30 results on '"Cockman ME"'
Search Results
2. Ogfod1 deletion increases cardiac beta-alanine levels and protects mice against ischaemia- reperfusion injury.
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Harris M, Sun J, Keeran K, Aponte A, Singh K, Springer D, Gucek M, Pirooznia M, Cockman ME, Murphy E, and Kennedy LM
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- Animals, Mice, beta-Alanine metabolism, Chromatography, Liquid, Infarction, Inosine, Iron, Ischemia, Ketoglutaric Acids, Mice, Knockout, Nucleotides, Oxygenases, Phosphates, Proline, Proteome, Purine Nucleotides, Pyrimidines, Ribosomal Proteins, Tandem Mass Spectrometry, Uric Acid, Carnosine pharmacology, Myocardial Reperfusion Injury genetics, Myocardial Reperfusion Injury prevention & control, Myocardial Reperfusion Injury metabolism, Nuclear Proteins genetics, Carrier Proteins genetics
- Abstract
Aims: Prolyl hydroxylation is a post-translational modification that regulates protein stability, turnover, and activity. The proteins that catalyze prolyl hydroxylation belong to the 2-oxoglutarate- and iron-dependent oxygenase family of proteins. 2-oxoglutarate- and iron-dependent oxygenase domain-containing protein 1 (Ogfod1), which hydroxylates a proline in ribosomal protein s23 is a newly described member of this family. The aims of this study were to investigate roles for Ogfod1 in the heart, and in the heart's response to stress., Methods and Results: We isolated hearts from wild-type (WT) and Ogfod1 knockout (KO) mice and performed quantitative proteomics using tandem mass Tag labelling coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) to identify protein changes. Ingenuity pathway analysis identified 'Urate Biosynthesis/Inosine 5'-phosphate Degradation' and 'Purine Nucleotides Degradation II (Aerobic)' as the most significantly enriched pathways. We performed metabolomics analysis and found that both purine and pyrimidine pathways were altered with the purine nucleotide inosine 5'-monophosphate showing a 3.5-fold enrichment in KO hearts (P = 0.011) and the pyrimidine catabolism product beta-alanine showing a 1.7-fold enrichment in KO hearts (P = 0.014). As changes in these pathways have been shown to contribute to cardioprotection, we subjected isolated perfused hearts to ischaemia and reperfusion (I/R). KO hearts showed a 41.4% decrease in infarct size and a 34% improvement in cardiac function compared to WT hearts. This protection was also evident in an in vivo I/R model. Additionally, our data show that treating isolated perfused WT hearts with carnosine, a metabolite of beta-alanine, improved protection in the context of I/R injury, whereas treating KO hearts with carnosine had no impact on recovery of function or infarct size., Conclusions: Taken together, these data show that Ogfod1 deletion alters the myocardial proteome and metabolome to confer protection against I/R injury., Competing Interests: Conflict of interest: The authors have no conflict of interest to declare., (Published by Oxford University Press on behalf of the European Society of Cardiology 2021. This work is written by a US Government employee and is in the public domain in the US.)
- Published
- 2022
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3. Widespread hydroxylation of unstructured lysine-rich protein domains by JMJD6.
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Cockman ME, Sugimoto Y, Pegg HB, Masson N, Salah E, Tumber A, Flynn HR, Kirkpatrick JM, Schofield CJ, and Ratcliffe PJ
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- Cell Cycle Proteins metabolism, Humans, Hydroxylation, Lysine metabolism, Protein Domains, Transcription Factors metabolism, Intrinsically Disordered Proteins metabolism, Jumonji Domain-Containing Histone Demethylases chemistry, Jumonji Domain-Containing Histone Demethylases metabolism
- Abstract
The Jumonji domain-containing protein JMJD6 is a 2-oxoglutarate-dependent dioxygenase associated with a broad range of biological functions. Cellular studies have implicated the enzyme in chromatin biology, transcription, DNA repair, mRNA splicing, and cotranscriptional processing. Although not all studies agree, JMJD6 has been reported to catalyze both hydroxylation of lysine residues and demethylation of arginine residues. However, despite extensive study and indirect evidence for JMJD6 catalysis in many cellular processes, direct assignment of JMJD6 catalytic substrates has been limited. Examination of a reported site of proline hydroxylation within a lysine-rich region of the tandem bromodomain protein BRD4 led us to conclude that hydroxylation was in fact on lysine and catalyzed by JMJD6. This prompted a wider search for JMJD6-catalyzed protein modifications deploying mass spectrometric methods designed to improve the analysis of such lysine-rich regions. Using lysine derivatization with propionic anhydride to improve the analysis of tryptic peptides and nontryptic proteolysis, we report 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, including 19 sites of hydroxylation on BRD4. Most hydroxylations were within lysine-rich regions that are predicted to be unstructured; in some, multiple modifications were observed on adjacent lysine residues. Almost all of the JMJD6 substrates defined in these studies have been associated with membraneless organelle formation. Given the reported roles of lysine-rich regions in subcellular partitioning by liquid-liquid phase separation, our findings raise the possibility that JMJD6 may play a role in regulating such processes in response to stresses, including hypoxia.
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- 2022
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4. Factor inhibiting HIF can catalyze two asparaginyl hydroxylations in VNVN motifs of ankyrin fold proteins.
