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Methyltransferase
Kinds of Methyltransferase Terms modified by Methyltransferase Selected AbstractsChanging transcriptional initiation sites and alternative 5,- and 3,-splice site selection of the first intron deploys Arabidopsis PROTEIN ISOASPARTYL METHYLTRANSFERASE2 variants to different subcellular compartmentsTHE PLANT JOURNAL, Issue 1 2008Randy D. Dinkins Summary Arabidopsis thaliana (L.) Heynh. possesses two PROTEIN-L-ISOASPARTATE METHYLTRANSFERASE (PIMT) genes encoding enzymes (EC 2.1.1.77) capable of converting uncoded l -isoaspartyl residues, arising spontaneously at l -asparaginyl and l -aspartyl sites in proteins, to l -aspartate. PIMT2 produces at least eight transcripts by using four transcriptional initiation sites (TIS; resulting in three different initiating methionines) and both 5,- and 3,-alternative splice site selection of the first intron. The transcripts produce mature proteins capable of converting l -isoaspartate to l -aspartate in small peptide substrates. PIMT:GFP fusion proteins generated a detectable signal in the nucleus. However, whether the protein was also detectable in the cytoplasm, endo-membrane system, chloroplasts, and/or mitochondria, depended on the transcript from which it was produced. On-blot-methylation of proteins, prior to the completion of germination, indicated that cruciferin subunits contain isoaspartate. The implications of using transcriptional mechanisms to expand a single gene's repertoire to protein variants capable of entry into the cell's various compartments are discussed in light of PIMT's presumed role in repairing the proteome. [source] The ratio of campesterol to sitosterol that modulates growth in Arabidopsis is controlled by STEROL METHYLTRANSFERASE 2;1THE PLANT JOURNAL, Issue 6 2001Aurélie Schaeffer Summary The Arabidopsis genome contains three distinct genes encoding sterol-C24-methyltransferases (SMTs) involved in sterol biosynthesis. The expression of one of them, STEROL METHYLTRANSFERASE 2;1, was modulated in 35S::SMT2;1 Arabidopsis in order to study its physiological function. Plants overexpressing the transgene accumulate sitosterol, a 24-ethylsterol which is thought to be the typical plant membrane reinforcer, at the expense of campesterol. These plants displayed a reduced stature and growth that could be restored by brassinosteroid treatment. Plants showing co-suppression of SMT2;1 were characterized by a predominant 24-methylsterol biosynthetic pathway leading to a high campesterol content and a depletion in sitosterol. Pleiotropic effects on development such as reduced growth, increased branching, and low fertility of high-campesterol plants were not modified by exogenous brassinosteroids, indicating specific sterol requirements to promote normal development. Thus SMT2;1 has a crucial role in balancing the ratio of campesterol to sitosterol in order to fit both growth requirements and membrane integrity. [source] A 3-D QSAR Study of Catechol- O -Methyltransferase Inhibitors Using CoMFA and CoMSIAMOLECULAR INFORMATICS, Issue 10 2008Chunzhi Ai Abstract Inhibitors of Catechol- O -Methyltransferase (COMT) play an important role in the treatment of Parkinson's Disease (PD). A new Three-Dimensional Quantitative Structure,Activity Relationship (3-D QSAR) analysis was performed on 36 previously reported COMT inhibitors employing Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) methodologies to correlate the molecular fields and percent inhibition values and three predictive models were derived. The CoMFA and CoMSIA models with steric and electrostatic field yielded cross-validated rs of 0.585 and 0.528, respectively whereas the conventional rs were 0.979 and 0.891, respectively. The CoMSIA model with hydrophobic field exhibited a r of 0.544 and a r of 0.930. The individual inspection of 3-D contours generated from these models helps in understanding the possible region for structural modification of molecules to improve the inhibitory bioactivity. These 3-D QSAR models are also useful for designing and predicting novel COMT inhibitors. [source] Pneumocystis carinti erg6 Gene: Sequencing and Expression of Recombinant SAM:Sterol Methyltransferase in Heterologous SystemsTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2001EDNA S. KANESHIRO [source] Halomethane Biosynthesis: Structure of a SAM-Dependent Halide Methyltransferase from Arabidopsis thaliana,ANGEWANDTE CHEMIE, Issue 21 2010Jason Aus der Pflanze in die Luft: Die Struktur des Halogenmethan erzeugenden pflanzlichen Enzyms Halogenid/Thiocyanat-Methyltransferase wurde bestimmt. Modellieren eines Halogenidions und der Methylgruppe von S -Adenosyl- L -methionin (SAM) im aktiven Zentrum (siehe Bild; Chlorid: grüne Kugel; SAM: C,grün, O,rot, S,gelb, N,blau) zeigt deren Ausrichtung für die Reaktion. [source] Biocatalytic Friedel,Crafts Alkylation Using Non-natural Cofactors,ANGEWANDTE CHEMIE, Issue 50 2009Harald Stecher Dipl.