RESUMO
Protein arginine N-methyltransferases (PRMTs) are a family of 9 enzymes that catalyze mono- or di-methylation of arginine residues using S-adenosyl-l-methionine (SAM). Arginine methylation is an important post-translational modification that can regulate the activity and structure of target proteins. Altered PRMT activity can lead to a variety of health issues including neurodevelopmental disease, autoimmune disorders, cancer, and cardiovascular disease. Thus, developing a robust mechanistic understanding of PRMT function may provide insight into these various disease states and enable the development of potential therapeutic agents. Although PRMTs have been studied for nearly two decades, a consensus regarding the mechanism of action for this class of enzymes has remained noticeably elusive. To address this shortcoming, differential scanning fluorimetry (DSF) was used to gain mechanistic insight into the order of PRMT substrate and cofactor binding. This methodology confirms that PRMT cofactor binding precedes target substrate binding and supports the use of DSF to study bisubstrate enzymatic reaction mechanisms.
Assuntos
Arginina/química , Varredura Diferencial de Calorimetria/métodos , Coenzimas/química , Ensaios Enzimáticos/métodos , Fluorometria/métodos , Proteína-Arginina N-Metiltransferases/química , S-Adenosilmetionina/química , Arginina/metabolismo , Catálise , Dimerização , Corantes Fluorescentes/química , Expressão Gênica , Cinética , Metilação , Conformação Proteica em alfa-Hélice , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Recombinantes , S-Adenosilmetionina/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
Protein arginine methyltransferase (PRMT) is an important epigenetic regulator in eukaryotic cells. During encystation, an essential process for Acanthamoeba survival, the expression of a lot of genes involved in the encystation process has to be regulated in order to be induced or inhibited. However, the regulation mechanism of these genes is yet unknown. In this study, the full-length 1,059 bp cDNA sequence of Acanthamoeba castellanii PRMT1 (AcPRMT1) was cloned for the first time. The AcPRMT1 protein comprised of 352 amino acids with a SAM-dependent methyltransferase PRMT-type domain. The expression level of AcPRMT1 was highly increased during encystation of A. castellanii. The EGFP-AcPRMT1 fusion protein was distributed over the cytoplasm, but it was mainly localized in the nucleus of Acanthamoeba. Knock down of AcPRMT1 by synthetic siRNA with a complementary sequence failed to form mature cysts. These findings suggested that AcPRMT1 plays a critical role in the regulation of encystation of A. castellanii. The target gene of AcPRMT1 regulation and the detailed mechanisms need to be investigated by further studies.
Assuntos
Acanthamoeba castellanii/enzimologia , Acanthamoeba castellanii/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Encistamento de Parasitas/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Acanthamoeba castellanii/citologia , Acanthamoeba castellanii/crescimento & desenvolvimento , Citoplasma/genética , Citoplasma/metabolismo , DNA de Protozoário/genética , Expressão Gênica/genética , Fusão Gênica , Proteínas de Fluorescência Verde , Encistamento de Parasitas/fisiologia , Proteína-Arginina N-Metiltransferases/químicaRESUMO
The transmethylation to arginine residues of proteins is catalyzed by protein arginine methyltransferases (PRMTs) that form monomethylarginine (MMA), asymmetric (ADMA) and symmetric dimethylarginines (SDMA). Although we previously demonstrated that the generation of ADMA residues in whole proteins is driven by PRMT-1 in Caenorhabditis elegans, much less is known about MMA and SDMA in vivo. In this study, we measured the amounts of different methylarginines in whole protein extracts made from wild-type (N2) C. elegans and from prmt-1 and prmt-5 null mutants using liquid chromatography-tandem mass spectrometry. Interestingly, we found that the amounts of MMA and SDMA are about fourfold higher than those of ADMA in N2 protein lysates using acid hydrolysis. We were unable to detect SDMA residues in the prmt-5 null mutant. In comparison with N2, an increase in SDMA and decrease in MMA were observed in prmt-1 mutant worms with no ADMA, but ADMA and MMA levels were unchanged in prmt-5 mutant worms. These results suggest that PRMT-1 contributes, at least in part, to MMA production, but that PRMT-5 catalyzes the symmetric dimethylation of substrates containing MMA residues in vivo.