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Leissing TM, Hardy AP, Chan H, Wang Y, Tumber A, Chowdhury R, Feng T, Coleman ML, Cockman ME, Kramer HB, Berridge G, Fischer R, Kessler BM, Ratcliffe PJ, Lu X, and Schofield CJ
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- Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Apoptosis Regulatory Proteins, Catalysis, Humans, Hydroxylation, Hypoxia, Ankyrin Repeat, Mixed Function Oxygenases metabolism, Repressor Proteins metabolism
- Abstract
The aspariginyl hydroxylase human factor inhibiting hypoxia-inducible factor (FIH) is an important regulator of the transcriptional activity of hypoxia-inducible factor. FIH also catalyzes the hydroxylation of asparaginyl and other residues in ankyrin repeat domain-containing proteins, including apoptosis stimulating of p53 protein (ASPP) family members. ASPP2 is reported to undergo a single FIH-catalyzed hydroxylation at Asn-986. We report biochemical and crystallographic evidence showing that FIH catalyzes the unprecedented post-translational hydroxylation of both asparaginyl residues in "VNVN" and related motifs of ankyrin repeat domains in ASPPs (i.e., ASPP1, ASPP2, and iASPP) and the related ASB11 and p18-INK4C proteins. Our biochemical results extend the substrate scope of FIH catalysis and may have implications for its biological roles, including in the hypoxic response and ASPP family function., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2022
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5. Corrigendum: Contrasting effects on HIF-1α regulation by disease-causing pVHL mutations correlate with patterns of tumourigenesis in von Hippel-Lindau disease.
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Clifford SC, Cockman ME, Smallwood AC, Mole DR, Woodward ER, Maxwell PH, Ratcliffe PJ, and Maher ER
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- 2021
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6. Lack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates.
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Cockman ME, Lippl K, Tian YM, Pegg HB, Figg WD Jnr, Abboud MI, Heilig R, Fischer R, Myllyharju J, Schofield CJ, and Ratcliffe PJ
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- Hydroxylation, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Hypoxia-Inducible Factor-Proline Dioxygenases genetics, Oxygen metabolism, Recombinant Proteins genetics, Substrate Specificity, Hypoxia-Inducible Factor-Proline Dioxygenases metabolism, Peptides metabolism, Protein Processing, Post-Translational, Recombinant Proteins metabolism
- Abstract
Human and other animal cells deploy three closely related dioxygenases (PHD 1, 2 and 3) to signal oxygen levels by catalysing oxygen regulated prolyl hydroxylation of the transcription factor HIF. The discovery of the HIF prolyl-hydroxylase (PHD) enzymes as oxygen sensors raises a key question as to the existence and nature of non-HIF substrates, potentially transducing other biological responses to hypoxia. Over 20 such substrates are reported. We therefore sought to characterise their reactivity with recombinant PHD enzymes. Unexpectedly, we did not detect prolyl-hydroxylase activity on any reported non-HIF protein or peptide, using conditions supporting robust HIF-α hydroxylation. We cannot exclude PHD-catalysed prolyl hydroxylation occurring under conditions other than those we have examined. However, our findings using recombinant enzymes provide no support for the wide range of non-HIF PHD substrates that have been reported., Competing Interests: MC, KL, YT, HP, WF, MA, RH, RF No competing interests declared, JM equity holder in FibroGen Inc, CS, PR scientific co-founder and equity holder in ReOx, (© 2019, Cockman et al.)
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- 2019
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7. Selective Inhibitors of a Human Prolyl Hydroxylase (OGFOD1) Involved in Ribosomal Decoding.
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Thinnes CC, Lohans CT, Abboud MI, Yeh TL, Tumber A, Nowak RP, Attwood M, Cockman ME, Oppermann U, Loenarz C, and Schofield CJ
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- Carrier Proteins antagonists & inhibitors, Drug Design, Humans, Nuclear Proteins antagonists & inhibitors, Ribosomes metabolism, Structure-Activity Relationship, Substrate Specificity, Prolyl Hydroxylases metabolism, Prolyl-Hydroxylase Inhibitors chemistry, Prolyl-Hydroxylase Inhibitors metabolism, Prolyl-Hydroxylase Inhibitors pharmacology, Ribosomes drug effects
- Abstract
Human prolyl hydroxylases are involved in the modification of transcription factors, procollagen, and ribosomal proteins, and are current medicinal chemistry targets. To date, there are few reports on inhibitors selective for the different types of prolyl hydroxylases. We report a structurally informed template-based strategy for the development of inhibitors selective for the human ribosomal prolyl hydroxylase OGFOD1. These inhibitors did not target the other human oxygenases tested, including the structurally similar hypoxia-inducible transcription factor prolyl hydroxylase, PHD2., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)
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- 2019
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8. Inherent DNA-binding specificities of the HIF-1α and HIF-2α transcription factors in chromatin.
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Smythies JA, Sun M, Masson N, Salama R, Simpson PD, Murray E, Neumann V, Cockman ME, Choudhry H, Ratcliffe PJ, and Mole DR
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- Cell Line, Chromatin genetics, DNA genetics, DNA-Binding Proteins genetics, Epigenomics, Gene Expression Regulation genetics, Humans, Promoter Regions, Genetic, Protein Isoforms genetics, Basic Helix-Loop-Helix Transcription Factors genetics, Cell Hypoxia genetics, Hypoxia-Inducible Factor 1, alpha Subunit genetics, Transcription, Genetic
- Abstract
Hypoxia-inducible factor (HIF) is the major transcriptional regulator of cellular responses to hypoxia. The two principal HIF-α isoforms, HIF-1α and HIF-2α, are progressively stabilized in response to hypoxia and form heterodimers with HIF-1β to activate a broad range of transcriptional responses. Here, we report on the pan-genomic distribution of isoform-specific HIF binding in response to hypoxia of varying severity and duration, and in response to genetic ablation of each HIF-α isoform. Our findings reveal that, despite an identical consensus recognition sequence in DNA, each HIF heterodimer loads progressively at a distinct repertoire of cell-type-specific sites across the genome, with little evidence of redistribution under any of the conditions examined. Marked biases towards promoter-proximal binding of HIF-1 and promoter-distant binding of HIF-2 were observed under all conditions and were consistent in multiple cell type. The findings imply that each HIF isoform has an inherent property that determines its binding distribution across the genome, which might be exploited to therapeutically target the specific transcriptional output of each isoform independently., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)
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- 2019
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9. Publisher Correction: The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases.