-Ing. Eine neuartige biokatalytische C-C-Verknüpfung, die äquivalent zur Friedel-Crafts-Alkylierung ist, wird vorgestellt. S -Adenosyl- L -methionin (SAM), der Haupt-Methyldonor bei Methyltransferase(Mtase)-katalysierten biologischen Methylierungen, kann Alkylierungen bewirken (siehe Schema). Diese Enzyme akzeptieren nichtnatürliche Cofaktoren und können andere Funktionalitäten als Me auf aromatische Substrate übertragen. [source] Molekulare Erkennung in der aktiven Tasche der Catechol- O - Methyltransferase: energetisch günstige Verdrängung eines von einem Bisubstratinhibitor importierten Wassermoleküls,ANGEWANDTE CHEMIE, Issue 48 2009Manuel Ellermann Verdrängungswettbewerb: Der Bindungsmodus hochpotenter Bisubstratinhibitoren der Catechol- O -Methyltransferase (COMT) wurde durch Kristallstrukturanalysen des ternären Komplexes mit COMT und Mg2+ bestimmt. Ein einzelnes, vom Liganden importiertes Wassermolekül wird verdrängt, wobei mindestens eine freie Bindungsenthalpie in Höhe von ,1.8,kcal,mol,1 gewonnen wird, welche die energetisch ungünstige Konformation des Liganden im gebundenen Zustand kompensiert. [source] Crystallization and preliminary X-ray crystallographic studies of O -methyltransferase from Anabaena PCC 7120ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Guoming Li 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, , = , = , = 90°. There are eight molecules per asymmetric unit. [source] Inactivation of O6 -Methylguanine-DNA Methyltransferase in Human Lung Adenocarcinoma Relates to High-grade Histology and Worse Prognosis among SmokersCANCER SCIENCE, Issue 2 2002Hiroyuki Hayashi To evaluate the significance of O6 -methylguanine-DNA methyltransferase (MGMT) activity in the development of human lung adenocarcinoma (AC), we investigated promoter hypermethylation of the MGMTx gene by methylation-specific PCR, and the expression of MGMT protein by immuno-histochemistry in relation to smoking history of the patients. In total, 31 of 87 AC patients (35.5%) showed hypermethylation of the MGMT gene, and no significant difference was observed between smokers (37.3%) and non-smokers (33.3%). However, hypermethylation of the MGMT gene increased in parallel with lesser differentiation grade of tumors among smokers (well, 16.7%; moderately, 42.1%; poorly, 57.1%; P=0.022), although this trend was not observed among non-smokers. Almost all the tumors with promoter hypermethylation of the MGMT gene showed consistently negative MGMT staining by immunohistochemistry. When the prognosis of stage-I patients was compared among smokers, it was apparent that the prognosis of patients with inactivated MGMT was worse than that of MGMT-positive patients (P=0.036). Such differences in the prognoses were not observed among non-smokers. In conclusion, MGMT inactivation is related to the differentiation grade and the prognosis of lung AC patients among smokers. Although further studies are required, we speculate that smoking may induce hypermethylation, not only of the MGMT gene, but also of other important tumor suppressor genes. [source] Protection against Malignant Progression of Spontaneously Developing Liver Tumors in Transgenic Mice Expressing O6 -Methylguanine-DNA MethyltransferaseCANCER SCIENCE, Issue 11 2000Xiusheng Qin To study the effect of O6 -methylguanine-DNA methyltransferase (MGMT) on carcinogenesis, we have previously generated MGMT transgenic mice overexpressing the bacterial MGMT gene, ada, and demonstrated that high MGMT levels in the liver suppress induction of liver tumors after treatment with an alkylating hepatocarcinogen. To examine the effects of life-long elevation of MGMT activity on mouse spontaneous liver tumor development, ada-transgenic and control nontransgenic mice were compared. We also examined mutations at codon 61 of the H-ras oncogene, reported as a hot spot in mouse liver tumors, using a direct DNA sequencing method. The results revealed no significant difference in tumor incidence or mutation spectrum, but interestingly, ada-transgenic