Assuntos
Arginina/análogos & derivados , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Arginina/análise , Arginina/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/isolamento & purificação , Cromatografia Líquida , Metilação , Mutação , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Espectrometria de Massas em TandemRESUMO
Protein arginine methyltransferase 7 (PRMT7) is a unique but less characterized member of the family of protein arginine methyltransferases (PRMTs) that plays a role in male germline gene imprinting. PRMT7 is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem. PRMT7 genes from five different species were cloned and expressed in Escherichia coli and Sf21 insect cells. Four gave soluble proteins from Sf21 cells, of which two were homogeneous and one gave crystals. The mouse PRMT7 structure was solved by the single anomalous dispersion method using a crystal soaked with thimerosal that diffracted to beyond 2.1â Å resolution. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 97.4, c = 168.1â Å and one PRMT7 monomer in the asymmetric unit. The structure of another crystal form belonging to space group I222 was solved by molecular replacement.
Assuntos
Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Sequência de Aminoácidos , Animais , Proteínas de Arabidopsis/metabolismo , Cromatografia em Gel , Clonagem Molecular , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Humanos , Luz , Masculino , Camundongos , Dados de Sequência Molecular , Espalhamento de Radiação , TransfecçãoRESUMO
Reactive oxygen species (ROS) play an important role in normal biological functions and pathological processes. ROS is one of the driving forces for oxidizing proteins, especially on cysteine thiols. The labile, transient, and dynamic nature of oxidative modifications poses enormous technical challenges for both accurate modification site determination and quantitation of cysteine thiols. The present study describes a mass spectrometry-based approach that allows effective discovery and quantification of irreversible cysteine modifications. The utilization of a long reverse phase column provides high-resolution chromatography to separate different forms of modified cysteine thiols from protein complexes or cell lysates. This Fourier transform mass spectrometry (FT-MS) approach enabled detection and quantitation of ataxia telangiectasia mutated (ATM) complex cysteine sulfoxidation states using Skyline MS1 filtering. When we applied the long column ultra high pressure liquid chromatography (UPLC)-MS/MS analysis, 61 and 44 peptides from cell lysates and cells were identified with cysteine modifications in response to in vitro and in vivo H2O2 oxidation, respectively. Long column ultra high pressure liquid chromatography pseudo selected reaction monitoring (UPLC-pSRM) was then developed to monitor the oxidative level of cysteine thiols in cell lysate under varying concentrations of H2O2 treatment. From UPLC-pSRM analysis, the dynamic conversion of sulfinic (S-O2H) and sulfonic acid (S-O3H) was observed within nucleoside diphosphate kinase (Nm23-H1) and heat shock 70 kDa protein 8 (Hsc70). These methods are suitable for proteome-wide studies, providing a highly sensitive, straightforward approach to identify proteins containing redox-sensitive cysteine thiols in biological systems.
Assuntos
Cisteína/metabolismo , Proteoma/metabolismo , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/isolamento & purificação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Dados de Sequência Molecular , Oxidantes/metabolismo , Oxidantes/farmacologia , Oxirredução , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteoma/química , Proteoma/isolamento & purificação , Padrões de Referência , Sulfóxidos/isolamento & purificação , Sulfóxidos/metabolismo , Espectrometria de Massas em Tandem/normasRESUMO
Coactivator-associated arginine methyl transferase 1 (CARM1) is a protein arginine methyltransferase (PRMT) family member that functions as a coactivator in androgen and estrogen signaling pathways and plays a role in the progression of prostate and breast cancer. CARM1 catalyzes methylation of diverse protein substrates. Prior attempts to purify the full-length mouse CARM1 protein have proven unsatisfactory. The full-length protein expressed in Escherichia coli forms insoluble inclusion bodies that are difficult to denature and refold. The presented results demonstrate the use of a novel HaloTag™ technology to purify full-length CARM1 from both E. coli and mammalian HEK293T cells. A small amount of CARM1 was purified from E. coli; however, the protein was truncated on the N-terminus by 10-50 amino acids, most likely due to endogenous proteolytic activity. In contrast, substantial quantities of soluble full-length CARM1 were purified from transiently transfected HEK293T cells. The CARM1 from HEK293T cells was isolated alongside a number of co-purifying interacting proteins. The covalent bond formed between the HaloTag and the HaloLink resin allowed the use of stringent wash conditions without risk of eluting the CARM1 protein. The results also illustrate a highly effective approach for purifying and enriching both CARM1-associated proteins as well as substrates for CARM1's methyltransferase activity.