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Markolovic S, Zhuang Q, Wilkins SE, Eaton CD, Abboud MI, Katz MJ, McNeil HE, Leśniak RK, Hall C, Struwe WB, Konietzny R, Davis S, Yang M, Ge W, Benesch JLP, Kessler BM, Ratcliffe PJ, Cockman ME, Fischer R, Wappner P, Chowdhury R, Coleman ML, and Schofield CJ
- Abstract
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.
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- 2018
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10. The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases.
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Markolovic S, Zhuang Q, Wilkins SE, Eaton CD, Abboud MI, Katz MJ, McNeil HE, Leśniak RK, Hall C, Struwe WB, Konietzny R, Davis S, Yang M, Ge W, Benesch JLP, Kessler BM, Ratcliffe PJ, Cockman ME, Fischer R, Wappner P, Chowdhury R, Coleman ML, and Schofield CJ
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- GTP Phosphohydrolases chemistry, Humans, Hydroxylation, Jumonji Domain-Containing Histone Demethylases chemistry, Models, Molecular, Biocatalysis, GTP Phosphohydrolases metabolism, Jumonji Domain-Containing Histone Demethylases metabolism
- Abstract
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.
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- 2018
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11. A Ribosomopathy Reveals Decoding Defective Ribosomes Driving Human Dysmorphism.
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Paolini NA, Attwood M, Sondalle SB, Vieira CMDS, van Adrichem AM, di Summa FM, O'Donohue MF, Gleizes PE, Rachuri S, Briggs JW, Fischer R, Ratcliffe PJ, Wlodarski MW, Houtkooper RH, von Lindern M, Kuijpers TW, Dinman JD, Baserga SJ, Cockman ME, and MacInnes AW
- Subjects
- Autism Spectrum Disorder genetics, Carrier Proteins genetics, Cells, Cultured, Child, Child, Preschool, Codon genetics, Developmental Disabilities genetics, Exome, Female, Fibroblasts cytology, Fibroblasts metabolism, Genetic Variation, Hearing Loss genetics, Humans, Intellectual Disability genetics, Male, Microcephaly genetics, Mutation, Mutation, Missense, Nuclear Proteins genetics, Oxidative Stress, Protein Biosynthesis genetics, Sequence Alignment, Sequence Analysis, DNA, Ribosomal Proteins genetics, Ribosomes genetics
- Abstract
Ribosomal protein (RP) gene mutations, mostly associated with inherited or acquired bone marrow failure, are believed to drive disease by slowing the rate of protein synthesis. Here de novo missense mutations in the RPS23 gene, which codes for uS12, are reported in two unrelated individuals with microcephaly, hearing loss, and overlapping dysmorphic features. One individual additionally presents with intellectual disability and autism spectrum disorder. The amino acid substitutions lie in two highly conserved loop regions of uS12 with known roles in maintaining the accuracy of mRNA codon translation. Primary cells revealed one substitution severely impaired OGFOD1-dependent hydroxylation of a neighboring proline residue resulting in 40S ribosomal subunits that were blocked from polysome formation. The other disrupted a predicted pi-pi stacking interaction between two phenylalanine residues leading to a destabilized uS12 that was poorly tolerated in 40S subunit biogenesis. Despite no evidence of a reduction in the rate of mRNA translation, these uS12 variants impaired the accuracy of mRNA translation and rendered cells highly sensitive to oxidative stress. These discoveries describe a ribosomopathy linked to uS12 and reveal mechanistic distinctions between RP gene mutations driving hematopoietic disease and those resulting in developmental disorders., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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12. Hydroxylation of the eukaryotic ribosomal decoding center affects translational accuracy.
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Loenarz C, Sekirnik R, Thalhammer A, Ge W, Spivakovsky E, Mackeen MM, McDonough MA, Cockman ME, Kessler BM, Ratcliffe PJ, Wolf A, and Schofield CJ
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- Chlorophyta, Codon, Terminator genetics, Humans, Hydroxylation, Mass Spectrometry, Oxygenases genetics, Oxygenases metabolism, Ribosomes metabolism, Saccharomyces cerevisiae, Schizosaccharomyces, Species Specificity, Protein Biosynthesis physiology, Protein Processing, Post-Translational physiology, Ribosomal Proteins metabolism, Ribosomes physiology
- Abstract
The mechanisms by which gene expression is regulated by oxygen are of considerable interest from basic science and therapeutic perspectives. Using mass spectrometric analyses of Saccharomyces cerevisiae ribosomes, we found that the amino acid residue in closest proximity to the decoding center, Pro-64 of the 40S subunit ribosomal protein Rps23p (RPS23 Pro-62 in humans) undergoes posttranslational hydroxylation. We identify RPS23 hydroxylases as a highly conserved eukaryotic subfamily of Fe(II) and 2-oxoglutarate dependent oxygenases; their catalytic domain is closely related to transcription factor prolyl trans-4-hydroxylases that act as oxygen sensors in the hypoxic response in animals. The RPS23 hydroxylases in S. cerevisiae (Tpa1p), Schizosaccharomyces pombe and green algae catalyze an unprecedented dihydroxylation modification. This observation contrasts with higher eukaryotes, where RPS23 is monohydroxylated; the human Tpa1p homolog OGFOD1 catalyzes prolyl trans-3-hydroxylation. TPA1 deletion modulates termination efficiency up to ∼10-fold, including of pathophysiologically relevant sequences; we reveal Rps23p hydroxylation as its molecular basis. In contrast to most previously characterized accuracy modulators, including antibiotics and the prion state of the S. cerevisiae translation termination factor eRF3, Rps23p hydroxylation can either increase or decrease translational accuracy in a stop codon context-dependent manner. We identify conditions where Rps23p hydroxylation status determines viability as a consequence of nonsense codon suppression. The results reveal a direct link between oxygenase catalysis and the regulation of gene expression at the translational level. They will also aid in the development of small molecules altering translational accuracy for the treatment of genetic diseases linked to nonsense mutations.
