Maximiliano J. Katz

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

Significance Emerging evidence indicates that posttranslational hydroxylation of intracellularly localized proteins is more prevalent than once thought. We identify Drosophila melanogaster sudestada1 (sud1) as a gene that is needed for normal growth in the fly and show that sud1 encodes a prolyl-hydroxylase that catalyzes posttranslational hydroxylation of a conserved residue in the small ribosomal subunit protein RPS23. Knockdown of Sud1 results in growth impairment and reduced RPS23 hydroxylation, which is associated with activation of the unfolded protein response, induction of apoptosis, and increased autophagy. Together with findings in humans and yeast reported in the companion articles, the work reveals a new type of posttranslational ribosome modification that is highly conserved in eukaryotes. 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.

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

Background: Peptidylglycine α-Amidating Monooxygenase (PAM) is solely responsible for catalysis of amidation, a biologically important post-translational modification. Results: Modification-specific antibodies reveal that peptide substrate amidation is strikingly sensitive to the exposure of cells to moderate hypoxia. Conclusion: PAM-dependent amidation has the potential to signal oxygen levels in the same range as the hypoxia-inducible factor (HIF) system. Significance: Physiological effects of hypoxia may be PAM-dependent. 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.

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

The Drosophila insulin-degrading enzyme (dIDE) is a negative modulator of the PI3K pathway that restrains tissue growth in an autonomous manner. Larvae reared in high sucrose exhibit reduced growth and delayed developmental timing due to insulin resistance; dIDE loss of function exacerbates these phenotypes.

Hydroxylation and translational adaptation to stress: some answers lie beyond the STOP codon

Regulation of protein synthesis contributes to maintenance of homeostasis and adaptation to environmental changes. mRNA translation is controlled at various levels including initiation, elongation and termination, through post-transcriptional/translational modifications of components of the protein synthesis machinery. Recently, protein and RNA hydroxylation have emerged as important enzymatic modifications of tRNAs, elongation and termination factors, as well as ribosomal proteins. These modifications enable a correct STOP codon recognition, ensuring translational fidelity. Recent studies are starting to show that STOP codon read-through is related to the ability of the cell to cope with different types of stress, such as oxidative and chemical insults, while correlations between defects in hydroxylation of protein synthesis components and STOP codon read-through are beginning to emerge. In this review we will discuss our current knowledge of protein synthesis regulation through hydroxylation of components of the translation machinery, with special focus on STOP codon recognition. We speculate on the possibility that programmed STOP codon read-through, modulated by hydroxylation of components of the protein synthesis machinery, is part of a concerted cellular response to stress.

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.

Musashi mediates translational repression of the Drosophila hypoxia inducible factor

Adaptation to hypoxia depends on a conserved α/β 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.

The Jumonji-C oxygenase JMJD7 catalyzes (3 S )-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.Structural, biochemical and cellular studies reveal JMJD7 to be a Jumonji-C oxygenase that catalyzes (3S)-lysyl hydroxylation of the translation factor family of GTPases, DRG1 and DRG2.

Publisher Correction: The Jumonji-C oxygenase JMJD7 catalyzes (3 S )-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.

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

Zonda, a novel Drosophila immunophilin, is an early component of the autophagy machinery necessary for Vps34-mediated phosphatidylinositol 3-phosphate deposition prior to omegasome formation. We propose that Zonda is critically required for the initiation of autophagosome biogenesis.

PS5.103Role of the Notch pathway in differentiation of Drosophila blood cells

diverse genetic backgrounds. We have developed culture conditions that support long-term self-renewal of multipotent pancreatic progenitors, which are developmentally much closer to the specialised cells of the adult pancreas. These cultured Pancreatic Progenitor (cPP) cells express key pancreatic transcription factors, including PDX1 and SOX9, and exhibit transcriptomes closely related to their in vivo counterparts. Exposure to differentiation cues directs cells towards the pancreatic endocrine, acinar and ductal lineages, indicating multi-lineage potency. Furthermore, cPP cells give rise to Insulin+ beta-like cells in vitro and in vivo, suggesting they offer a convenient alternative to pluripotent cells as a source of pancreatic cell types for modelling pancreatic development and diabetes.