Assuntos
Cromatografia de Afinidade/métodos , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Western Blotting , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Proteínas Imobilizadas/metabolismo , Camundongos , Microscopia de Fluorescência , Proteína-Arginina N-Metiltransferases/biossíntese , Proteína-Arginina N-Metiltransferases/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Solubilidade , TransfecçãoRESUMO
Protein arginine methyltransferases (PRMTs) are SAM-dependent enzymes that catalyze the mono- and dimethylation of peptidyl arginine residues. Although all PRMTs produce monomethyl arginine (MMA), type 1 PRMTs go on to form asymmetrically dimethylated arginine (ADMA), while type 2 enzymes form symmetrically dimethylated arginine (SDMA). PRMT1 is the major type 1 PRMT in vivo, thus it is the primary producer of the competitive NOS inhibitor, ADMA. Hence, potent inhibitors, which are highly selective for this particular isozyme, could serve as excellent therapeutics for heart disease. However, the design of such inhibitors is impeded by a lack of information regarding this enzyme's kinetic and catalytic mechanisms. Herein we report an analysis of the kinetic mechanism of human PRMT1 using both an unmethylated and a monomethylated substrate peptide based on the N-terminus of histone H4. The results of initial velocity and product and dead-end inhibition experiments indicate that PRMT1 utilizes a rapid equilibrium random mechanism with the formation of dead-end EAP and EBQ complexes. This mechanism is gratifyingly consistent with previous results demonstrating that PRMT1 catalyzes substrate dimethylation in a partially processive manner.
Assuntos
Proteína-Arginina N-Metiltransferases/metabolismo , Catálise , Cardiopatias/enzimologia , Humanos , Isoenzimas/metabolismo , Cinética , Metilação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/isolamento & purificaçãoRESUMO
Protein arginine methylation is a posttranslational modification involved in various cellular functions including cell signaling, protein subcellular localization and transcriptional regulation. We analyze the protein arginine methyltransferases (PRMTs) that catalyze the formation of methylarginines in porcine brain. We fractionated the brain extracts and determined the PRMT activities as well as the distribution of different PRMT proteins in subcellular fractions of porcine brain. The majority of the type I methyltransferase activities that catalyze the formation of asymmetric dimethylarginines was in the cytosolic S3 fraction. High specific activity of the methyltransferase was detected in the S4 fraction (high-salt stripping of the ultracentrifugation precipitant P3 fraction), indicating that part of the PRMT was peripherally associated with membrane and ribosomal fractions. The amount and distribution of PRMT1 are consistent with the catalytic activity. The elution patterns from gel filtration and anion exchange chromatography also indicate that the type I activity in S3 and S4 are mostly from PRMT1. Our results suggest that part of the type I arginine methyltransferases in brains, mainly PRMT1, are sequestered in an inactive form as they associated with membranes or large subcellular complexes. Our biochemical analyses confirmed the complex distribution of different PRMTs and implicate their regulation and catalytic activities in brain.
Assuntos
Encéfalo/enzimologia , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Western Blotting , Cromatografia em Gel , Cromatografia por Troca Iônica , Ativação Enzimática/efeitos dos fármacos , Metilação/efeitos dos fármacos , Cloreto de Potássio/farmacologia , SuínosRESUMO
Human PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) encodes a type II protein arginine (Arg) methyltransferase and its homologs in animals and yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe) are known to regulate RNA processing, signal transduction, and gene expression. However, PRMT5 homologs in higher plants have not yet been reported and the biological roles of these proteins in plant development remain elusive. Here, using conventional biochemical approaches, we purified a plant histone Arg methyltransferase from cauliflower (Brassica oleracea) that was nearly identical to AtPRMT5, an Arabidopsis (Arabidopsis thaliana) homolog of human PRMT5. AtPRMT5 methylated histone H4, H2A, and myelin basic protein in vitro. Western blot using symmetric dimethyl histone H4 Arg 3-specific antibody and thin-layer chromatography analysis demonstrated that AtPRMT5 is a type II enzyme. Mutations in AtPRMT5 caused pleiotropic developmental defects, including growth retardation, dark green and curled leaves, and FlOWERING LOCUS C (FLC)-dependent delayed flowering. Therefore, the type II protein Arg methyltransferase AtPRMT5 is involved in promotion of vegetative growth and FLC-dependent flowering time regulation in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Histonas/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Plântula/crescimento & desenvolvimento , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Arginina/metabolismo , Brassica/química , Metilação , Dados de Sequência Molecular , Mutação , Fenótipo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Fatores de TempoRESUMO
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. CARM1(28-507) and two structural states of CARM1(140-480) were expressed, purified and crystallized. Crystals of CARM1(28-507) belong to space group P6(2)22, with unit-cell parameters a = b = 136.0, c = 125.3 A; they diffract to beyond 2.5 A resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM1(28-507) was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1(140-480) belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 A; they diffract to beyond 2.7 A resolution and contain two monomers in the asymmetric unit. Crystals of CARM1(140-480) in complex with S-adenosyl-L-homocysteine belong to space P2(1)2(1)2, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 A; they diffract to beyond 2.6 A resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1(140-480) were solved by molecular-replacement techniques from the structure of CARM1(28-507).
