit. Protein-protein interface calculations in the crystal lattices suggest that the dimer interface has the specific characteristics for homodimer protein-protein interactions, while the two active sites are spatially...

  7. O6-methylguanine DNA methyltransferase as a promising target for the treatment of temozolomide-resistant gliomas

    OpenAIRE

    Fan, C-H; Liu, W-L; Cao, H.; Wen, C; Chen, L.; G. Jiang

    2013-01-01

    Temozolomide (TMZ) is an alkylating agent currently used as first-line therapy for gliomas treatment due to its DNA-damaging effect. However, drug resistance occurs, preventing multi-cycle use of this chemotherapeutic agent. One of the major mechanisms of cancer drug resistance is enhanced activity of a DNA repair enzyme, O6-methylguanine-DNA-methyltransferase (MGMT), which counteracts chemotherapy-induced DNA alkylation and is a key component of chemoresistance. MGMT repairs TMZ-induced DNA ...

  8. A novel route to product specificity in the Suv4-20 family of histone H4K20 methyltransferases

    OpenAIRE

    Southall, Stacey M.; Cronin, Nora B.; Wilson, Jon R.

    2013-01-01

    The delivery of site-specific post-translational modifications to histones generates an epigenetic regulatory network that directs fundamental DNA-mediated processes and governs key stages in development. Methylation of histone H4 lysine-20 has been implicated in DNA repair, transcriptional silencing, genomic stability and regulation of replication. We present the structure of the histone H4K20 methyltransferase Suv4-20h2 in complex with its histone H4 peptide substrate and S-adenosyl methion...

  9. Effects of Active‐Site Modification and Quaternary Structure on the Regioselectivity of Catechol‐O‐Methyltransferase

    OpenAIRE

    Law, Brian J. C.; Bennett, Matthew R.; Thompson, Mark L; Levy, Colin; Shepherd, Sarah A; Leys, David; Micklefield, Jason

    2016-01-01

    Abstract Catechol‐O‐methyltransferase (COMT), an important therapeutic target in the treatment of Parkinson's disease, is also being developed for biocatalytic processes, including vanillin production, although lack of regioselectivity has precluded its more widespread application. By using structural and mechanistic information, regiocomplementary COMT variants were engineered that deliver either meta‐ or para‐methylated catechols. X‐ray crystallography further revealed how the active‐site r...

  10. Noncompetitive Inhibition of Indolethylamine-N-methyltransferase by N,N-Dimethyltryptamine and N,N-Dimethylaminopropyltryptamine

    OpenAIRE

    Chu, Uyen B.; Vorperian, Sevahn K.; Satyshur, Kenneth; Eickstaedt, Kelsey; Cozzi, Nicholas V; Mavlyutov, Timur; Hajipour, Abdol R.; Ruoho, Arnold E.

    2014-01-01

    Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic m...

  11. Protein Isoaspartate Methyltransferase Prevents Apoptosis Induced by Oxidative Stress in Endothelial Cells: Role of Bcl-Xl Deamidation and Methylation

    OpenAIRE

    Amelia Cimmino; Rosanna Capasso; Fabbri Muller; Irene Sambri; Lucia Masella; Marianna Raimo; Maria Luigia De Bonis; Stefania D'Angelo; Vincenzo Zappia; Patrizia Galletti; Diego Ingrosso

    2008-01-01

    BACKGROUND: Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free alpha-carboxyl group at the isoaspartyl site, thus initiating the repair ...

  12. Downregulation of Histone H3 Lysine 9 Methyltransferase G9a Induces Centrosome Disruption and Chromosome Instability in Cancer Cells

    OpenAIRE

    Kondo, Yutaka; Shen, Lanlan; Ahmed, Saira; Boumber, Yanis; Sekido, Yoshitaka; Haddad, Bassem R; Issa, Jean-Pierre J.

    2008-01-01

    Background Modifications of the histone amino-terminal tails affect access of regulatory factors and complexes to chromatin and thereby influence biological processes. Cancer cells are characterized by prominent epigenetic dysregulation, including histone modifications. However, the functional roles of the histone methyltransferases (HMT) in cancer remain unclear. Methodology/Principal Findings We studied RNAi-based inhibition (knockdown, KD) of 2 different H3K9 HMTs, SUV39H1 and G9a. Knockdo...