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- 2014
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13. OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation.
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Singleton RS, Liu-Yi P, Formenti F, Ge W, Sekirnik R, Fischer R, Adam J, Pollard PJ, Wolf A, Thalhammer A, Loenarz C, Flashman E, Yamamoto A, Coleman ML, Kessler BM, Wappner P, Schofield CJ, Ratcliffe PJ, and Cockman ME
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- Analysis of Variance, Carrier Proteins genetics, Computational Biology, Fluorescent Antibody Technique, Gene Knockdown Techniques, Humans, Hydroxylation, Immunoblotting, Immunoprecipitation, Ketoglutaric Acids metabolism, Luciferases, Nuclear Proteins genetics, Proline metabolism, Protein Biosynthesis genetics, Yeasts, Carrier Proteins metabolism, Nuclear Proteins metabolism, Prolyl Hydroxylases metabolism, Protein Biosynthesis physiology, Protein Processing, Post-Translational physiology, Ribosomal Proteins metabolism
- Abstract
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.
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- 2014
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14. Sudestada1, a Drosophila ribosomal prolyl-hydroxylase required for mRNA translation, cell homeostasis, and organ growth.
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Katz MJ, Acevedo JM, Loenarz C, Galagovsky D, Liu-Yi P, Pérez-Pepe M, Thalhammer A, Sekirnik R, Ge W, Melani M, Thomas MG, Simonetta S, Boccaccio GL, Schofield CJ, Cockman ME, Ratcliffe PJ, and Wappner P
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- Animals, Animals, Genetically Modified, Apoptosis genetics, Autophagy genetics, Blotting, Western, Body Weights and Measures, Chromatography, Liquid, DNA Primers genetics, Drosophila Proteins genetics, Fat Body cytology, Female, Gene Knockdown Techniques, Hydroxylation, Prolyl Hydroxylases genetics, Protein Processing, Post-Translational physiology, RNA Interference, Real-Time Polymerase Chain Reaction, Ribosomal Proteins genetics, Tandem Mass Spectrometry, Unfolded Protein Response genetics, Drosophila enzymology, Drosophila Proteins metabolism, Homeostasis physiology, Prolyl Hydroxylases metabolism, Protein Biosynthesis physiology, Ribosomal Proteins metabolism
- Abstract
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.
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- 2014
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15. Optimal translational termination requires C4 lysyl hydroxylation of eRF1.
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Feng T, Yamamoto A, Wilkins SE, Sokolova E, Yates LA, Münzel M, Singh P, Hopkinson RJ, Fischer R, Cockman ME, Shelley J, Trudgian DC, Schödel J, McCullagh JS, Ge W, Kessler BM, Gilbert RJ, Frolova LY, Alkalaeva E, Ratcliffe PJ, Schofield CJ, and Coleman ML
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- Amino Acid Sequence, Animals, Catalysis, Cell Line, Tumor, Codon, Terminator, HeLa Cells, Humans, Hydrolysis, Hydroxylation, Jumonji Domain-Containing Histone Demethylases, Models, Molecular, Molecular Sequence Data, Protein Processing, Post-Translational, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Gene Expression Regulation, Histone Demethylases metabolism, Mixed Function Oxygenases chemistry, Peptide Chain Termination, Translational genetics, Peptide Termination Factors chemistry, Protein Biosynthesis
- Abstract
Efficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation., (Copyright © 2014 Elsevier Inc. All rights reserved.)
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- 2014
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16. Oxygenase-catalyzed ribosome hydroxylation occurs in prokaryotes and humans.
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Ge W, Wolf A, Feng T, Ho CH, Sekirnik R, Zayer A, Granatino N, Cockman ME, Loenarz C, Loik ND, Hardy AP, Claridge TDW, Hamed RB, Chowdhury R, Gong L, Robinson CV, Trudgian DC, Jiang M, Mackeen MM, Mccullagh JS, Gordiyenko Y, Thalhammer A, Yamamoto A, Yang M, Liu-Yi P, Zhang Z, Schmidt-Zachmann M, Kessler BM, Ratcliffe PJ, Preston GM, Coleman ML, and Schofield CJ
- Subjects
- Animals, Arginine metabolism, Chromosomal Proteins, Non-Histone metabolism, Dioxygenases, Enzyme Inhibitors pharmacology, Escherichia coli metabolism, Escherichia coli Proteins antagonists & inhibitors, Histidine metabolism, Histone Demethylases, Humans, Hydroxylation, Magnetic Resonance Spectroscopy, Mixed Function Oxygenases antagonists & inhibitors, Nuclear Proteins metabolism, Oxygenases antagonists & inhibitors, Ribosomal Proteins metabolism, Escherichia coli Proteins metabolism, Mixed Function Oxygenases metabolism, Oxygenases metabolism, Prokaryotic Cells metabolism, Ribosomes metabolism
- Abstract
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.
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- 2012
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17. Quantitative mass spectrometry reveals dynamics of factor-inhibiting hypoxia-inducible factor-catalyzed hydroxylation.