Assuntos
Proteína-Arginina N-Metiltransferases/química , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Primers do DNA , Camundongos , Conformação Proteica , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/isolamento & purificação , SpodopteraRESUMO
OBJECTIVE: To identify a new antibactrial polypeptide HCP-1 isolated from human cervical mucus. METHODS: HCP-1 was isolated and purified from human cervical mucus, and N-terminal sequence of HCP-1 was determined. A degenerate primer was designed according to CodeHop methods, and an "anchor-oliga-dT primer" was used for the synthesis of cDNA. Using the degenerate primer and anchor-primer, cDNAs were amplified by PCR. The PCR products were cloned, sequenced, and analyzed by biological software. RESULTS: N-terminat sequence of HCP-1 was PKRKAEGDAK. The full length of HCP-1 cDNA was isolated and of which the sequence was the same as HMG-17. OMIGA software analysis indicated that this molecule contained an alpha-helix region. CONCLUSION: The new antibacterial polypeptide isolated from human cervical mucus is HMG-17. It may play a role in the human cervical mucus and the alpha-helix domain may be its antibacterial activity region.
Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Muco do Colo Uterino/química , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Proteína-Arginina N-Metiltransferases/farmacologia , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/farmacologia , Adulto , Antibacterianos/química , Muco do Colo Uterino/fisiologia , Feminino , Humanos , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Proteína-Arginina N-Metiltransferases/química , Proteínas Repressoras/químicaRESUMO
PRMT3 (protein arginine methyltransferase 3) is one of four type I arginine methyltransferases that catalyse the formation of asymmetric dimethylarginine. PRMT3 is unique in that its N-terminus harbours a C2H2 zinc-finger domain that is proposed to confer substrate specificity. In addition, PRMT3 is the only type I enzyme that is restricted to the cytoplasm. Known in vitro substrates for PRMT3 include GST-GAR (a glutathione S-transferase fusion protein containing the glycine- and arginine-rich N-terminal region of fibrillarin), Sam68 (Src-associated substrate during mitosis 68 kDa) and PABP-N1 [poly(A)-binding protein-N1; PABP2]. Here we report the identification of an in vivo substrate for mammalian PRMT3. We found that FLAG-tagged PRMT3 can 'pull down' a protein with a molecular mass of 30 kDa from HeLa cell extracts. MS identified this PRMT3-interacting protein as rpS2 (ribosomal protein S2). In vitro studies showed that the zinc-finger domain of PRMT3 is necessary and sufficient for binding to rpS2. In addition, rpS2 is methylated by PRMT3 in vitro and is also methylated in cell lines. Deletion analysis of the rpS2 amino acid sequence identified a N-terminal Arg-Gly repeat as the methylation site. Furthermore, both PRMT3 and rpS2 co-sediment with free ribosomal subunits. These studies implicate PRMT3 in ribosomal function and in the regulation of protein synthesis.
