Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dnmt3a and Dnmt3b are de novo DNA methyltransferases that also act as transcriptional repressors independent of methyltransferase activity. To elucidate the underlying mechanism of transcriptional repression, Dnmt3a was purified from mouse lymphosarcoma cells (P1798) by extensive fractionation on five different chromatographic matrices followed by glycerol density gradient centrifugation. Liquid chromatography electrospray tandem mass spectrometry analysis of Dnmt3a-associated polypeptides identified the methyl CpG binding protein Mbd3, histone deacetylase 1(Hdac1), and components of Brg1 complex (Brg1, Baf155, and Baf57) in the purified preparation. Association of Dnmt3a with Mbd3 and Brg1 was confirmed by coimmunoprecipitation and coimmunolocalization studies. Glutathione S-transferase pulldown assay showed that the NH2-terminal ATRX homology domain of Dnmt3a interacts with the methyl CpG binding domain of Mbd3 and with both bromo and ATPase domains of Brg1. Chromatin immunoprecipitation assay revealed that all three proteins are associated with transcriptionally silent methylated metallothionein (MT-I) promoter in the mouse lymphosarcoma cells. To understand the functional significance of their association with the promoter, their role on the MT-I promoter activity was analyzed by transient transfection assay. The results showed that Mbd3 and Dnmt3a specifically inhibited the methylated promoter, and the catalytic activity of Dnmt3a was dispensable for the suppression. In contrast, the wild-type but not the ATPase-inactive mutant of Brg1 suppressed MT-I promoter irrespective of its methylation status, implicating involvement of ATP-dependent chromatin remodeling in the process. Coexpression of two of the three interacting proteins at a time augmented their repressor function. This study shows physical and functional interaction of Dnmt3a with components of nucleosome remodeling machinery.
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PMID:Physical and functional interaction of DNA methyltransferase 3A with Mbd3 and Brg1 in mouse lymphosarcoma cells. 1632 36

The m7GpppN cap structure of eukaryotic mRNA is formed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase. In trypanosomatid protozoa, the m7GpppN is further modified by seven methylation steps within the first four transcribed nucleosides to form the cap 4 structure. The RNA triphosphatase and guanylyltransferase components have been characterized in Trypanosoma brucei. Here we describe the identification and characterization of a T. brucei (guanine N-7) methyltransferase (TbCmt1). Sequence alignment of the 324-amino acid TbCmt1 with the corresponding enzymes from human (Hcm1), fungal (Abd1), and microsporidian (Ecm1) revealed the presence of conserved residues known to be essential for methyltransferase activity. Purified recombinant TbCmt1 catalyzes the transfer of a methyl group from S-adenosylmethionine to the N-7 position of the cap guanine in GpppN-terminated RNA to form the m7GpppN cap. TbCmt1 also methylates GpppG and GpppA but not GTP or dGTP. Mutational analysis of individual residues of TbCmt1 that were predicted-on the basis of the crystal structure of Ecm1--to be located at or near the active site identified six conserved residues in the putative AdoMet- or cap-binding pocket that caused significant reductions in TbCmt1 methyltransferase activity. We also report the identification of a second T. brucei RNA (guanine N-7) cap methyltransferase (named TbCgm1). The 1050-amino acid TbCgm1 consists of a C-terminal (guanine N-7) methyltransferase domain, which is homologous with TbCmt1, and an N-terminal guanylyltransferase domain, which contains signature motifs found in the nucleotidyl transferase superfamily.
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PMID:Characterization of a Trypanosoma brucei RNA cap (guanine N-7) methyltransferase. 1643 85