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Singleton RS, Trudgian DC, Fischer R, Kessler BM, Ratcliffe PJ, and Cockman ME
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- Animals, Ankyrin Repeat, Cell Hypoxia, HEK293 Cells, Humans, Hydroxylation, Hypoxia-Inducible Factor 1 genetics, Mass Spectrometry, Mice, Mixed Function Oxygenases genetics, Protein Binding, Protein Structure, Tertiary, Repressor Proteins genetics, Hypoxia-Inducible Factor 1 metabolism, Mixed Function Oxygenases metabolism, Oxygen metabolism, Repressor Proteins metabolism, Transcription, Genetic
- Abstract
The asparaginyl hydroxylase, factor-inhibiting hypoxia-inducible factor (HIF), is central to the oxygen-sensing pathway that controls the activity of HIF. Factor-inhibiting HIF (FIH) also catalyzes the hydroxylation of a large set of proteins that share a structural motif termed the ankyrin repeat domain (ARD). In vitro studies have defined kinetic properties of FIH with respect to different substrates and have suggested FIH binds more tightly to certain ARD proteins than HIF and that ARD hydroxylation may have a lower K(m) value for oxygen than HIF hydroxylation. However, regulation of asparaginyl hydroxylation on ARD substrates has not been systematically studied in cells. To address these questions, we employed isotopic labeling and mass spectrometry to monitor the accrual, inhibition, and decay of hydroxylation under defined conditions. Under the conditions examined, hydroxylation was not reversed but increased as the protein aged. The extent of hydroxylation on ARD proteins was increased by addition of ascorbate, whereas iron and 2-oxoglutarate supplementation had no significant effect. Despite preferential binding of FIH to ARD substrates in vitro, when expressed as fusion proteins in cells, hydroxylation was found to be more complete on HIF polypeptides compared with sites within the ARD. Furthermore, comparative studies of hydroxylation in graded hypoxia revealed ARD hydroxylation was suppressed in a site-specific manner and was as sensitive as HIF to hypoxic inhibition. These findings suggest that asparaginyl hydroxylation of HIF-1 and ARD proteins is regulated by oxygen over a similar range, potentially tuning the HIF transcriptional response through competition between the two types of substrate.
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- 2011
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18. Factor-inhibiting hypoxia-inducible factor (FIH) catalyses the post-translational hydroxylation of histidinyl residues within ankyrin repeat domains.
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Yang M, Chowdhury R, Ge W, Hamed RB, McDonough MA, Claridge TD, Kessler BM, Cockman ME, Ratcliffe PJ, and Schofield CJ
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- Amino Acid Sequence, HEK293 Cells, Humans, Hydroxylation, Mass Spectrometry methods, Mixed Function Oxygenases, Models, Molecular, Molecular Sequence Data, Protein Structure, Tertiary, Repressor Proteins genetics, Sequence Alignment, Tankyrases chemistry, Tankyrases genetics, Tankyrases metabolism, Ankyrin Repeat genetics, Histidine metabolism, Protein Processing, Post-Translational, Repressor Proteins metabolism
- Abstract
Factor-inhibiting hypoxia-inducible factor (FIH) is an Fe(II)/2-oxoglutarate-dependent dioxygenase that acts as a negative regulator of the hypoxia-inducible factor (HIF) by catalysing β-hydroxylation of an asparaginyl residue in its C-terminal transcriptional activation domain (CAD). In addition to the hypoxia-inducible factor C-terminal transcriptional activation domain (HIF-CAD), FIH also catalyses asparaginyl hydroxylation of many ankyrin repeat domain-containing proteins, revealing a broad sequence selectivity. However, there are few reports on the selectivity of FIH for the hydroxylation of specific residues. Here, we report that histidinyl residues within the ankyrin repeat domain of tankyrase-2 can be hydroxylated by FIH. NMR and crystallographic analyses show that the histidinyl hydroxylation occurs at the β-position. The results further expand the scope of FIH-catalysed hydroxylations., (© 2011 The Authors Journal compilation © 2011 FEBS.)
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- 2011
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19. FIH-dependent asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.
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Cockman ME, Webb JD, and Ratcliffe PJ
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- Animals, Asparagine metabolism, Humans, Hydroxylation, Mixed Function Oxygenases, Ankyrin Repeat, Proteins chemistry, Proteins metabolism, Repressor Proteins metabolism
- Abstract
Studies on hypoxia-sensitive pathways have identified a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The asparaginyl hydroxylase factor inhibiting HIF (FIH) targets a conserved asparaginyl residue in the C-terminal transactivation domain of HIF-alpha. This modification suppresses HIF transcriptional activity by inhibiting co-activator recruitment. Recent work has demonstrated that FIH targets an alternative class of substrate. Proteins containing a common interaction motif known as the ankyrin repeat domain (ARD) have been shown to be efficiently hydroxylated by FIH. This review aims to summarize what is currently known regarding ARD hydroxylation, including the kinetics and determinants of FIH-mediated ARD hydroxylation, the structural and functional consequences of ARD hydroxylation, and the potential for cross-talk between ARD proteins and HIF signaling.
- Published
- 2009
- Full Text
- View/download PDF
20. Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.