  13. Epistasis between catechol-O-methyltransferase and type II metabotropic glutamate receptor 3 genes on working memory brain function

    OpenAIRE

    Tan, Hao-Yang; Chen, Qiang; Sust, Steven; Joshua W Buckholtz; Meyers, John D.; Egan, Michael F.; Mattay, Venkata S.; Meyer-Lindenberg, Andreas; Weinberger, Daniel R.; Callicott, Joseph H.

    2007-01-01

    Dopaminergic and glutamatergic systems are critical components responsible for prefrontal signal-to-noise tuning in working memory. Recent functional MRI (fMRI) studies of genetic variation in these systems in catechol-O-methyltransferase (COMT) and in metabotropic glutamate receptor mgluR3 (GRM3), respectively, suggest that these genes influence prefrontal physiological signal-to-noise in humans. Here, using fMRI, we extend these individual gene findings to examine the combined effects of CO...

  14. Therapy and progression – induced O6-methylguanine-DNA methyltransferase and mismatch repair alterations in recurrent glioblastoma multiforme

    OpenAIRE

    Agarwal, S.; Suri, V.; M C Sharma; C. Sarkar

    2015-01-01

    Despite multimodality treatment protocol including surgical resection, radiotherapy, and chemotherapy in patients with glioblastoma multiforme (GBM), most suffer from treatment failure and tumor recurrence within a few months of initial surgery. The effectiveness of temozolomide (TMZ), the most commonly used chemotherapeutic agent, is largely dependent on the methylation status of the promoter of the gene O6-methylguanine-DNA methyltransferase (MGMT) and the integrity of the mismatch repair (...

  15. Affect-Modulated Startle: Interactive Influence of Catechol-O-Methyltransferase Val158Met Genotype and Childhood Trauma

    OpenAIRE

    Klauke, Benedikt; Winter, Bernward; Gajewska, Agnes; Zwanzger, Peter; Reif, Andreas; Herrmann, Martin J.; Dlugos, Andrea; Warrings, Bodo; Jacob, Christian; Mühlberger, Andreas; Arolt, Volker; Pauli, Paul; Deckert, Jürgen; Domschke, Katharina

    2012-01-01

    The etiology of emotion-related disorders such as anxiety or affective disorders is considered to be complex with an interaction of biological and environmental factors. Particular evidence has accumulated for alterations in the dopaminergic and noradrenergic system – partly conferred by catechol-O-methyltransferase (COMT) gene variation – for the adenosinergic system as well as for early life trauma to constitute risk factors for those conditions. Applying a multi-level approach, in a sample...

  16. Catechol O-Methyltransferase Haplotype Predicts Immediate Musculoskeletal Neck Pain and Psychological Symptoms after Motor Vehicle Collision

    OpenAIRE

    McLean, Samuel A.; Diatchenko, Luda; Lee, Young M.; Swor, Robert A.; Domeier, Robert M; Jones, Jeffrey S.; Jones, Christopher W.; Reed, Caroline; Harris, Richard E; Maixner, William; Clauw, Daniel J.; Liberzon, Israel

    2010-01-01

    Genetic variations in the catechol-o-methyltransferase (COMT) gene have been associated with experimental pain and risk of chronic pain development, but no studies have examined genetic predictors of neck pain intensity and other patient characteristics after motor vehicle collision (MVC). We evaluated the association between COMT genotype and acute neck pain intensity and other patient characteristics in 89 Caucasian individuals presenting to the emergency department (ED) after MVC. In the E...

  17. Investigating the Potential Role of Genetic and Epigenetic Variation of DNA Methyltransferase Genes in Hyperplastic Polyposis Syndrome

    OpenAIRE

    Drini, Musa; Nicholas C. Wong; Hamish S Scott; Craig, Jeffrey M; Dobrovic, Alexander; Chelsee A Hewitt; Dow, Christofer; Young, Joanne P; Jenkins, Mark A; Saffery, Richard; Macrae, Finlay A.

    2011-01-01

    Background Hyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC). At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP), potentially linked to aberrant DNA methyltransferase (DNMT) activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). In...