mice were found to have fewer malignant tumors and survived longer, indicating a possible protective role of MGMT against malignant conversion. [source] Bisubstrate Inhibitors of the Enzyme Catechol O -Methyltransferase (COMT): Efficient Inhibition Despite the Lack of a Nitro GroupCHEMBIOCHEM, Issue 9 2004Ralph Paulini A new generation of bisubstrate inhibitors for the S -adenosylmethionine- and magnesium ion-dependent enzyme catechol O -methyltransferase (COMT), feature binding affinities (IC50 values) in the double-digit nanomolar range despite the lack of a nitro group on the catechol moiety. Inhibitor potency does not directly correlate with the pKa value of the catechol HO groups and is strongly enhanced by hydrophobic aromatic substituents attached in a biaryl-type fashion to position 5 of the catechol ring. [source] Sequence-specific Methyltransferase- Induced Labeling of DNA (SMILing DNA)CHEMBIOCHEM, Issue 3 2004Goran Pljevalj Abstract A new concept for sequence-specific labeling of DNA by using chemically modified cofactors for DNA methyltransferases is presented. Replacement of the amino acid side chain of the natural cofactor S -adenosyl- L -methionine with an aziridine group leads to a cofactor suitable for DNA methyltransferase-catalyzed sequence-specific coupling with DNA. Sequence-specifically fluorescently labeled plasmid DNA was obtained by using the DNA methyltransferase from Thermus aquaticus (M.TaqI) as catalyst and attaching a fluorophore to the aziridine cofactor. First results suggest that all classes of DNA methyltransferases with different recognition sequences can be used. In addition, this novel method for DNA labeling should be applicable to a wide variety of reporter groups. [source] Search for Histamine H3 Receptor Antagonists with Combined Inhibitory Potency at N, -Methyltransferase: Ether Derivatives.CHEMINFORM, Issue 22 2005J. Apelt Abstract For Abstract see ChemInform Abstract in Full Text. [source] The Emerging Therapeutic Potential of Histone Methyltransferase and Demethylase InhibitorsCHEMMEDCHEM, Issue 10 2009Astrid Spannhoff Dr. Abstract Epigenetics is defined as heritable changes to the transcriptome that are independent of changes in the genome. The biochemical modifications that govern epigenetics are DNA methylation and posttranslational histone modifications. Among the histone modifications, acetylation and deacetylation are well characterized, whereas the fields of histone methylation and especially demethylation are still in their infancy. This is particularly true with regard to drug discovery. There is strong evidence that these modifications play an important role in the maintenance of transcription as well as in the development of certain diseases. This article gives an overview of the mechanisms of action of histone methyltransferases and demethylases, their role in the formation of certain diseases, and available inhibitors. Special emphasis is placed on the strategies that led to the first inhibitors which are currently available and the screening approaches that were used in that process. [source] Candidate genes and the behavioral phenotype in 22q11.2 deletion syndromeDEVELOPMENTAL DISABILITIES RESEARCH REVIEW, Issue 1 2008Sarah E. Prasad Abstract There is an overwhelming evidence that children and adults with 22q11.2 deletion syndrome (22q11.2DS) have a characteristic behavioral phenotype. In particular, there is a growing body of evidence that indicates an unequivocal association between 22q11.2DS and schizophrenia, especially in adulthood. Deletion of 22q11.2 is the third highest risk for the development of schizophrenia, with only a greater risk conferred by being the child of two parents with schizophrenia or the monozygotic co-twin of an affected individual. Both linkage and association studies of people with schizophrenia have implicated several susceptibility genes, of which three are in the 22q11.2 region; catechol- o -methyltransferase (COMT), proline dehydrogenase (PRODH), and Gnb1L. In addition, variation in Gnb1L is associated with the presence of psychosis in males with 22q11.2DS. In mouse models of 22q11.2DS, haploinsufficiency of Tbx1 and Gnb1L is associated with reduced prepulse inhibition, a schizophrenia endophenotype. The study of 22q11.2DS provides an attractive model to increase our understanding of the development and pathogenesis of schizophrenia and other psychiatric disorders in 22q11.2DS and in wider population. © 2008 Wiley-Liss, Inc. Dev Disabil