- Author
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Cockman ME, Webb JD, Kramer HB, Kessler BM, and Ratcliffe PJ
- Subjects
- Amino Acid Sequence, Amino Acids, Dicarboxylic pharmacology, Cell Line, Tumor, Endoribonucleases chemistry, Endoribonucleases metabolism, Humans, Hydroxylation drug effects, Immunoblotting, Mass Spectrometry, Mixed Function Oxygenases, Molecular Sequence Data, Protein Binding drug effects, Repressor Proteins chemistry, Reproducibility of Results, Substrate Specificity drug effects, Tankyrases chemistry, Tankyrases metabolism, Ankyrin Repeat, Asparagine metabolism, Proteomics methods, Repressor Proteins metabolism
- Abstract
Post-translational hydroxylation has been considered an unusual modification on intracellular proteins. However, following the recognition that oxygen-sensitive prolyl and asparaginyl hydroxylation are central to the regulation of the transcription factor hypoxia-inducible factor (HIF), interest has centered on the possibility that these enzymes may have other substrates in the proteome. In support of this certain ankyrin repeat domain (ARD)-containing proteins, including members of the IkappaB and Notch families, have been identified as alternative substrates of the HIF asparaginyl hydroxylase factor inhibiting HIF (FIH). Although these findings imply a potentially broad range of substrates for FIH, the precise extent of this range has been difficult to determine because of the difficulty of capturing transient enzyme-substrate interactions. Here we describe the use of pharmacological "substrate trapping" together with stable isotope labeling by amino acids in cell culture (SILAC) technology to stabilize and identify potential FIH-substrate interactions by mass spectrometry. To pursue these potential FIH substrates we used conventional data-directed tandem MS together with alternating low/high collision energy tandem MS to assign and quantitate hydroxylation at target asparaginyl residues. Overall the work has defined 13 new FIH-dependent hydroxylation sites with a degenerate consensus corresponding to that of the ankyrin repeat and a range of ARD-containing proteins as actual and potential substrates for FIH. Several ARD-containing proteins were multiply hydroxylated, and detailed studies of one, Tankyrase-2, revealed eight sites that were differentially sensitive to FIH-catalyzed hydroxylation. These findings indicate that asparaginyl hydroxylation is likely to be widespread among the approximately 300 ARD-containing species in the human proteome.
- Published
- 2009
- Full Text
- View/download PDF
21. The VHL tumor suppressor inhibits expression of the IGF1R and its loss induces IGF1R upregulation in human clear cell renal carcinoma.
- Author
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Yuen JS, Cockman ME, Sullivan M, Protheroe A, Turner GD, Roberts IS, Pugh CW, Werner H, and Macaulay VM
- Subjects
- Humans, Kidney metabolism, RNA, Messenger metabolism, Sp1 Transcription Factor physiology, Transcription, Genetic, Tumor Cells, Cultured, Carcinoma, Renal Cell genetics, Kidney Neoplasms genetics, Receptor, IGF Type 1 metabolism, Up-Regulation, Von Hippel-Lindau Tumor Suppressor Protein physiology
- Abstract
Clear cell renal cell cancer (CC-RCC) is a highly chemoresistant tumor characterized by frequent inactivation of the von Hippel-Lindau (VHL) gene. The prognosis is reportedly worse in patients whose tumors express immunoreactive type I insulin-like growth factor receptor (IGF1R), a key mediator of tumor cell survival. We aimed to investigate how IGF1R expression is regulated, and found that IGF1R protein levels were unaffected by hypoxia, but were higher in CC-RCC cells harboring mutant inactive VHL than in isogenic cells expressing wild-type (WT) VHL. IGF1R mRNA and promoter activities were significantly lower in CC-RCC cells expressing WT VHL, consistent with a transcriptional effect. In Sp1-null Drosophila Schneider cells, IGF1R promoter activity was dependent on exogenous Sp1, and was suppressed by full-length VHL protein (pVHL) but only partially by truncated VHL lacking the Sp1-binding motif. pVHL also reduced the stability of IGF1R mRNA via sequestration of HuR protein. Finally, IGF1R mRNA levels were significantly higher in CC-RCC biopsies than benign kidney, confirming the clinical relevance of these findings. Thus, we have identified a new hypoxia-independent role for VHL in suppressing IGF1R transcription and mRNA stability. VHL inactivation leads to IGF1R upregulation, contributing to renal tumorigenesis and potentially also to chemoresistance.
- Published
- 2007
- Full Text
- View/download PDF
22. Asparaginyl hydroxylation of the Notch ankyrin repeat domain by factor inhibiting hypoxia-inducible factor.
- Author
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Coleman ML, McDonough MA, Hewitson KS, Coles C, Mecinovic J, Edelmann M, Cook KM, Cockman ME, Lancaster DE, Kessler BM, Oldham NJ, Ratcliffe PJ, and Schofield CJ
- Subjects
- Asparagine metabolism, Crystallography, X-Ray, Humans, Hydroxylation, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Mixed Function Oxygenases, Protein Structure, Tertiary, Receptor, Notch1 metabolism, Receptor, Notch2, Receptor, Notch3, Receptors, Notch chemistry, Repressor Proteins chemistry, Transcription Factors chemistry, Ankyrin Repeat, Receptors, Notch metabolism, Repressor Proteins metabolism, Transcription Factors metabolism
- Abstract
The stability and activity of hypoxia-inducible factor (HIF) are regulated by the post-translational hydroxylation of specific prolyl and asparaginyl residues. We show that the HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also catalyzes hydroxylation of highly conserved asparaginyl residues within ankyrin repeat (AR) domains (ARDs) of endogenous Notch receptors. AR hydroxylation decreases the extent of ARD binding to FIH while not affecting signaling through the canonical Notch pathway. ARD proteins were found to efficiently compete with HIF for FIH-dependent hydroxylation. Crystallographic analyses of the hydroxylated Notch ARD (2.35A) and of Notch peptides bound to FIH (2.4-2.6A) reveal the stereochemistry of hydroxylation on the AR and imply that significant conformational changes are required in the ARD fold in order to enable hydroxylation at the FIH active site. We propose that ARD proteins function as natural inhibitors of FIH and that the hydroxylation status of these proteins provides another oxygen-dependent interface that modulates HIF signaling.