  18. Methyltransferase PRMT1 Is a Binding Partner of HBx and a Negative Regulator of Hepatitis B Virus Transcription

    OpenAIRE

    Benhenda, Shirine; Ducroux, Aurélie; Rivière, Lise; Sobhian, Bijan; Ward, Michael D.; Dion, Sarah; Hantz, Olivier; Protzer, Ulrike; Michel, Marie-Louise; Benkirane, Monsef; Semmes, Oliver J.; Buendia, Marie-Annick; Neuveut, Christine

    2013-01-01

    The hepatitis B virus X protein (HBx) is essential for virus replication and has been implicated in the development of liver cancer. HBx is recruited to viral and cellular promoters and activates transcription by interacting with transcription factors and coactivators. Here, we purified HBx-associated factors in nuclear extracts from HepG2 hepatoma cells and identified protein arginine methyltransferase 1 (PRMT1) as a novel HBx-interacting protein. We showed that PRMT1 overexpression reduced ...

  19. The Role of the Methyltransferase Domain of Bifunctional Restriction Enzyme RM.BpuSI in Cleavage Activity

    OpenAIRE

    Arthur Sarrade-Loucheur; Shuang-yong Xu; Siu-Hong Chan

    2013-01-01

    Restriction enzyme (REase) RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS), bifunctional polypeptide possessing both methyltransferase (MTase) and endonuclease activities (Type IIC) and endonuclease activity stimulated by S-adenosyl-L-methionine (SAM) (Type IIG). The stimulatory effect of SAM on cleavage activity presents a major paradox: a co-factor of the MTase activity that renders the substrate unsusceptible to cleavag...

  20. Genetic contribution of catechol-O-methyltransferase variants in treatment outcome of low back pain: a prospective genetic association study

    OpenAIRE

    Omair, Ahmad; Benedicte A Lie; Reikeras, Olav; Holden, Marit; Brox, Jens I

    2012-01-01

    Background Treatment outcome of low back pain (LBP) is associated with inter-individual variations in pain relief and functional disability. Genetic variants of catechol-O-methyltransferase (COMT) gene have previously been shown to be associated with pain sensitivity and pain medication. This study examines the association between COMT polymorphisms and 7–11 year change in Oswestry Disability Index (ODI) and Visual Analog Score (VAS) for LBP as clinical outcome variables in...

  1. Genetic contribution of catechol-O-methyltransferase variants in treatment outcome of low back pain: a prospective genetic association study

    OpenAIRE

    Omair Ahmad; Lie Benedicte; Reikeras Olav; Holden Marit; Brox Jens

    2012-01-01

    Abstract Background Treatment outcome of low back pain (LBP) is associated with inter-individual variations in pain relief and functional disability. Genetic variants of catechol-O-methyltransferase (COMT) gene have previously been shown to be associated with pain sensitivity and pain medication. This study examines the association between COMT polymorphisms and 7–11 year change in Oswestry Disability Index (ODI) and Visual Analog Score (VAS) for LBP as clinical outcome variables in patients ...

  2. Genetic contribution of catechol-O-methyltransferase variants in treatment outcome of low back pain: a prospective genetic association study

    OpenAIRE

    Omair, Ahmad; Lie, Benedicte Alexandra; Reikeras, Olav; Holden, Marit; Brox, Jens Ivar

    2012-01-01

    Background Treatment outcome of low back pain (LBP) is associated with inter-individual variations in pain relief and functional disability. Genetic variants of catechol-O-methyltransferase (COMT) gene have previously been shown to be associated with pain sensitivity and pain medication. This study examines the association between COMT polymorphisms and 7–11 year change in Oswestry Disability Index (ODI) and Visual Analog Score (VAS) for LBP as clinical outcome variables in patients treated w...

  3. MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells | Office of Cancer Genomics

    Science.gov (United States)

    The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A.

  4. The Histone Methyltransferase SMYD2 Methylates PARP1 and Promotes Poly(ADP-ribosyl)ation Activity in Cancer Cells

    OpenAIRE

    Lianhua Piao; Daechun Kang; Takehiro Suzuki; Akiko Masuda; Naoshi Dohmae; Yusuke Nakamura; Ryuji Hamamoto

    2014-01-01

    Poly(ADP-ribose) polymerase-1 (PARP1) catalyzes the poly(ADP-ribosyl)ation of protein acceptors using NAD+ as the substrate is now considered as an important target for development of anticancer therapy. PARP1 is known to be post-translationally modified in various ways including phosphorylation and ubiquitination, but the physiological role of PARP1 methylation is not well understood. Herein we demonstrated that the histone methyltransferase SMYD2, which plays critical roles in human carcino...