Assuntos
Biossíntese de Proteínas/fisiologia , Proteína-Arginina N-Metiltransferases/fisiologia , Proteínas Ribossômicas/metabolismo , Animais , Arginina/metabolismo , Cromatografia de Afinidade , Células HeLa , Humanos , Metilação , Camundongos , Células NIH 3T3 , Mapeamento de Interação de Proteínas , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Ribossomos/metabolismo , Dedos de Zinco/fisiologiaRESUMO
We have identified a mammalian arginine N-methyltransferase, PRMT7, that can catalyze the formation of omega-NG-monomethylarginine in peptides. This protein is encoded by a gene on human chromosome 16q22.1 (human locus AK001502). We expressed a full-length human cDNA construct in Escherichia coli as a glutathione S-transferase (GST) fusion protein. We found that GST-tagged PRMT7 catalyzes the S-adenosyl-[methyl-3H]-l-methionine-dependent methylation of the synthetic peptide GGPGGRGGPGG-NH2 (R1). The radiolabeled peptide was purified by high-pressure liquid chromatography and acid hydrolyzed to free amino acids. When the hydrolyzed products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species which co-migrated with an omega-NG-monomethylarginine standard. Surprisingly, GST-PRMT7 was not able to catalyze the in vitro methylation of a GST-fibrillarin (amino acids 1-148) fusion protein (GST-GAR), a methyl-accepting substrate for the previously characterized PRMT1, PRMT3, PRMT4, PRMT5, and PRMT6 enzymes. Nor was it able to methylate myelin basic protein or histone H2A, in vitro substrates of PRMT5. This specificity distinguishes PRMT7 from all of the other known arginine methyltransferases. An additional unique feature of PRMT7 is that it seems to have arisen from a gene duplication event and contains two putative AdoMet-binding motifs. To see if both motifs were necessary for activity, each putative domain was expressed as a GST-fusion and tested for activity with peptides R1 and R2 (acetyl-GGRGG-NH2). These truncated proteins were enzymatically inactive, suggesting that both domains are required for functionality.
Assuntos
Cromossomos Humanos Par 16 , Metiltransferases/genética , Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases , Sequência de Aminoácidos , Mapeamento Cromossômico , Humanos , Metiltransferases/isolamento & purificação , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Proteína-Arginina N-Metiltransferases/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The recruitment of coactivators by nuclear hormone receptors (NRs) promotes transcription by subverting chromatin-mediated repression. Although the histone methylation enzyme CARM1 and an ATP-remodeling complex have been individually implicated in nuclear receptor-dependent transcription, neither a functional nor mechanistic linkage between these systems has been identified. In the process of purifying endogenous CARM1-interacting proteins, we identified an associated complex, nucleosomal methylation activator complex (NUMAC), which includes at least eight components of SWI/SNF, including the ATPase BRG1. In the NUMAC complex, the methylase, CARM1, acquires the ability to covalently modify nucleosomal histones, and the directed nucleosome versus free core histone methylation-specificity change is increased dramatically. Reciprocally, CARM1 stimulates the ATPase activity of BRG1, a key component in nucleosome remodeling. In vivo, CARM1 and BRG1 coassemble on an estrogen receptor (ER)-target gene to cooperatively activate ER-dependent transcription. This association of ATP-remodeling factors with HMT CARM1 defines a new component of regulation in the nuclear hormone-signaling pathway.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Substâncias Macromoleculares , Metilação , Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Especificidade por SubstratoRESUMO
N(G)-Methylation of arginine residues in many nucleic-acid-binding proteins are formed post-translationally, catalysed by S-adenosylmethionine:protein-arginine N-methyltransferase in their glycine-rich and arginine-rich motifs. The amino acid sequences of the stimulator of HIV-1 TAR (Tat-responsive element) RNA-binding protein (SRB) and fibronectin also show the presence of the internal -Gly-Arg-Gly- (-GRG-) sequence, which is potentially methylatable by the methyltransferase. To investigate the sequence requirement for methylation of these proteins, several synthetic oligopeptides with different chain lengths and sequences similar to the -GRG- regions of SRB and fibronectin were synthesized. Whereas the heptapeptide AGGRGKG (residues 16-22 in SRB) served as the methyl acceptor for the methyltransferase with a K(m) of 50 microM, the 19-mer peptide (residues 10-28 in SRB) was methylated with a K(m) of 8.3 microM, indicating that a greater peptide chain length yields a better methyl acceptor. Product analysis of the methylated [methyl-(14)C]SRB-peptide by HPLC indicated the formation of N(G)-monomethylarginine and N(G),N(G)-dimethyl(asymmetric)arginine. Synthetic peptides containing the cell attachment sequence [Arg-Gly-Asp ('RGD')] in fibronectin, GRGDSPK, GGRGDSPK and GGGRGDSPK, were also studied; whereas GRGDSPK was a poor methyl acceptor, the longer peptides were better methyl acceptors. To provide an understanding of the effect of methylation on fibronectin peptide, arginine-unmethylated and methylated GGRGDSPK were compared for their effect on the mitogenesis induced by beta-hexosaminidase A and an agonistic antibody (mAb(15)) in bovine tracheal smooth-muscle cells; whereas the former inhibited 35-67% of mitogenesis at a concentration of 5-10 microM, the latter did not block mitogenesis. This lack of inhibition by the insertion of a methyl group on the arginyl residue of the cell attachment sequence might be due to the hindrance of the binding of fibronectin peptide to integrins.