Res Rev 2008;14:26,34. [source] Nuclear proteome analysis of undifferentiated mouse embryonic stem and germ cellsELECTROPHORESIS, Issue 11 2008Nicolas Buhr Abstract Embryonic stem cells (ESCs) and embryonic germ cells (EGCs) provide exciting models for understanding the underlying mechanisms that make a cell pluripotent. Indeed, such understanding would enable dedifferentiation and reprogrammation of any cell type from a patient needing a cell therapy treatment. Proteome analysis has emerged as an important technology for deciphering these biological processes and thereby ESC and EGC proteomes are increasingly studied. Nevertheless, their nuclear proteomes have only been poorly investigated up to now. In order to investigate signaling pathways potentially involved in pluripotency, proteomic analyses have been performed on mouse ESC and EGC nuclear proteins. Nuclei from ESCs and EGCs at undifferentiated stage were purified by subcellular fractionation. After 2-D separation, a subtractive strategy (subtracting culture environment contaminating spots) was applied and a comparison of ESC, (8.5 day post coïtum (dpc))-EGC and (11.5,dpc)-EGC specific nuclear proteomes was performed. A total of 33 ESC, 53 (8.5,dpc)-EGC, and 36 (11.5,dpc)-EGC spots were identified by MALDI-TOF-MS and/or nano-LC-MS/MS. This approach led to the identification of two isoforms (with and without N -terminal acetylation) of a known pluripotency marker, namely developmental pluripotency associated 5 (DPPA5), which has never been identified before in 2-D gel-MS studies of ESCs and EGCs. Furthermore, we demonstrated the efficiency of our subtracting strategy, in association with a nuclear subfractionation by the identification of a new protein (protein arginine N -methyltransferase 7; PRMT7) behaving as proteins involved in pluripotency. [source] Protection of hematopoietic cells from O6 -alkylation damage by O6 -methylguanine DNA methyltransferase gene transfer: studies with different O6 -alkylating agents and retroviral backbonesEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2001Michael Jansen Abstract: Overexpression of O6 -methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O6 -alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O6 -alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8,12 µg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer. [source] Defective DNA methylation and CD70 overexpression in CD4+ T cells in MRL/lpr lupus-prone miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2007Abstract We have determined that abnormal DNA methylation in T cells coincides with the development of autoimmunity, using a mouse model that exhibits an age-dependent lupus-like disease (MRL/lpr mice). Splenic CD4+ T cells were isolated from these mice at 5 and 16,wk of age (before and after autoimmunity is established) and the expression of DNA methyltransferase,1 (Dnmt1) and the methylation-sensitive gene Tnfsf7 (CD70) was measured. Bisulfite DNA sequencing was used to monitor the methylation status of the Tnfsf7 gene. We found that Dnmt1 steady-state mRNA levels were significantly lower in 16-wk-old MRL/lpr mice, which had established autoimmunity, compared to the 5-wk-old MRL/lpr mice. Furthermore, the expression of CD70 was higher in MRL/lpr mice at 16,wk. CD70 was overexpressed in MRL/lpr mice compared to age- and sex-matched MRL+/+ controls. Bisulfite DNA sequencing of the Tnfsf7 gene in MRL/lpr mice revealed that at 16,wk, CG pairs were hypomethylated compared to 5-wk-old mice, and that Tnfsf7 from MRL/lpr mice was hypomethylated at 16,wk relative to age-matched MRL+/+ controls. Our data indicate that decreased expression of Dnmt1 and the corresponding T cell DNA hypomethylation correlate with the development of age-dependent autoimmunity in MRL/lpr mice. [source] Effects of dopamine-related gene,gene interactions on working memory component processesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2009Christine Stelzel Abstract Dopamine modulates complex cognitive functions like working memory and cognitive control. It is widely accepted that an optimal level of prefrontal dopamine supports working memory performance. In the present study we used a molecular genetic approach to test whether the optimal activity of the dopamine system for different component processes of working memory is additionally related to the availability of dopamine D2 receptors. We sought evidence for this assumption by investigating the interaction effect (epistasis) of variations in two dopaminergic candidate genes: the catechol- O -methyltransferase (COMT) Val158Met polymorphism, which has been shown to influence prefrontal dopamine concentration, and the DRD2/ANKK1-Taq-Ia polymorphism, which has been related to the density of D2 receptors. Our results show that COMT effects on working memory performance are modulated by the DRD2/ANKK1-TAQ-Ia polymorphism and the specific working memory component process under investigation. Val, participants , supposedly characterized by increased prefrontal dopamine concentrations , outperformed Val+ participants in the manipulation of working memory contents, but only when D2 receptor density could be considered to be high. No such effect was present for passive maintenance of working memory contents or for maintenance in the face of distracting information. This beneficial effect of a balance between prefrontal dopamine availability and D2 receptor density reveals the importance of considering epistasis effects and different working memory subprocesses in genetic association studies. [source] Polymorphisms located in the region containing BHMT and BHMT2 genes as maternal protective factors for orofacial cleftsEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 4 2010Adrianna Mostowska Mostowska A, Hozyasz KK, Biedziak B, Misiak J, Jagodzinski PP. Polymorphisms located in the region containingBHMTandBHMT2genes as maternal protective factors for orofacial clefts. Eur J Oral Sci 2010; 118: 325,332. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci Nonsyndromic cleft lip with or without cleft palate (NCL/P) is one of the most common craniofacial malformations; however, its aetiology is still unclear. Because the effects of maternal nutrition on fetal development are well known, we decided to pursue the question of whether polymorphic variants of genes encoding enzymes involved in choline metabolism might be associated with the maternal risk of having a baby with NCL/P. Analysis of 18 single nucleotide polymorphisms (SNPs) of betaine-homocysteine methyltransferase (BHMT), betaine-homocysteine methyltransferase-2 (BHMT2), choline dehydrogenase (CHDH), choline kinase (CHKA), dimethylglycine dehydrogenase (DMGDH), choline-phosphate cytidylyltransferase A (PCYT1A), and phosphatidylethanolamine N -methyltransferase (PEMT) provided evidence that polymorphisms located in the region containing BHMT and BHMT2 were protective factors against NCL/P affected pregnancies in our population. The strongest signal was found for the SNP located in the intronic sequence of BHMT2. Women carrying two copies of the rs625879 T allele had a significantly decreased risk of having offspring with orofacial clefts. These results were significant, even after correction for multiple comparisons. Moreover, the gene,gene interaction analysis revealed a significant epistatic interaction of BHMT2 (rs673752), PEMT (rs12325817), and PCYT1A (rs712012) with maternal NCL/P susceptibility. Altogether, our study identified a novel gene, the nucleotide variants of which were be associated with a decreased risk of having a baby with NCL/P. [source] Structure,activity relationships of wheat flavone O -methyltransferase , a homodimer of convenienceFEBS JOURNAL, Issue 9 2008Jack A. Kornblatt Wheat flavone O -methyltransferase catalyzes three sequential methylations of the flavone tricetin. Like other flavonoid O -methyltransferases, the protein is a homodimer. We demonstrate, using analytical ultracentrifugation, that perchlorate dissociates the dimer into monomers. The resulting monomers retain all their catalytic capacity, including the ability to catalyze the three successive methylations. We show, using isothermal titration calorimetry, that the binding constant for S -adenosyl- l -methionine does not change significantly as the protein dissociates. The second substrate, tricetin, binds to the dimers but could not be tested with the monomers. CD, UV and fluorescence spectroscopy show that there are substantial changes in the structure of the protein as it dissociates. The fact that there are differences between the monomers and dimers even as the monomers maintain activity may be the result of the very low catalytic capacity of this enzyme. Maximal turnover numbers for the dimers and monomers are only about 6,7 per minute. Even though the binding pockets for S -adenosyl- l -methionine, tricetin, selgin and tricin are intact, selection of a catalytically competent structure may be