- Published
- 2007
- Full Text
- View/download PDF
23. Interaction of hydroxylated collagen IV with the von hippel-lindau tumor suppressor.
- Author
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Grosfeld A, Stolze IP, Cockman ME, Pugh CW, Edelmann M, Kessler B, Bullock AN, Ratcliffe PJ, and Masson N
- Subjects
- Animals, Basic Helix-Loop-Helix Transcription Factors metabolism, Binding Sites, Cell Line, Collagen Type IV genetics, Humans, Hydroxylation, Neoplasms genetics, Neoplasms metabolism, Proteasome Endopeptidase Complex metabolism, Protein Binding, Ubiquitin metabolism, Von Hippel-Lindau Tumor Suppressor Protein genetics, Collagen Type IV metabolism, Extracellular Matrix metabolism, Hydroxyproline metabolism, Von Hippel-Lindau Tumor Suppressor Protein metabolism
- Abstract
The von Hippel-Lindau tumor suppressor (pVHL) targets hydroxylated alpha-subunits of hypoxia-inducible factor (HIF) for ubiquitin-mediated proteasomal destruction through direct interaction with the hydroxyproline binding pocket in its beta-domain. Although disruption of this process may contribute to VHL-associated tumor predisposition by up-regulation of HIF target genes, genetic and biochemical analyses support the existence of additional functions, including a role in the assembly of extracellular matrix. In an attempt to delineate these pathways, we searched for novel pVHL-binding proteins. Here we report a direct, hydroxylation-dependent interaction with alpha-chains of collagen IV. Interaction with pVHL was also observed with fibrillar collagen chains, but not the folded collagen triple helix. The interaction was suppressed by a wide range of tumor-associated mutations, including those that do not disturb the regulation of HIF, supporting a role in HIF-independent tumor suppressor functions.
- Published
- 2007
- Full Text
- View/download PDF
24. Posttranslational hydroxylation of ankyrin repeats in IkappaB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH).
- Author
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Cockman ME, Lancaster DE, Stolze IP, Hewitson KS, McDonough MA, Coleman ML, Coles CH, Yu X, Hay RT, Ley SC, Pugh CW, Oldham NJ, Masson N, Schofield CJ, and Ratcliffe PJ
- Subjects
- Amino Acid Sequence, Decarboxylation, Humans, Hydroxylation, Ketoglutaric Acids metabolism, Mass Spectrometry, Molecular Sequence Data, NF-KappaB Inhibitor alpha, NF-kappa B p50 Subunit analysis, NF-kappa B p50 Subunit chemistry, NF-kappa B p50 Subunit metabolism, Protein Binding, Recombinant Proteins metabolism, Repressor Proteins chemistry, Transcription Factors chemistry, Ankyrin Repeat, Hypoxia-Inducible Factor 1 metabolism, I-kappa B Proteins chemistry, I-kappa B Proteins metabolism, Mixed Function Oxygenases metabolism, Protein Processing, Post-Translational, Repressor Proteins metabolism, Transcription Factors metabolism
- Abstract
Studies on hypoxia-sensitive pathways have revealed a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The recognition of these unprecedented signaling processes has led to a search for other substrates of the HIF hydroxylases. Here we show that the human HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also efficiently hydroxylates specific asparaginyl (Asn)-residues within proteins of the IkappaB family. After the identification of a series of ankyrin repeat domain (ARD)-containing proteins in a screen for proteins interacting with FIH, the ARDs of p105 (NFKB1) and IkappaBalpha were shown to be efficiently hydroxylated by FIH at specific Asn residues in the hairpin loops linking particular ankyrin repeats. The target Asn residue is highly conserved as part of the ankyrin consensus, and peptides derived from a diverse range of ARD-containing proteins supported FIH enzyme activity. These findings demonstrate that this type of protein hydroxylation is not restricted to HIF and strongly suggest that FIH-dependent ARD hydroxylation is a common occurrence, potentially providing an oxygen-sensitive signal to a diverse range of processes.
- Published
- 2006
- Full Text
- View/download PDF
25. Oxygen sensing.
- Author
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Cockman ME and Pugh CW
- Subjects
- Anaerobiosis genetics, Anaerobiosis physiology, Dioxygenases metabolism, Gene Expression physiology, Humans, Hypoxia genetics, Hypoxia-Inducible Factor-Proline Dioxygenases, Protein Binding, Hypoxia metabolism, Oxygen Consumption
- Published
- 2005
26. Gene array of VHL mutation and hypoxia shows novel hypoxia-induced genes and that cyclin D1 is a VHL target gene.
- Author
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Wykoff CC, Sotiriou C, Cockman ME, Ratcliffe PJ, Maxwell P, Liu E, and Harris AL
- Subjects
- Carcinoma, Renal Cell pathology, Cell Hypoxia, Humans, Kidney Neoplasms pathology, Tumor Cells, Cultured, Von Hippel-Lindau Tumor Suppressor Protein, Carcinoma, Renal Cell genetics, Cyclin D1 pharmacology, Gene Expression Profiling, Kidney Neoplasms genetics, Tumor Suppressor Proteins genetics, Ubiquitin-Protein Ligases genetics
- Abstract
Gene expression analysis was performed on a human renal cancer cell line (786-0) with mutated VHL gene and a transfectant with wild-type VHL to analyse genes regulated by VHL and to compare with the gene programme regulated by hypoxia. There was a highly significant concordance of the global gene response to hypoxia and genes suppressed by VHL. Cyclin D1 was the most highly inducible transcript and 14-3-3 epsilon was downregulated. There were some genes regulated by VHL but not hypoxia in the renal cell line, suggesting a VHL role independent of hypoxia. However in nonrenal cell lines they were hypoxia regulated. These included several new pathways regulated by hypoxia, including RNase 6PL, collagen type 1 alpha 1, integrin alpha 5, ferritin light polypeptide, JM4 protein, transgelin and L1 cell adhesion molecule. These were not found in a recent SAGE analysis of the same cell line. Hypoxia induced downregulation of Cyclin D1 in nonrenal cells via an HIF independent pathway. The selective regulation of Cyclin D1 by hypoxia in renal cells may therefore contribute to the tissue selectivity of VHL mutation.
