RESUMO
Oil bodies of similar sizes were observed in the cells of embryo and aleurone layer of rice seeds, and remained their structural integrity in vitro after isolation. Comparably, two abundant oleosin isoforms were found in both preparations of oil bodies isolated from the embryo and the aleurone layer. Immunological detection and mass spectrometric analyses indicated that the two oleosin isoforms, termed oleosin-H and oleosin-L, in the embryo and those in the aleurone layer were identical proteins encoded by the same genes (BAF12898.1 and BAF15387.1 for oleosin-H and oleosin-L, respectively). In contrast, one caleosin was found in oil bodies isolated from the embryo but not those isolated from the aleurone layer. Immunological staining of rice seeds confirms that oleosin is present in both embryo and aleurone layer while caleosin exists only in embryo. Caleosin extracted from oil bodies of rice embryo migrated faster on SDS-PAGE in the presence of Ca(2+), in a manner identical to caleosin extracted from sesame oil bodies. Similar to other known monocot caleosins, the rice caleosin possesses an N-terminal appendix that is absent in dicotyledonous caleosins.
Assuntos
Oryza/metabolismo , Óleos de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Sesamum/metabolismo , Sequência de Aminoácidos , Cálcio , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peso Molecular , Oryza/química , Oryza/ultraestrutura , Óleos de Plantas/química , Óleos de Plantas/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Isoformas de Proteínas , Estrutura Terciária de Proteína , Sementes/química , Sementes/ultraestrutura , Sesamum/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizAssuntos
Processamento de Proteína Pós-Traducional , Biocatálise , Escherichia coli/metabolismo , Glicoproteínas de Membrana/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Fosfoadenosina Fosfossulfato/química , Plasmídeos/metabolismo , Sulfato Adenililtransferase/química , Sulfato Adenililtransferase/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismoRESUMO
Protein arginine methylation is found in many nucleic acid binding proteins affecting numerous cellular functions. In this study we identified methylarginine-containing proteins in HeLa cell extracts by two-dimensional electrophoresis and immunoblotting with a methylarginine-specific antibody. Protein spots with matched protein stain and blotting signals were analyzed by mass spectrometry. The identities of 12 protein spots as 11 different proteins were suggested. Known methylarginine-containing proteins such as hnRNP A2/B1, hnRNP A1, hnRNP G and FUS were identified, indicating the feasibility of our approach. However, four highly abundant metabolic enzymes that might co-electrophorese with methylarginine-containing proteins were also identified. Other nucleic acid binding proteins hnRNP M, hnRNP I and NonO protein were identified. Recombinant hnRNP M and a peptide with the RGG sequence in hnRNP M could be further methylated in vitro. The immunoblotting results of immunoprecipitated hnRNP I and NonO protein are consistent with arginine methylation in both proteins. In this study we identified methylarginine-containing proteins in HeLa cells through proteomic approaches and the method is fast and robust for further applications.
Assuntos
Arginina/análogos & derivados , Eletroforese em Gel Bidimensional/métodos , Immunoblotting/métodos , Sequência de Aminoácidos , Animais , Anticorpos/química , Especificidade de Anticorpos , Arginina/análise , Arginina/metabolismo , Proteínas de Ligação a DNA , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/análise , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/análise , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Humanos , Metilação , Dados de Sequência Molecular , Proteínas Associadas à Matriz Nuclear/análise , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/análise , Fatores de Transcrição de Octâmero/metabolismo , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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
Adenosine dialdehyde (AdOx) is an indirect methyltransferase inhibitor broadly used in cell culture to accumulate methyl-accepting proteins in hypomethylated states for in vitro protein methylation analyses. In this study we included a translation inhibitor, cycloheximide, in the AdOx treatment of HeLa cells. The methyl-accepting proteins disappeared in the double treatment, indicating that they were most likely newly synthesized in the AdOx incubation period. AdOx treatment could also be used in combination with in vivo methylation, another technique frequently used to study protein methylation. AdOx treatment prior to in vivo methylation accumulated methyl-accepting proteins for the labeling reaction. The continued presence of AdOx in the in vivo labeling period decreased the methylation of the majority of in vivo methyl-accepting polypeptides. The level and pattern of the in vivo methylated polypeptides did not change after a 12-h chase, supporting the notion that the methylated polypeptide as well as the methyl groups on the modified polypeptides are stable. On the other hand, methylarginine-specific antibodies detected limited but consistent reduction of the methylarginine-containing proteins in AdOx-treated samples compared to the untreated ones. Thus, AdOx treatment probably only blocked a small fraction of stable protein methylation. Overall, it is likely that base-stable methylation are formed soon after the synthesis of the polypeptide and remain stable after the modification.
Assuntos
Adenosina/análogos & derivados , Adenosina/farmacologia , Cicloeximida/química , Adenosina/química , Arginina/química , Western Blotting , Cicloeximida/farmacologia , Metilação de DNA , Detergentes/farmacologia , Células HeLa , Humanos , Metilação , Oxigênio/metabolismo , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/química , Inibidores da Síntese de Proteínas/farmacologia , Proteínas/química , Fatores de Tempo , Tripsina/farmacologiaRESUMO
We investigated the global distribution of methylaccepting proteins in lymphoblastoid cells by two-dimensional (2-D) gel electrophoresis. The 2-D electrophoreograms of normal and hypo-methylation (cells grown with a methyltransferase inhibitor adenosine dialdehyde) protein extracts did not exhibit significant differences. However, in vitro methylation of the hypomethylated extracts in the presence of the methyl-group donor S-adenosyl-[methyl-3H]-methionine revealed close to a hundred signals. Less than one-fifth of the signals could be correlated with protein stains, indicating that most of the methylaccepting proteins are low abundant ones. We analyzed six of the spots that can be correlated with protein stains and suggested their identities. Among these putative protein methylacceptors, three are heterogeneous nuclear ribonucleoproteins (hnRNPA2/B1 and hnRNP K) that are reportedly methylated in their arginine- and glycine-rich RGG motifs.