a very slow step during catalysis. [source] Crystal structures of CbiL, a methyltransferase involved in anaerobic vitamin B12 biosynthesis, and CbiL in complex with S -adenosylhomocysteine , implications for the reaction mechanismFEBS JOURNAL, Issue 2 2007Kei Wada During anaerobic cobalamin (vitamin B12) biosynthesis, CbiL catalyzes methylation at the C-20 position of a cyclic tetrapyrrole ring using S -adenosylmethionine as a methyl group source. This methylation is a key modification for the ring contraction process, by which a porphyrin-type tetrapyrrole ring is converted to a corrin ring through elimination of the modified C-20 and direct bonding of C-1 to C-19. We have determined the crystal structures of Chlorobium tepidum CbiL and CbiL in complex with S -adenosylhomocysteine (the S -demethyl form of S -adenosylmethionine). CbiL forms a dimer in the crystal, and each subunit consists of N-terminal and C-terminal domains. S -Adenosylhomocysteine binds to a cleft between the two domains, where it is specifically recognized by extensive hydrogen bonding and van der Waals interactions. The orientation of the cobalt-factor II substrate was modeled by simulation, and the predicted model suggests that the hydroxy group of Tyr226 is located in close proximity to the C-20 atom as well as the C-1 and C-19 atoms of the tetrapyrrole ring. These configurations allow us to propose a catalytic mechanism: the conserved Tyr226 residue in CbiL catalyzes the direct transfer of a methyl group from S -adenosylmethionine to the substrate through an SN2-like mechanism. Furthermore, the structural model of CbiL binding to its substrate suggests the axial residue coordinated to the central cobalt of cobalt-factor II. [source] Modulation of glucocorticoid receptor-interacting protein 1 (GRIP1) transactivation and co-activation activities through its C-terminal repression and self-association domainsFEBS JOURNAL, Issue 10 2006Pei-Yao Liu Glucocorticoid receptor-interacting protein 1 (GRIP1), a p160 family nuclear receptor co-activator, possesses at least two autonomous activation domains (AD1 and AD2) in the C-terminal region. AD1 activity appears to be mediated by CBP/p300, whereas AD2 activity is apparently mediated through co-activator-associated arginine methyltransferase 1 (CARM1). The mechanisms responsible for regulating the activities of AD1 and AD2 are not well understood. We provide evidence that the GRIP1 C-terminal region may be involved in regulating its own transactivation and nuclear receptor co-activation activities through primary self-association and a repression domain. We also compared the effects of the GRIP1 C terminus with those of other factors that functionally interact with the GRIP1 C terminus, such as CARM1. Based on our results, we propose a regulatory mechanism involving conformational changes to GRIP1 mediated through its intramolecular and intermolecular interactions, and through modulation of the effects of co-repressors on its repression domains. These are the first results to indicate that the structural components of GRIP1, especially those of the C terminus, might functionally modulate its putative transactivation activities and nuclear receptor co-activator functions. [source] Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylaseFEBS JOURNAL, Issue 5 2003Structural, functional implications Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302,6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19,33) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15,K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein- l -isoaspartic acid O -methyltransferase (PIMT; EC 2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 °C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR,,34 min to ,,31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR,,34 min species decreased with a biphasic time-course with t0.5 -values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of ,,1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 °C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial ,-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32,Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition. [source] Regulation of transcription of the Dnmt1 gene by Sp1 and Sp3 zinc finger proteinsFEBS JOURNAL, Issue 12 2002Shotaro Kishikawa The Sp family is a family of transcription factors that bind to cis -elements in the promoter regions of various genes. Regulation of transcription by Sp proteins is based on interactions