Assuntos
Arginina/metabolismo , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Replicação do DNA , Fibronectinas/química , Fibronectinas/metabolismo , Cinética , Fígado/enzimologia , Metilação , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Proteína-Arginina N-Metiltransferases/metabolismo , RatosRESUMO
Type I protein arginine methyltransferases catalyze the formation of asymmetric omega-N(G),N(G)-dimethylarginine residues by transferring methyl groups from S-adenosyl-L-methionine to guanidino groups of arginine residues in a variety of eucaryotic proteins. The predominant type I enzyme activity is found in mammalian cells as a high molecular weight complex (300-400 kDa). In a previous study, this protein arginine methyltransferase activity was identified as an additional activity of 10-formyltetrahydrofolate dehydrogenase (FDH) protein. However, immunodepletion of FDH activity in RAT1 cells and in murine tissue extracts with antibody to FDH does not diminish type I methyltransferase activity toward the methyl-accepting substrates glutathione S-transferase fibrillarin glycine arginine domain fusion protein or heterogeneous nuclear ribonucleoprotein A1. Similarly, immunodepletion with anti-FDH antibody does not remove the endogenous methylating activity for hypomethylated proteins present in extracts from adenosine dialdehyde-treated RAT1 cells. In contrast, anti-PRMT1 antibody can remove PRMT1 activity from RAT1 extracts, murine tissue extracts, and purified rat liver FDH preparations. Tissue extracts from FDH(+/+), FDH(+/-), and FDH(-/-) mice have similar protein arginine methyltransferase activities but high, intermediate, and undetectable FDH activities, respectively. Recombinant glutathione S-transferase-PRMT1, but not purified FDH, can be cross-linked to the methyl-donor substrate S-adenosyl-L-methionine. We conclude that PRMT1 contributes the major type I protein arginine methyltransferase enzyme activity present in mammalian cells and tissues.
Assuntos
Arginina/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Proteínas Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Metilação , Camundongos , Camundongos Mutantes , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/isolamento & purificação , Proteínas Metiltransferases/classificação , Proteínas Metiltransferases/isolamento & purificação , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/classificação , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Ratos , S-Adenosilmetionina/metabolismoRESUMO
Protein-arginine N-methyltransferase (protein methylase I) catalyzes methylation of arginyl residues on substrate protein posttranslationally utilizing S-adenosyl-L-methionine as the methyl donor and yields NG-methylarginine residues. Arginyl-fructose and arginyl-fructosyl-glucose from Korean red ginseng were found to inhibit protein methylase I activity in vitro. This inhibitory activity was shown to be due to arginyl moiety in the molecules, rather than that of carbohydrates. Several basic amino acids as well as polyamines were also found to inhibit protein methylase I activity. Interestingly, the intensity of the inhibitory activity was correlated with the number of amino-group in polyamines, thus, in the order of spermine > spermidine > putrescine > agmatine-sulfate, with IC50 at approximately 15 mM, 25 mM, 35 mM, and 50 mM, respectively. On the other hand, neutral amino acids or NaCl did not inhibit the enzyme activity. Lineweaver-Burk plot analysis of the protein methylase I activity in the presence of arginine and spermidine indicated that the inhibition was competitive in nature in respect to protein substrate, with the Ki values of 24.8 mM and 11.5 mM, respectively.