The benzylisoquinoline alkaloids of opium poppy, including the narcotic analgesics morphine and codeine, accumulate in the multinucleate cytoplasm of specialized laticifers that accompany vascular tissues throughout the plant. In mature opium poppy plants, immunofluorescence labeling using specific antibodies showed that four alkaloid biosynthetic enzymes, (S)-norcoclaurine 6-O-methyltransferase (6OMT), (S)-coclaurine N-methyltransferase (CNMT), (S)-3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase (4'OMT) and salutaridinol-7-O-acetyltransferase (SAT) were restricted to sieve elements of the phloem adjacent or proximal to laticifers. The identity of sieve elements was confirmed by (i) the specific immunogold labeling of the characteristic cytoplasm of this cell type, (ii) the co-localization of a sieve element-specific H(+)-ATPase with all biosynthetic enzymes and (iii) the strict association of sieve plates with immunofluorescent cells. The localization of laticifers was demonstrated antibodies specific to major latex protein (MLP), which is characteristic of this cell type. In situ hybridization using antisense RNA probes for 6OMT, CNMT, 4'OMT and SAT showed that the corresponding gene transcripts were found in the companion cell paired with each sieve element. Seven benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B1), berberine bridge enzyme, codeinone reductase, 6OMT, CNMT, 4'OMT and SAT were localized by immunofluorescence labeling to the sieve elements in the root and hypocotyl of opium poppy seedlings. The abundance of these enzymes increased rapidly between 1 and 3 days after seed germination. The localization of seven biosynthetic enzymes to the sieve elements provides strong support for the unique, cell type-specific biosynthesis of benzylisoquinoline alkaloids in the opium poppy.
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PMID:The role of phloem sieve elements and laticifers in the biosynthesis and accumulation of alkaloids in opium poppy. 1681 79

Guanidinoacetate methyltransferase (GAMT) deficiency is an inherited neurometabolic disorder biochemically characterized by tissue accumulation of guanidinoacetate (GAA) and depletion of creatine. Affected patients present epilepsy and mental retardation whose etiopathogeny is unclear. In a previous study we showed that instrastriatal administration of GAA caused a reduction of Na(+),K(+)-ATPase and creatine kinase (CK) activities, as well as an increase in TBARS (an index of lipid peroxidation). In the present study we investigated the in vitro and in vivo effects of GAA on glucose uptake from [U-(14)C] acetate (citric acid cycle activity) and on the activities of complexes II, II-III, III and IV of the respiratory chain in striatum of rats. Results showed that 50 and 100 microM GAA (in vitro studies) and GAA administration (in vivo studies) significantly inhibited complexes II and II-III, respectively, but did not alter complexes III and IV, as well as CO(2) production. We also studied the influence of taurine or vitamins E and C on the inhibitory effects caused by intrastriatal administration of GAA on complexes II and II-III, Na(+),K(+)-ATPase and CK activities, and on TBARS in rat striatum. Pre-treatment with taurine and vitamins E and C revealed that taurine prevents the effects of intrastriatal administration of GAA on the inhibition of complex II, complex II-III, and Na(+),K(+)-ATPase activities. Vitamins E and C prevent the effects of intrastriatal administration of GAA on the inhibition of CK and Na(+),K(+)-ATPase activities, and on the increase of TBARS. The data suggest that GAA in vivo and in vitro treatment disturbs important parameters of striatum energy metabolism and that oxidative damage may be mediating these effects. It is presumed that defects in striatum bioenergetics might be involved in the pathophysiology of striatum damage characteristic of patients with GAMT-deficiency.
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PMID:Evidence that the inhibitory effects of guanidinoacetate on the activities of the respiratory chain, Na+,K+-ATPase and creatine kinase can be differentially prevented by taurine and vitamins E and C administration in rat striatum in vivo. 1740 7

Capping of the pre-mRNA 5' end by addition a monomethylated guanosine cap (m(7)G) is an essential and the earliest modification in the biogenesis of mRNA. The reaction is catalyzed by three enzymes: triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase. Whereas this modification occurs co-transcriptionally in most eukaryotic organisms, trypanosomatid protozoa mRNAs acquire the m(7)G cap by trans-splicing, which entails the transfer of the capped spliced leader (SL) from the SL RNA to the mRNA. Intriguingly, the genomes of all trypanosomatid protozoa sequenced to date possess two distinct proteins with the signature motifs of guanylyltransferases: TbCGM1 and the previously characterized TbCE1. Here we provide biochemical evidence that TbCgm1 is a capping enzyme. Whereas RNAi-induced downregulation of TbCe1 had no phenotypic consequences, we found that TbCGM1 is essential for trypanosome viability and is required for SL RNA capping. Furthermore, consistent with co-transcriptional addition of the m(7)G cap, chromatin immunoprecipitation revealed recruitment of TbCgm1 to the SL RNA genes.
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PMID:Evidence for a capping enzyme with specificity for the trypanosome spliced leader RNA. 1794 28