between a GC-rich binding site (GGGCGG) in DNA and C-terminal zinc finger motifs in the proteins. In this study, we characterized the GC-rich promoter of the gene for the DNA methyltransferase (Dnmt1) that is responsible for methylation of cytosine residues in mammals and plays a role in gene silencing. We found that a cis -element (nucleotides ,161 to ,147) was essential for the expression of the mouse gene for Dnmt1. DNA-binding assays indicated that transcription factors Sp1 and Sp3 bound to the same cis -element in this region in a dose-dependent manner. In Drosophila SL2 cells, which lack the Sp family of transcription factors, forced expression of Sp1 or Sp3 enhanced transcription from the Dnmt1 promoter. Stimulation by Sp1 and Sp3 were independent phenomena. Furthermore, cotransfection reporter assays with a p300-expression plasmid revealed the activation of the promoter of the Dnmt1 gene in the presence of Sp3. The transcriptional coactivator p300 interacted with Sp3 in vivo and in vitro. Our results indicate that expression of the Dnmt1 gene is controled by Sp1 and Sp3 and that p300 is involved in the activation by Sp3. [source] Ski co-repressor complexes maintain the basal repressed state of the TGF-, target gene, SMAD7, via HDAC3 and PRMT5GENES TO CELLS, Issue 1 2009Takanori Tabata The products encoded by ski and its related gene, sno, (Ski and Sno) act as transcriptional co-repressors and interact with other co-repressors such as N-CoR/SMRT and mSin3A. Ski and Sno mediate transcriptional repression by various repressors, including Mad, Rb and Gli3. Ski/Sno also suppress transcription induced by multiple activators, such as Smads and c-Myb. In particular, the inhibition of TGF-,-induced transcription by binding to Smads is correlated with the oncogenic activity of Ski and Sno. However, the molecular mechanism by which Ski and Sno mediate transcriptional repression remains unknown. In this study, we report the purification and characterization of Ski complexes. The Ski complexes purified from HeLa cells contained histone deacetylase 3 (HDAC3) and protein arginine methyltransferase 5 (PRMT5), in addition to multiple Smad proteins (Smad2, Smad3 and Smad4). Chromatin immunoprecipitation assays indicated that these components of the Ski complexes were localized on the SMAD7 gene promoter, which is the TGF-, target gene, in TGF-,-untreated HepG2 cells. Knockdown of these components using siRNA led to up-regulation of SMAD7 mRNA. These results indicate that Ski complexes serve to maintain a TGF-,-responsive promoter at a repressed basal level via the activities of histone deacetylase and histone arginine methyltransferase. [source] Genome-wide and locus-specific DNA hypomethylation in G9a deficient mouse embryonic stem cellsGENES TO CELLS, Issue 1 2007Kohta Ikegami In the mammalian genome, numerous CpG-rich loci define tissue-dependent and differentially methylated regions (T-DMRs). Euchromatin from different cell types differs in terms of its tissue-specific DNA methylation profile as defined by these T-DMRs. G9a is a euchromatin-localized histone methyltransferase (HMT) and catalyzes methylation of histone H3 at lysines 9 and 27 (H3-K9 and -K27). To test whether HMT activity influences euchromatic cytosine methylation, we analyzed the DNA methylation status of approximately 2000 CpG-rich loci, which are predicted in silico, in G9a,/, embryonic stem cells by restriction landmark genomic scanning (RLGS). While the RLGS profile of wild-type cells contained about 1300 spots, 32 new spots indicating DNA demethylation were seen in the profile of G9a,/, cells. Virtual-image RLGS (Vi-RLGS) allowed us to identify the genomic source of ten of these spots. These were confirmed to be cytosine demethylated, not just at the Not I site detected by the RLGS but extending over several kilobase pairs in cis. Chromatin immunoprecipitation (ChIP) confirmed these loci to be targets of G9a, with decreased H3-K9 and/or -K27 dimethylation in the G9a,/, cells. These data indicate that G9a site-selectively contributes to DNA methylation. [source] PCNA clamp facilitates action of DNA cytosine methyltransferase 1 on hemimethylated DNAGENES TO CELLS, Issue 10 2002Tetsuo Iida Background: Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein known as a processivity factor of DNA polymerase ,. In addition to this role, PCNA interacts with a number of other proteins to increase their local concentration at replicated DNA sites. DNA cytosine methyltransferase 