  5. The Histone Methyltransferase SMYD2 Methylates PARP1 and Promotes Poly(ADP-ribosyl)ation Activity in Cancer Cells12

    OpenAIRE

    Piao, Lianhua; Kang, Daechun; Suzuki, Takehiro; Masuda, Akiko; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2014-01-01

    Poly(ADP-ribose) polymerase-1 (PARP1) catalyzes the poly(ADP-ribosyl)ation of protein acceptors using NAD+ as the substrate is now considered as an important target for development of anticancer therapy. PARP1 is known to be post-translationally modified in various ways including phosphorylation and ubiquitination, but the physiological role of PARP1 methylation is not well understood. Herein we demonstrated that the histone methyltransferase SMYD2, which plays critical roles in human carcino...

  6. The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia.

    Directory of Open Access Journals (Sweden)

    William N Pappano

    Full Text Available Histone methyltransferases are epigenetic regulators that modify key lysine and arginine residues on histones and are believed to play an important role in cancer development and maintenance. These epigenetic modifications are potentially reversible and as a result this class of enzymes has drawn great interest as potential therapeutic targets of small molecule inhibitors. Previous studies have suggested that the histone lysine methyltransferase G9a (EHMT2 is required to perpetuate malignant phenotypes through multiple mechanisms in a variety of cancer types. To further elucidate the enzymatic role of G9a in cancer, we describe herein the biological activities of a novel peptide-competitive histone methyltransferase inhibitor, A-366, that selectively inhibits G9a and the closely related GLP (EHMT1, but not other histone methyltransferases. A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2. Additionally, the selectivity profile of A-366 has aided in the discovery of a potentially important role for G9a/GLP in maintenance of leukemia. Treatment of various leukemia cell lines in vitro resulted in marked differentiation and morphological changes of these tumor cell lines. Furthermore, treatment of a flank xenograft leukemia model with A-366 resulted in growth inhibition in vivo consistent with the profile of H3K9me2 reduction observed. In summary, A-366 is a novel and highly selective inhibitor of G9a/GLP that has enabled the discovery of a role for G9a/GLP enzymatic activity in the growth and differentiation status of leukemia cells.

  7. Association Between the Catechol-O-Methyltransferase Val158Met Polymorphism and Self-Perceived Social Acceptance in Adolescent Girls

    OpenAIRE

    Waugh, Christian E.; Dearing, Karen F.; Joormann, Jutta; GOTLIB, IAN H.

    2009-01-01

    Low perceived social acceptance is a significant risk factor for emotional difficulties in children. No studies, however, have examined genetic factors that may underlie individual differences in perceived social acceptance. In the present study we examined the relation between polymorphisms on the catechol-O-methyltransferase (COMT) Val158Met and serotonin transporter promoter (5-HTTLPR) genes and perceived social acceptance in 103 adolescent girls. Only the COMT polymorphism was related to ...

  8. Association between the Catechol-O-Methyltransferase (COMT) Val158Met Polymorphism and Manual Aiming Control in Healthy Subjects

    OpenAIRE

    Lage, Guilherme M.; Miranda, Débora M.; Romano-Silva, Marco A.; Simone B. Campos; Albuquerque, Maicon R.; Corrêa, Humberto; Leandro F. Malloy-Diniz

    2014-01-01

    Background Prefrontal dopamine is catabolized by the catechol-O-methyltransferase (COMT) enzyme. Current evidence suggests that the val/met single nucleotide polymorphism in the COMT gene can predict the efficiency of executive cognition in humans. Individuals carrying the val allele perform more poorly because less synaptic dopamine is available. Methodology/Principal Findings We investigated the influence of the COMT polymorphism on motor performance in a task that requires different execut...

