RESUMO
2-Oxoglutarate (2OG) and Fe(II)-dependent oxygenase domain-containing protein 1 (OGFOD1) is predicted to be a conserved 2OG oxygenase, the catalytic domain of which is related to hypoxia-inducible factor prolyl hydroxylases. OGFOD1 homologs in yeast are implicated in diverse cellular functions ranging from oxygen-dependent regulation of sterol response genes (Ofd1, Schizosaccharomyces pombe) to translation termination/mRNA polyadenylation (Tpa1p, Saccharomyces cerevisiae). However, neither the biochemical activity of OGFOD1 nor the identity of its substrate has been defined. Here we show that OGFOD1 is a prolyl hydroxylase that catalyzes the posttranslational hydroxylation of a highly conserved residue (Pro-62) in the small ribosomal protein S23 (RPS23). Unusually OGFOD1 retained a high affinity for, and forms a stable complex with, the hydroxylated RPS23 substrate. Knockdown or inactivation of OGFOD1 caused a cell type-dependent induction of stress granules, translational arrest, and growth impairment in a manner complemented by wild-type but not inactive OGFOD1. The work identifies a human prolyl hydroxylase with a role in translational regulation.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Prolil Hidroxilases/metabolismo , Biossíntese de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Ribossômicas/metabolismo , Análise de Variância , Proteínas de Transporte/genética , Biologia Computacional , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Hidroxilação , Immunoblotting , Imunoprecipitação , Ácidos Cetoglutáricos/metabolismo , Luciferases , Proteínas Nucleares/genética , Prolina/metabolismo , Biossíntese de Proteínas/genética , LevedurasRESUMO
Genome sequences predict the presence of many 2-oxoglutarate (2OG)-dependent oxygenases of unknown biochemical and biological functions in Drosophila. Ribosomal protein hydroxylation is emerging as an important 2OG oxygenase catalyzed pathway, but its biological functions are unclear. We report investigations on the function of Sudestada1 (Sud1), a Drosophila ribosomal oxygenase. As with its human and yeast homologs, OGFOD1 and Tpa1p, respectively, we identified Sud1 to catalyze prolyl-hydroxylation of the small ribosomal subunit protein RPS23. Like OGFOD1, Sud1 catalyzes a single prolyl-hydroxylation of RPS23 in contrast to yeast Tpa1p, where Pro-64 dihydroxylation is observed. RNAi-mediated Sud1 knockdown hinders normal growth in different Drosophila tissues. Growth impairment originates from both reduction of cell size and diminution of the number of cells and correlates with impaired translation efficiency and activation of the unfolded protein response in the endoplasmic reticulum. This is accompanied by phosphorylation of eIF2α and concomitant formation of stress granules, as well as promotion of autophagy and apoptosis. These observations, together with those on enzyme homologs described in the companion articles, reveal conserved biochemical and biological roles for a widely distributed ribosomal oxygenase.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/enzimologia , Homeostase/fisiologia , Prolil Hidroxilases/metabolismo , Biossíntese de Proteínas/fisiologia , Proteínas Ribossômicas/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/genética , Autofagia/genética , Western Blotting , Pesos e Medidas Corporais , Cromatografia Líquida , Primers do DNA/genética , Proteínas de Drosophila/genética , Corpo Adiposo/citologia , Feminino , Técnicas de Silenciamento de Genes , Hidroxilação , Prolil Hidroxilases/genética , Processamento de Proteína Pós-Traducional/fisiologia , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/genética , Espectrometria de Massas em Tandem , Resposta a Proteínas não Dobradas/genéticaRESUMO
The finding that oxygenase-catalyzed protein hydroxylation regulates animal transcription raises questions as to whether the translation machinery and prokaryotic proteins are analogously modified. Escherichia coli ycfD is a growth-regulating 2-oxoglutarate oxygenase catalyzing arginyl hydroxylation of the ribosomal protein Rpl16. Human ycfD homologs, Myc-induced nuclear antigen (MINA53) and NO66, are also linked to growth and catalyze histidyl hydroxylation of Rpl27a and Rpl8, respectively. This work reveals new therapeutic possibilities via oxygenase inhibition and by targeting modified over unmodified ribosomes.