1 (Dnmt1), which preserves epigenetic signals by completing the methylation of hemimethylated DNA after DNA replication, has been indicated as one of these PCNA binding proteins by a previous work. However, the molecular mechanisms and functional significance of their association have not yet been studied. Results: Dnmt1 can be readily isolated from nuclear extracts by PCNA affinity chromatography. Studies of the interactions between the two proteins demonstrate that the N-terminal region of Dnmt1, which contains a typical PCNA binding motif, has core PCNA binding activity, and that the remaining portion of the protein exerts a negative influence on the interaction of Dnmt1 with PCNA. The affinity of Dnmt1 for DNA is much higher for DNA bound by PCNA than for free DNA. Furthermore, DNA methylation assays with hemimethylated DNA as a substrate revealed that PCNA clamp-bound DNA is methylated more efficiently by Dnmt1 than is free DNA. Conclusion: These results provide the first biochemical evidence that physical interactions between PCNA and Dnmt1 facilitate the methylation of newly neplicated DNA, on which PCNA remains associated as a functional clamp. [source] Subtle gene,environment interactions driving paranoia in daily lifeGENES, BRAIN AND BEHAVIOR, Issue 1 2009C. J. P. Simons It has been suggested that genes impact on the degree to which minor daily stressors cause variation in the intensity of subtle paranoid experiences. The objective of the present study was to test the hypothesis that catechol- O -methyltransferase (COMT) Val158Met and brain-derived neurotrophic factor (BDNF) Val66Met in part mediate genetic effects on paranoid reactivity to minor stressors. In a general population sample of 579 young adult female twins, on the one hand, appraisals of (1) event-related stress and (2) social stress and, on the other hand, feelings of paranoia in the flow of daily life were assessed using momentary assessment technology for five consecutive days. Multilevel regression analyses were used to examine moderation of daily life stress-induced paranoia by COMT Val158Met and BDNF Val66Met genotypes. Catechol- O -methyltransferase Val carriers displayed more feelings of paranoia in response to event stress compared with Met carriers. Brain-derived neurotrophic factor Met carriers showed more social-stress-induced paranoia than individuals with the Val/Val genotype. Thus, paranoia in the flow of daily life may be the result of gene,environment interactions that can be traced to different types of stress being moderated by different types of genetic variation. [source] Chromosome 8 BAC array comparative genomic hybridization and expression analysis identify amplification and overexpression of TRMT12 in breast cancer,GENES, CHROMOSOMES AND CANCER, Issue 7 2007Virginia Rodriguez Genomic changes in chromosome 8 are commonly observed in breast cancer cell lines and tumors. To fine map such genomic changes by comparative genomic hybridization (CGH), a high resolution (100 kb) chromosome 8 array that can detect single copy changes was developed using Phi29 DNA polymerase amplified BAC (bacterial artificial chromosome) DNA. The BAC array CGH resolved the two known amplified regions (8q21 and 8q24) of a breast cancer cell line (SKBR3) into nine separate regions including six amplicons and three deleted regions, all of which were verified by Fluorescence in situ hybridization. The extent of the gain/loss for each region was validated by qPCR. CGH was performed with a total of 8 breast cancer cell lines, and common regions of genomic amplification/deletion were identified by segmentation analysis. A 1.2-Mb region (125.3,126.5 Mb) and a 1.0-Mb region (128.1,129.1 Mb) in 8q24 were amplified in 7/8 cell lines. A global expression analysis was performed to evaluate expression changes associated with genomic amplification/deletion: a novel gene, TRMT12 (at 125.5 Mb), amplified in 7/8 cell lines, showed highest expression in these cell lines. Further analysis by RT-qPCR using RNA from 30 breast tumors showed that TRMT12 was overexpressed >2 fold in 87% (26/30) of the tumors. TRMT12 is a homologue of a yeast gene encoding a tRNA methyltransferase involved in the posttranscriptional modification of tRNAPhe, and exploring the biological consequence of its altered expression, may reveal novel pathways in tumorigenesis. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. Published 2007 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||||||||