Assuntos
Aminoácidos/química , Arginina/química , Inibidores Enzimáticos/farmacologia , Panax/química , Extratos Vegetais/farmacologia , Plantas Medicinais , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Arginina/farmacologia , Cromatografia por Troca Iônica , Cinética , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Espermidina/farmacologiaRESUMO
Heterogenous nuclear ribonucleoproteins (hnRNPs) bind pre-mRNAs and facilitate their processing into mRNAs. Many of the hnRNPs undergo extensive posttranslational modifications including methylation on arginine residues. hnRNPs contain about 65% of the total NG,NG-dimethylarginine found in the cell nucleus. The role of this modification is not known. Here we identify the hnRNPs that are methylated in HeLa cells and demonstrate that most of the pre-mRNA-binding proteins receive this modification. Using recombinant human hnRNP A1 as a substrate, we have partially purified and characterized a protein-arginine N-methyltransferase specific for hnRNPs from HeLa cells. This methyltransferase can methylate the same subset of hnRNPs in vitro as are methylated in vivo. Furthermore, it can also methylate other RNA-binding proteins that contain the RGG motif RNA-binding domain. This activity is evolutionarily conserved from lower eukaryotes to mammals, suggesting that methylation has a significant role in the function of RNA-binding proteins.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Arginina/química , Arginina/metabolismo , Sítios de Ligação/genética , Citoplasma/enzimologia , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Metilação , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Proteína-Arginina N-Metiltransferases/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Especificidade por SubstratoRESUMO
A protein methylase I (S-adenosylmethionine-protein-arginine N-methyltransferase; EC 2.1.1.23), with a high specificity for recombinant heterogeneous nuclear ribonucleoprotein particle (hnRNP) protein A1, was purified from rat liver. The purification method is simple and rapid; a single initial step of DEAE-cellulose DE-52 chromatography resulted in a 114-fold enrichment from the cytosol, and subsequent Sephadex G-200 chromatography and f.p.l.c. yielded a homogeneous preparation. Ouchterlony double-immunodiffusion analysis indicated that the rat liver enzyme is immunologically different from an analogous enzyme from the calf brain, nuclear protein/histone-specific protein methylase I [Ghosh, Paik and Kim (1988) J. Biol. Chem. 263, 19024-19033; Rajpurohit, Lee, Park, Paik and Kim (1994) J. Biol. Chem. 269, 1075-1082]. The purified enzyme has a molecular mass of 450 kDa on Superose chromatography and 110 kDa on SDS/PAGE, indicating that it is composed of four identical-size subunits. The Km values for protein A1 and S-adenosyl-L-methionine were 0.54 x 10(-6) and 6.3 x 10(-6) M respectively. S-Adenosyl-L-homocysteine and sinefungin were effective inhibitors of the enzyme with Ki values of 8.4 x 10(-6) M and 0.65 x 10(-6) M respectively. Bivalent metal ions such as Zn2+, Mn2+ and Ni2+ were particularly toxic to the enzyme; at 1 mM Zn2+, 99% of the activity was inhibited. In addition, 50% of the enzyme activity was lost by treatment with 0.12 mM p-chloromercuribenzoate, indicating a requirement for a thiol group for enzyme activity. Glycerol, a compound often used to prevent enzyme inactivation, inhibited over 80% of the activity when present in the reaction mixture at a concentration of 20%.
Assuntos
Fígado/enzimologia , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Animais , Encéfalo/enzimologia , Bovinos , Cromatografia DEAE-Celulose , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Glicerol/farmacologia , Metilação , Peso Molecular , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , Ratos , Especificidade por SubstratoRESUMO
1. Protein methylase I (S-adenosylmethionine[:]protein-arginine N-methyltransferase; EC 2.1.1.23) which methylates protein-bound arginine residues has been purified from human term placenta 400-fold with an approximate yield of 6%. 2. When histone was used as in vitro substrate, the methylation products were found to be NG-mono-, NG, NG-di- and NG, N'G-dimethylarginine. The enzyme was found to be sensitive toward Cu2+ with Ki value of 8 x 10(-5) M. The Km value for S-adenosyl-L-methionine was 5 x 10(-6) M. 3. When this partially purified protein methylase I was incubated with isolated human placental nuclei and S-adenosyl-L-[methyl-3H]methionine, the major endogenous [methyl-3H]-labeled proteins were protein species of 23, 38, 45 and 68 kDa, the 23 kDa species being the most predominant. 4. The endogenous enzyme activity during the pregnancy increased significantly, reaching more than 4 times the initial activity at the end of term.