Publisher Correction: The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases.

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.

The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases.

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.

Zonda is a novel early component of the autophagy pathway in Drosophila.

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.

Musashi mediates translational repression of the Drosophila hypoxia inducible factor.

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.

miR-190 Enhances HIF-Dependent Responses to Hypoxia in Drosophila by Inhibiting the Prolyl-4-hydroxylase Fatiga.

Cellular and systemic responses to low oxygen levels are principally mediated by Hypoxia Inducible Factors (HIFs), a family of evolutionary conserved heterodimeric transcription factors, whose alpha- and beta-subunits belong to the bHLH-PAS family. In normoxia, HIFα is hydroxylated by specific prolyl-4-hydroxylases, targeting it for proteasomal degradation, while in hypoxia the activity of these hydroxylases decreases due to low oxygen availability, leading to HIFα accumulation and expression of HIF target genes. To identify microRNAs required for maximal HIF activity, we conducted an overexpression screen in Drosophila melanogaster, evaluating the induction of a HIF transcriptional reporter. miR-190 overexpression enhanced HIF-dependent biological responses, including terminal sprouting of the tracheal system, while in miR-190 loss of function embryos the hypoxic response was impaired. In hypoxic conditions, miR-190 expression was upregulated and required for induction of HIF target genes by directly inhibiting the HIF prolyl-4-hydroxylase Fatiga. Thus, miR-190 is a novel regulator of the hypoxia response that represses the oxygen sensor Fatiga, leading to HIFα stabilization and enhancement of hypoxic responses.

Phosphorylated Ribosomal Protein S6 Is Required for Akt-Driven Hyperplasia and Malignant Transformation, but Not for Hypertrophy, Aneuploidy and Hyperfunction of Pancreatic ß-Cells.

Constitutive expression of active Akt (Akttg) drives hyperplasia and hypertrophy of pancreatic ß-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a later stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6), an Akt effector, we generated mice that express constitutive Akt in ß-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6P-/-). rpS6 phosphorylation deficiency failed to block Akttg-induced hypertrophy and aneuploidy in ß-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akttg-driven hyperplasia and tumor formation in ß-cells. In vitro experiments with Akttg and rpS6P-/-;Akttg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akttg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation.

Striking Oxygen Sensitivity of the Peptidylglycine α-Amidating Monooxygenase (PAM) in Neuroendocrine Cells.

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.

Growing with the wind. Ribosomal protein hydroxylation and cell growth.

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.

Sudestada1, a Drosophila ribosomal prolyl-hydroxylase required for mRNA translation, cell homeostasis, and organ growth.

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.

The Drosophila insulin-degrading enzyme restricts growth by modulating the PI3K pathway in a cell-autonomous manner.

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.

Mice deficient in ribosomal protein S6 phosphorylation suffer from muscle weakness that reflects a growth defect and energy deficit.

BACKGROUND: Mice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all five phosphorylatable serine residues by alanines (rpS6(P-/-)), are viable and fertile. However, phenotypic characterization of these mice and embryo fibroblasts derived from them, has established the role of these modifications in the regulation of the size of several cell types, as well as pancreatic beta-cell function and glucose homeostasis. A relatively passive behavior of these mice has raised the possibility that they suffer from muscle weakness, which has, indeed, been confirmed by a variety of physical performance tests. METHODOLOGY/PRINCIPAL FINDINGS: A large variety of experimental methodologies, including morphometric measurements of histological preparations, high throughput proteomic analysis, positron emission tomography (PET) and numerous biochemical assays, were used in an attempt to establish the mechanism underlying the relative weakness of rpS6(P-/-) muscles. Collectively, these experiments have demonstrated that the physical inferiority appears to result from two defects: a) a decrease in total muscle mass that reflects impaired growth, rather than aberrant differentiation of myofibers, as well as a diminished abundance of contractile proteins; and b) a reduced content of ATP and phosphocreatine, two readily available energy sources. The abundance of three mitochondrial proteins has been shown to diminish in the knockin mouse. However, the apparent energy deficiency in this genotype does not result from a lower mitochondrial mass or compromised activity of enzymes of the oxidative phosphorylation, nor does it reflect a decline in insulin-dependent glucose uptake, or diminution in storage of glycogen or triacylglycerol (TG) in the muscle. CONCLUSIONS/SIGNIFICANCE: This study establishes rpS6 phosphorylation as a determinant of muscle strength through its role in regulation of myofiber growth and energy content. Interestingly, a similar role has been assigned for ribosomal protein S6 kinase 1, even though it regulates myoblast growth in an rpS6 phosphorylation-independent fashion.