RESUMO
Complex I of the mitochondrial respiratory chain is a large multisubunit enzyme that assembles from nuclear and mtDNA-encoded components. Several complex I assembly factors have been identified, but their precise functions are not well understood. Here, we have investigated the function of one of these, NDUFAF7, a soluble matrix protein comprised of a DUF185 domain that harbors a methyltransferase motif. Knockdown of NDUFAF7 by siRNA in human fibroblasts produced a specific complex I assembly defect, as did morpholino-mediated knockdown of the zebrafish ortholog. Germline disruption of the murine ortholog was an early embryonic lethal. The complex I assembly defect was characterized by rapid, AFG3L2-dependent, turnover of newly synthesized ND1, the subunit that seeds the assembly pathway, and by decreased steady-state levels of several other structural subunits including NDUFS2, NDUFS1 and NDUFA9. Expression of an NDUFAF7 mutant (G124V), predicted to disrupt methyltransferase activity, impaired complex I assembly, suggesting an assembly factor or structural subunit as a substrate for methylation. To identify the NDUFAF7 substrate, we used an anti-ND1 antibody to immunoprecipitate complex I and its associated assembly factors, followed by mass spectrometry to detect posttranslational protein modifications. Analysis of an NDUFAF7 methyltransferase mutant showed a 10-fold reduction in an NDUFS2 peptide containing dimethylated Arg85, but a 5-fold reduction in three other NDUFS2 peptides. These results show that NDUFAF7 functions to methylate NDUFS2 after it assembles into a complex I, stabilizing an early intermediate in the assembly pathway, and that this function is essential for normal vertebrate development.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Desenvolvimento Embrionário/genética , NADH Desidrogenase/genética , Motivos de Aminoácidos , Animais , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/genética , Linhagem Celular , Fibroblastos , Técnicas de Silenciamento de Genes , Genes Letais , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , NADH Desidrogenase/química , NADH Desidrogenase/metabolismo , Fenótipo , Domínios e Motivos de Interação entre Proteínas , Proteólise , Interferência de RNA , Especificidade por Substrato , Vertebrados , Peixe-ZebraRESUMO
RATIONALE: The renin-angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1-7). OBJECTIVE: To characterize a novel component of the RAS, alamandine. METHODS AND RESULTS: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1-7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1-7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein-coupled receptor, member D. Binding of alamandine to Mas-related G-protein-coupled receptor, member D is blocked by D-Pro(7)-angiotensin-(1-7), the Mas-related G-protein-coupled receptor, member D ligand ß-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/ß-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. CONCLUSIONS: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders.
Assuntos
Angiotensina I/química , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Descoberta de Drogas , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Sistema Renina-Angiotensina/fisiologia , Angiotensina I/fisiologia , Angiotensina II/análogos & derivados , Angiotensina II/química , Angiotensina II/fisiologia , Enzima de Conversão de Angiotensina 2 , Animais , Anti-Hipertensivos/isolamento & purificação , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Descoberta de Drogas/métodos , Humanos , Masculino , Oligopeptídeos/fisiologia , Fragmentos de Peptídeos/fisiologia , Peptidil Dipeptidase A/fisiologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/fisiologia , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos SHR , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/fisiologiaRESUMO
Este estudo teve como objetivo verificar a adequação físico-estrutural e de boas práticas de fabricação de alimentos de 12 panificadoras localizadas em grandes supermercados da cidade de Anápolis-GO.