Effective turnover of many incorrectly processed RNAs in yeast, including hypomodified tRNA(iMet), requires the TRAMP complex, which appends a short poly(A) tail to RNA designated for decay. The poly(A) tail stimulates degradation by the exosome. The TRAMP complex contains the poly(A) polymerase Trf4p, the RNA-binding protein Air2p, and the DExH RNA helicase Mtr4p. The role of Mtr4p in RNA degradation processes involving the TRAMP complex has been unclear. Here we show through a genetic analysis that MTR4 is required for degradation but not for polyadenylation of hypomodified tRNA(iMet). A suppressor of the trm6-504 mutation in the tRNA m(1)A58 methyltransferase (Trm6p/Trm61p), which causes a reduced level of tRNA(iMet), was mapped to MTR4. This mtr4-20 mutation changed a single amino acid in the conserved helicase motif VI of Mtr4p. The mutation stabilizes hypomodified tRNA(iMet) in vivo but has no effect on TRAMP complex stability or polyadenylation activity in vivo or in vitro. We further show that purified recombinant Mtr4p displays RNA-dependent ATPase activity and unwinds RNA duplexes with a 3'-to-5' polarity in an ATP-dependent fashion. Unwinding and RNA-stimulated ATPase activities are strongly reduced in the recombinant mutant Mtr4-20p, suggesting that these activities of Mtr4p are critical for degradation of polyadenylated hypomodified tRNA(iMet).
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PMID:Degradation of hypomodified tRNA(iMet) in vivo involves RNA-dependent ATPase activity of the DExH helicase Mtr4p. 1800 32

The RNA triphosphatase (RTPase) components of the mRNA capping apparatus are a bellwether of eukaryal taxonomy. Fungal and protozoal RTPases belong to the triphosphate tunnel metalloenzyme (TTM) family, exemplified by yeast Cet1. Several large DNA viruses encode metal-dependent RTPases unrelated to the cysteinyl-phosphatase RTPases of their metazoan host organisms. The origins of DNA virus RTPases are unclear because they are structurally uncharacterized. Mimivirus, a giant virus of amoeba, resembles poxviruses in having a trifunctional capping enzyme composed of a metal-dependent RTPase module fused to guanylyltransferase (GTase) and guanine-N7 methyltransferase domains. The crystal structure of mimivirus RTPase reveals a minimized tunnel fold and an active site strikingly similar to that of Cet1. Unlike homodimeric fungal RTPases, mimivirus RTPase is a monomer. The mimivirus TTM-type RTPase-GTase fusion resembles the capping enzymes of amoebae, providing evidence that the ancestral large DNA virus acquired its capping enzyme from a unicellular host.
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PMID:Characterization of a trifunctional mimivirus mRNA capping enzyme and crystal structure of the RNA triphosphatase domain. 1840 Jan 73