  9. The Catechol-O-Methyltransferase (COMT) val158met Polymorphism Affects Brain Responses to Repeated Painful Stimuli

    OpenAIRE

    Marco L. Loggia; Jensen, Karin; Gollub, Randy L.; Wasan, Ajay D; Edwards, Robert R.; Kong, Jian

    2011-01-01

    Despite the explosion of interest in the genetic underpinnings of individual differences in pain sensitivity, conflicting findings have emerged for most of the identified “pain genes”. Perhaps the prime example of this inconsistency is represented by catechol-O-methyltransferase (COMT), as its substantial association to pain sensitivity has been reported in various studies, but rejected in several others. In line with findings from behavioral studies, we hypothesized that the effect of COMT o...

  10. Effects of the Val(158)Met catechol-O-methyltransferase gene polymorphism on olfactory processing in schizophrenia

    OpenAIRE

    Kamath, Vidyulata; Moberg, Paul J.; Gur, Raquel E.; Doty, Richard L.; Turetsky, Bruce I.

    2011-01-01

    The catechol-O-methyltransferase (COMT) val158met polymorphism has received attention in schizophrenia due to its role in prefrontal dopamine catabolism. Given the rich dopaminergic innervations of the olfactory bulb and the influence of dopamine on the transmission of olfactory signals, we examined the influence of COMT genotype status on the olfactory processing impairment observed in schizophrenia. The University of Pennsylvania Smell Identification Test was administered unirhinally to ind...

  11. Association between Catechol-O-methyltransferase rs4680 (G > A) polymorphism and lung cancer risk

    OpenAIRE

    Tan, Xiang; CHEN, MINGWU

    2014-01-01

    Background The association between the Val158Met polymorphism in the catechol-O-methyltransferase (COMT) gene and lung cancer risk remains controversial and inconclusive. Therefore, the meta-analysis was performed to provide a quality reevaluation of the association between the COMT Val158Met polymorphism and the risk of lung cancer. Methods Two major public databases (Pubmed and Embase) and several Chinese databases were searched for eligible studies. Pooled odds ratios (OR) and 95% confiden...

  12. Genetic Dissection of the Role of Catechol-O-Methyltransferase in Cognition and Stress Reactivity in Mice

    OpenAIRE

    Papaleo, Francesco; Crawley, Jacqueline N.; Song, Jian; Lipska, Barbara K.; Pickel, Jim; Weinberger, Daniel R.; Chen, Jingshan

    2008-01-01

    The COMT (catechol-O-methyltransferase) gene has been linked to a spectrum of human phenotypes, including cognition, anxiety, pain sensitivity and psychosis. Doubts about its clinical impact exist, however, because of the complexity of human COMT polymorphism and clinical variability. We generated transgenic mice overexpressing a human COMT-Val polymorphism (Val-tg), and compared them with mice containing a null COMT mutation. Increased COMT enzyme activity in Val-tg mice resulted in disrupte...

  13. Catechol O-Methyltransferase (COMT) VAL158MET Functional Polymorphism, Dental Mercury Exposure, and Self-Reported Symptoms and Mood

    OpenAIRE

    Heyer, Nicholas J.; Echeverria, Diana; Martin, Michael D.; Farin, Federico M.; Woods, James S.

    2009-01-01

    Associations were evaluated between a functional single nucleotide polymorphism (Val158Met) in the gene encoding the catecholamine catabolic enzyme catechol O-methyltransferase (COMT), dental mercury exposure, and self-reported symptoms and mood among 183 male dentists and 213 female dental assistants. Self-reported symptoms, mood, and detailed work histories were obtained by computerized questionnaire. Spot urine samples were collected and analyzed for mercury concentrations to evaluate rece...

  14. Catechol-O-Methyltransferase (COMT) Val 108/158 Met polymorphism does not modulate executive function in children with ADHD

    OpenAIRE

    Stepanian Marina; Torkaman-Zehi Adam; Deguzman Rosherie; Mbekou Valentin; Lageix Philippe; Amor Leila; Grizenko Natalie; Taerk Evan; Baron Chantal; Joober Ridha

    2004-01-01

    Abstract Background An association has been observed between the catechol-O-methyltransferase (COMT) gene, the predominant means of catecholamine catabolism within the prefrontal cortex (PFC), and neuropsychological task performance in healthy and schizophrenic adults. Since several of the cognitive functions typically deficient in children with Attention Deficit Hyperactivity Disorder (ADHD) are mediated by prefrontal dopamine (DA) mechanisms, we investigated the relationship between a funct...

