RESUMEN
In the version of this article initially published, authors Sarah E. Wilkins, Charlotte D. Eaton, Martine I. Abboud and Maximiliano J. Katz were incorrectly included in the equal contributions footnote in the affiliations list. Footnote number seven linking to the equal contributions statement should be present only for Suzana Markolovic and Qinqin Zhuang, and the statement should read "These authors contributed equally: Suzana Markolovic, Qinqin Zhuang." The error has been corrected in the HTML and PDF versions of the article.
RESUMEN
Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(II)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.
Asunto(s)
Biocatálisis , GTP Fosfohidrolasas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , GTP Fosfohidrolasas/química , Humanos , Hidroxilación , Histona Demetilasas con Dominio de Jumonji/química , Modelos MolecularesRESUMEN
Adaptation to hypoxia depends on a conserved α/ß heterodimeric transcription factor called Hypoxia Inducible Factor (HIF), whose α-subunit is regulated by oxygen through different concurrent mechanisms. In this study, we have identified the RNA binding protein dMusashi, as a negative regulator of the fly HIF homologue Sima. Genetic interaction assays suggested that dMusashi participates of the HIF pathway, and molecular studies carried out in Drosophila cell cultures showed that dMusashi recognizes a Musashi Binding Element in the 3' UTR of the HIFα transcript, thereby mediating its translational repression in normoxia. In hypoxic conditions dMusashi is downregulated, lifting HIFα repression and contributing to trigger HIF-dependent gene expression. Analysis performed in mouse brains revealed that murine Msi1 protein physically interacts with HIF-1α transcript, suggesting that the regulation of HIF by Msi might be conserved in mammalian systems. Thus, Musashi is a novel regulator of HIF that inhibits responses to hypoxia specifically when oxygen is available.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Animales , Secuencia de Bases , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Sitios Genéticos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Mamíferos , Modelos Biológicos , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/genética , Tráquea/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/enzimología , Homeostasis/fisiología , Prolil Hidroxilasas/metabolismo , Biosíntesis de Proteínas/fisiología , Proteínas Ribosómicas/metabolismo , Animales , Animales Modificados Genéticamente , Apoptosis/genética , Autofagia/genética , Western Blotting , Pesos y Medidas Corporales , Cromatografía Liquida , Cartilla de ADN/genética , Proteínas de Drosophila/genética , Cuerpo Adiposo/citología , Femenino , Técnicas de Silenciamiento del Gen , Hidroxilación , Prolil Hidroxilasas/genética , Procesamiento Proteico-Postraduccional/fisiología , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Ribosómicas/genética , Espectrometría de Masas en Tándem , Respuesta de Proteína Desplegada/genéticaRESUMEN
Interactions between biological pathways and molecular oxygen require robust mechanisms for detecting and responding to changes in cellular oxygen availability, to support oxygen homeostasis. Peptidylglycine α-amidating monooxygenase (PAM) catalyzes a two-step reaction resulting in the C-terminal amidation of peptides, a process important for their stability and biological activity. Here we show that in human, mouse, and insect cells, peptide amidation is exquisitely sensitive to hypoxia. Different amidation events on chromogranin A, and on peptides processed from proopiomelanocortin, manifest similar striking sensitivity to hypoxia in a range of neuroendocrine cells, being progressively inhibited from mild (7% O2) to severe (1% O2) hypoxia. In developing Drosophila melanogaster larvae, FMRF amidation in thoracic ventral (Tv) neurons is strikingly suppressed by hypoxia. Our findings have thus defined a novel monooxygenase-based oxygen sensing mechanism that has the capacity to signal changes in oxygen availability to peptidergic pathways.
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Oxigenasas de Función Mixta/metabolismo , Complejos Multienzimáticos/metabolismo , Células Neuroendocrinas/metabolismo , Oxígeno/metabolismo , Amidas/metabolismo , Secuencia de Aminoácidos , Animales , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Cromogranina A/farmacología , Drosophila melanogaster/enzimología , Humanos , Ratones , Oxigenasas de Función Mixta/química , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Células Neuroendocrinas/efectos de los fármacosRESUMEN
Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. Here we report that a novel Drosophila immunophilin, which we have named Zonda, is critically required for starvation-induced autophagy. We show that Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions and, soon after starvation, nucleates in endoplasmic reticulum-associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13, and Atg17 but does not require Vps34, Vps15, Atg6, or Atg14. Zonda interacts physically with Atg1 through its kinase domain, as well as with Atg6 and Vps34. We propose that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation.
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Autofagia/fisiología , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Inmunofilinas/metabolismo , Animales , Proteínas Relacionadas con la Autofagia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Inmunofilinas/genética , Fagosomas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transducción de SeñalRESUMEN
In this Extra View we comment on our recent work on Sudestada1 (Sud1), a Drosophila 2-oxoglutarate (2OG)-dependent dioxygenase that belongs to the Ribosomal Oxygenase (ROX) subfamily. Sud1 is required for normal growth in Drosophila, and is conserved in yeast and mammals. We reported that Sud1 hydroxylates the ribosomal protein S23 (RPS23), and that its loss of function restricts growth and provokes activation of the unfolded protein response, apoptosis and autophagy. In this Extra View we speculate on the role that RPS23 hydroxylation might play in stop codon recognition and on the possible link between Sud1 loss-of-function and activation of the Unfolded Protein Response, Stress Granules formation and growth impairment.
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Procesos de Crecimiento Celular , Proteínas de Drosophila/metabolismo , Drosophila/enzimología , Regulación de la Expresión Génica , Prolil Hidroxilasas/metabolismo , Proteínas Ribosómicas/metabolismo , Animales , HidroxilaciónRESUMEN
Mammalian insulin-degrading enzyme (IDE) cleaves insulin, among other peptidic substrates, but its function in insulin signaling is elusive. We use the Drosophila system to define the function of IDE in the regulation of growth and metabolism. We find that either loss or gain of function of Drosophila IDE (dIDE) can restrict growth in a cell-autonomous manner by affecting both cell size and cell number. dIDE can modulate Drosophila insulin-like peptide 2 levels, thereby restricting activation of the phosphatidylinositol-3-phosphate kinase pathway and promoting activation of Drosophila forkhead box, subgroup O transcription factor. Larvae reared in high sucrose exhibit delayed developmental timing due to insulin resistance. We find that dIDE loss of function exacerbates this phenotype and that mutants display increased levels of circulating sugar, along with augmented expression of a lipid biosynthesis marker. We propose that dIDE is a modulator of insulin signaling and that its loss of function favors insulin resistance, a hallmark of diabetes mellitus type II.