Snf2SR, a suppressor of rna1(ts), which is a temperature-sensitive mutation in Schizosaccharomyces pombe RanGAP (GTPase activating protein), possesses both the SNF2 and the helicase domains conserved in the chromatin remodeling SNF2 ATPase/helicase protein family. We have now clarified a function of Snf2SR. Snf2SR indeed showed DNA-stimulated ATPase activity, proving that it is a member of the SNF2 ATPase/helicase family. Consistent with this role, Snf2SR was localized in the nucleus and cell fractionation analysis revealed that Snf2SR was tightly associated with the nuclear matrix. The disruption of snf2SR(+) was detrimental for a cell proliferation of S. pombe. Snf2SR that did not enhance RanGAP activity by itself, but abolished histone-H3-mediated RanGAP inhibition, as previously reported for the histone H3 methyltransferase, Clr4, another rna1(ts) suppressor. In contrast to Clr4, Snf2SR directly bound to the GDP-bound form of the S. pombe Ran homologue Spi1 and enhanced the nucleotide exchange activity of Pim1, the S. pombe RanGEF (guanine nucleotide exchange factor). Over-expression of Spi1-G18V, a Ran GTPase mutant fixed in the GTP-bound form, was lethal to S. pombe Deltasnf2SR. Together, our results indicate that Snf2SR is involved in the Ran GTPase cycle in vivo.
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PMID:Schizosaccharomyces pombe Snf2SR, a novel SNF2 family protein, interacts with Ran GTPase and modulates both RanGEF and RanGAP activities. 1842 2

The plus-strand RNA genome of Sindbis virus (SINV) encodes four nonstructural proteins (nsP1 to nsP4) that are involved in the replication of the viral RNA. The approximately 800-amino-acid nsP2 consists of an N-terminal domain with nucleoside triphosphatase and helicase activities and a C-terminal protease domain. Recently, the structure determined for Venezuelan equine encephalitis virus nsP2 indicated the presence of a previously unrecognized methyltransferase (MTase)-like domain within the C-terminal approximately 200 residues and raised a question about its functional importance. To assess the role of this MTase-like region in viral replication, highly conserved arginine and lysine residues were mutated to alanine. The plaque phenotypes of these mutants ranged from large/wild-type to small plaques with selected mutations demonstrating temperature sensitive lethality. The proteolytic polyprotein processing activity of nsP2 was unaffected in most of the mutants. Some of the temperature-sensitive mutants showed reduction in the minus-strand RNA synthesis, a function that has not yet been ascribed to nsP2. Mutation of SINV residue R615 rendered the virus noncytopathic and incapable of inhibiting the host cell translation but with no effects on the transcriptional inhibition. This property differentiated the mutation at R615 from previously described noncytopathic mutations. These results implicate nsP2 in regulation of minus-strand synthesis and suggest that different regions of the nsP2 MTase-like domain differentially modulate host defense mechanisms, independent of its role as the viral protease.
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PMID:Role for conserved residues of sindbis virus nonstructural protein 2 methyltransferase-like domain in regulation of minus-strand synthesis and development of cytopathic infection. 1849 73

Addition of a 5' cap to RNA polymerase II transcripts, the first step of pre-mRNA processing in eukaryotes from yeasts to mammals, is catalyzed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine-N-7)methyltransferase. The effects of knockdown of these capping enzymes in mammalian cells were investigated using T7 RNA polymerase-synthesized small interfering RNA and also a lentivirus-based inducible, short hairpin RNA system. Decreasing either guanylyltransferase or methyltransferase resulted in caspase-3 activation and elevated terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining characteristic of apoptosis. Induction of apoptosis was independent of p53 tumor suppressor but dependent on BAK or BAX. In addition, levels of the BH3 family member Bim increased, while Mcl-1 and Bik levels remained unchanged during apoptosis. In contrast to capping enzyme knockdown, apoptosis induced by cycloheximide inhibition of protein synthesis required BAK but not BAX. Both Bim and Mcl-1 levels decreased in cycloheximide-induced apoptosis while Bik levels were unchanged, suggesting that apoptosis in siRNA-treated cells is not a direct consequence of loss of mRNA translation. siRNA-treated BAK(-/-) BAX(-/-) double-knockout mouse embryonic fibroblasts failed to activate capase-3 or increase TUNEL staining but instead exhibited autophagy, as demonstrated by proteolytic processing of microtubule-associated protein 1 light chain 3 (LC3) and translocation of transfected green fluorescent protein-LC3 from the nucleus to punctate cytoplasmic structures.
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PMID:Apoptosis and autophagy induction in mammalian cells by small interfering RNA knockdown of mRNA capping enzymes. 1867 51


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