  15. Catechol-O-methyltransferase: Effects of the Val108Met polymorphism on protein turnover in human cells

    OpenAIRE

    Doyle, Anne E.; Yager, James D.

    2007-01-01

    A single nucleotide polymorphism in the human COMT (catechol-O-methyltransferase) gene has been associated with increased risk for breast cancer and several CNS diseases and disorders. The G to A polymorphism causes a valine (val) to methionine (met) substitution at codon 108 soluble- (S)/ 158 membrane- (MB) COMT, generating alleles encoding high and low activity forms of the enzyme, COMTH and COMTL, respectively. Tissues and cells with a COMTLL genotype have decreased COMT activity compared ...

  16. COMT Val158Met Genotype Determines the Direction of Cognitive Effects Produced by Catechol-O-Methyltransferase Inhibition

    OpenAIRE

    Farrell, Sarah M.; Tunbridge, Elizabeth M.; Braeutigam, Sven; Harrison, Paul J

    2012-01-01

    Background Catechol-O-methyltransferase (COMT) metabolizes dopamine. The COMT Val158Met polymorphism influences its activity, and multiple neural correlates of this genotype on dopaminergic phenotypes, especially working memory, have been reported. COMT activity can also be regulated pharmacologically by COMT inhibitors. The inverted-U relationship between cortical dopamine signaling and working memory predicts that the effects of COMT inhibition will differ according to COMT genotype. Method...

  17. Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis

    Directory of Open Access Journals (Sweden)

    Obarska Agnieszka

    2006-01-01

    Full Text Available Abstract Background Recently, HEN1 protein from Arabidopsis thaliana was discovered as an essential enzyme in plant microRNA (miRNA biogenesis. HEN1 transfers a methyl group from S-adenosylmethionine to the 2'-OH or 3'-OH group of the last nucleotide of miRNA/miRNA* duplexes produced by the nuclease Dicer. Previously it was found that HEN1 possesses a Rossmann-fold methyltransferase (RFM domain and a long N-terminal extension including a putative double-stranded RNA-binding motif (DSRM. However, little is known about the details of the structure and the mechanism of action of this enzyme, and about its phylogenetic origin. Results Extensive database searches were carried out to identify orthologs and close paralogs of HEN1. Based on the multiple sequence alignment a phylogenetic tree of the HEN1 family was constructed. The fold-recognition approach was used to identify related methyltransferases with experimentally solved structures and to guide the homology modeling of the HEN1 catalytic domain. Additionally, we identified a La-like predicted RNA binding domain located C-terminally to the DSRM domain and a domain with a peptide prolyl cis/trans isomerase (PPIase fold, but without the conserved PPIase active site, located N-terminally to the catalytic domain. Conclusion The bioinformatics analysis revealed that the catalytic domain of HEN1 is not closely related to any known RNA:2'-OH methyltransferases (e.g. to the RrmJ/fibrillarin superfamily, but rather to small-molecule methyltransferases. The structural model was used as a platform to identify the putative active site and substrate-binding residues of HEN and to propose its mechanism of action.

  18. Characterization of a DNA Adenine Methyltransferase Gene of Borrelia hermsii and Its Dispensability for Murine Infection and Persistence

    Science.gov (United States)

    James, Allison E.; Rogovskyy, Artem S.; Crowley, Michael A.; Bankhead, Troy

    2016-01-01

    DNA methyltransferases have been implicated in the regulation of virulence genes in a number of pathogens. Relapsing fever Borrelia species harbor a conserved, putative DNA methyltransferase gene on their chromosome, while no such ortholog can be found in the annotated genome of the Lyme disease agent, Borrelia burgdorferi. In the relapsing fever species Borrelia hermsii, the locus bh0463A encodes this putative DNA adenine methyltransferase (dam). To verify the function of the BH0463A protein product as a Dam, the gene was cloned into a Dam-deficient strain of Escherichia coli. Restriction fragment analysis subsequently demonstrated that complementation of this E. coli mutant with bh0463A restored adenine methylation, verifying bh0463A as a Dam. The requirement of bh0463A for B. hermsii viability, infectivity, and persistence was then investigated by genetically disrupting the gene. The dam- mutant was capable of infecting immunocompetent mice, and the mean level of spirochetemia in immunocompetent mice was not significantly different from wild type B. hermsii. Collectively, the data indicate that dam is dispensable for B. hermsii viability, infectivity, and persistence. PMID:27195796

  19. Structure of the corrinoid:coenzyme M methyltransferase MtaA from Methanosarcina mazei.

    Science.gov (United States)

    Hoeppner, Astrid; Thomas, Frank; Rueppel, Alma; Hensel, Reinhard; Blankenfeldt, Wulf; Bayer, Peter; Faust, Annette

    2012-11-01

    The zinc-containing corrinoid:coenzyme M methyltransferase MtaA is part of the methanol-coenzyme M-methyltransferase complex of Methanosarcina mazei. The whole complex consists of three subunits: MtaA, MtaB and MtaC. The MtaB-MtaC complex catalyses the cleavage of methanol (bound to MtaB) and the transfer of the methyl group onto the cobalt of cob(I)alamin (bound to MtaC). The MtaA-MtaC complex catalyses methyl transfer from methyl-cob(III)alamin (bound to MtaC) to coenzyme M (bound to MtaA). The crystal structure of the MtaB-MtaC complex from M. barkeri has previously been determined. Here, the crystal structures of MtaA from M. mazei in a substrate-free but Zn(2+)-bound state and in complex with Zn(2+) and coenzyme M (HS-CoM) are reported at resolutions of 1.8 and 2.1 Å, respectively. A search for homologous proteins revealed that MtaA exhibits 23% sequence identity to human uroporphyrinogen III decarboxylase, which has also the highest structural similarity (r.m.s.d. of 2.03 Å for 306 aligned amino acids). The main structural feature of MtaA is a TIM-barrel-like fold, which is also found in all other zinc enzymes that catalyse thiol-group alkylation. The active site of MtaA is situated at the narrow bottom of a funnel such that the thiolate group of HS-CoM points towards the Zn(2+) ion. The Zn(2+) ion in the active site of MtaA is coordinated tetrahedrally via His240, Cys242 and Cys319. In the substrate-free form the fourth ligand is Glu263. Binding of HS-CoM leads to exchange of the O-ligand of Glu263 for the S-ligand of HS-CoM with inversion of the zinc geometry. The interface between MtaA and MtaC for transfer of the methyl group from MtaC-bound methylcobalamin is most likely to be formed by the core complex of MtaB-MtaC and the N-terminal segment (a long loop containing three α-helices and a β-hairpin) of MtaA, which is not part of the TIM-barrel core structure of MtaA. PMID:23090404

  20. DNA-methyltransferase 3B 39179 G > T polymorphism and risk of sporadic colorectal cancer in a subset of Iranian population

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    Abdolreza Daraei

    2011-01-01

    Full Text Available Background: Epigenetic event is a biological regulation that influences the expression of various genes involved in cancer. DNA methylation is established by DNA methyltransferases, particularly DNAmethyltransferase 3B (DNMT3B. It seems to play an oncogenic role in the creation of abnormal methylation during tumorigenesis. The polymorphisms of the DNMT3B gene may influence DNMT3B activity in DNA methylation and increase the susceptibility to several cancers. These genetic polymorphisms have been studied in several cancers in different populations. Methods: In this study, we performed a case-control study with 125 colorectal cancer patients and 135 cancer-free controls to evaluate the association between DNMT3B G39179T polymorphism (rs1569686 in the promoter region and the risk of sporadic colorectal cancer. Up to now, few studies have investigated the role of this gene variant in sporadic colorectal cancer with no familial history. The genotypes of DNMT3B G39179T polymorphism was analyzed by PCR-RFLP. Results: We found that compared with G allele carriers, statistically the DNMT3B TT genotype (%34 was significantly associated with increased risk of colorectal cancer (adjusted OR, 3.993, 95% CI, 1.726-9.238, P = 0.001. Compared with DNMT3B TT genotype, the GT and GG genotypes had lower risk of developing sporadic colorectal cancer (OR = 0.848, 95% CI = 0.436-1.650. Conclusions: Our findings were consistent with that of previously reported case-control studies with colorectal cancer. These results suggest that the DNMT3B G39179T polymorphism influences DNMT3B expression, thus contributing to the genetic susceptibility to colorectal cancer. Further mechanistic studies are needed to unravel the causal molecular mechanisms.