- Published
- 2004
- Full Text
- View/download PDF
27. Selection of mutant CHO cells with constitutive activation of the HIF system and inactivation of the von Hippel-Lindau tumor suppressor.
- Author
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Vaux EC, Wood SM, Cockman ME, Nicholls LG, Yeates KM, Pugh CW, Maxwell PH, and Ratcliffe PJ
- Subjects
- Animals, Base Sequence, CHO Cells, Cell Fusion, Clone Cells, Cricetinae, DNA, Flow Cytometry, Genetic Complementation Test, Hydrolysis, Mice, Molecular Sequence Data, Mutation, Von Hippel-Lindau Tumor Suppressor Protein, DNA-Binding Proteins physiology, Gene Expression Regulation physiology, Genes, Tumor Suppressor, Ligases genetics, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases, von Hippel-Lindau Disease genetics
- Abstract
Hypoxia-inducible factor (HIF) mediates a widespread transcriptional response to hypoxia through binding to cis-acting DNA sequences termed hypoxia response elements (HREs). Activity of the transcriptional complex is suppressed in the presence of oxygen by processes that include the targeting of HIF-alpha subunits for ubiquitin-mediated proteolysis. To provide further insights into these processes we constructed Chinese hamster ovary (CHO) cells bearing stably integrated plasmids that expressed HRE-linked surface antigens and used these cells in genetic screens for mutants that demonstrated constitutive up-regulation of HRE activity. From mutagenized cultures, clones were isolated that demonstrated up-regulation of HRE activity and increased HIF-1alpha protein levels in normoxic culture. Transfection and cell fusion studies suggested that these cells possess recessive defects that affect one or more pathways involved in HIF-alpha proteolysis. Two lines were demonstrated to harbor truncating mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. In these cells, defects in ubiquitylation of exogenous human HIF-1alpha in vitro could be complemented by wild type pVHL, and re-expression of a wild type VHL gene restored a normal pattern of HIF/HRE activity, demonstrating the critical dependence of HIF regulation on pVHL in CHO cells. In contrast, other mutant cells had no demonstrable mutation in the VHL gene, and ubiquitylated exogenous HIF-1alpha normally, suggesting that they contain defects at other points in the oxygen-regulated processing of HIF-alpha subunits.
- Published
- 2001
- Full Text
- View/download PDF
28. Contrasting effects on HIF-1alpha regulation by disease-causing pVHL mutations correlate with patterns of tumourigenesis in von Hippel-Lindau disease.
- Author
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Clifford SC, Cockman ME, Smallwood AC, Mole DR, Woodward ER, Maxwell PH, Ratcliffe PJ, and Maher ER
- Subjects
- Adrenal Gland Neoplasms complications, Adrenal Gland Neoplasms genetics, Alleles, Brain Neoplasms complications, Brain Neoplasms genetics, Carcinoma, Renal Cell complications, Carcinoma, Renal Cell genetics, Cloning, Molecular, DNA-Binding Proteins metabolism, DNA-Binding Proteins physiology, Genotype, Hemangioblastoma complications, Hemangioblastoma genetics, Hypoxia-Inducible Factor 1, Hypoxia-Inducible Factor 1, alpha Subunit, Kidney Neoplasms complications, Kidney Neoplasms genetics, Nuclear Proteins metabolism, Nuclear Proteins physiology, Phenotype, Pheochromocytoma complications, Pheochromocytoma genetics, Protein Binding, Proteins genetics, Proteins metabolism, Transfection, Tumor Cells, Cultured, Ubiquitins metabolism, Von Hippel-Lindau Tumor Suppressor Protein, von Hippel-Lindau Disease complications, DNA-Binding Proteins genetics, Down-Regulation, Ligases, Mutation, Neoplasms genetics, Nuclear Proteins genetics, Proteins physiology, Transcription Factors, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases, von Hippel-Lindau Disease genetics
- Abstract
The von Hippel-Lindau tumour suppressor gene product (pVHL) associates with the elongin B and C and Cul2 proteins to form a ubiquitin-ligase complex (VCBC). To date, the only VCBC substrates identified are the hypoxia-inducible factor alpha subunits (HIF-1alpha and HIF-2alpha). However, pVHL is thought to have multiple functions and the significance of HIF-1alpha and HIF-2alpha regulation for tumour suppressor activity has not been defined. VHL disease is characterized by distinct clinical subtypes. Thus haemangioblastomas (HABs) and renal cell carcinoma (RCC) but not phaeochromocytoma (PHE) occur in type 1 VHL disease. Type 2 subtypes are characterized by PHE susceptibility but differ with respect to additional tumours (type 2A, PHE+HAB but not RCC; type 2B, PHE+ HAB+RCC; type 2C, PHE only). We investigated in detail the effect of 13 naturally occurring VHL mutations (11 missense), representing each phenotypic subclass, on HIF-alpha subunit regulation. Consistent effects on pVHL function were observed for all mutations within each subclass. Mutations associated with the PHE-only phenotype (type 2C) promoted HIF-alpha ubiquitylation in vitro and demonstrated wild-type binding patterns with pVHL interacting proteins, suggesting that loss of other pVHL functions are necessary for PHE susceptibility. Mutations causing HAB susceptibility (types 1, 2A and 2B) demonstrated variable effects on HIF-alpha subunit and elongin binding, but all resulted in defective HIF-alpha regulation and loss of p220 (fibronectin) binding. All RCC-associated mutations caused complete HIF-alpha dysregulation and loss of p220 (fibronectin) binding. Our findings are consistent with impaired ability to degrade HIF-alpha subunit being required for HAB development and RCC susceptibility.
- Published
- 2001
- Full Text
- View/download PDF
29. Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein.
- Author
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Cockman ME, Masson N, Mole DR, Jaakkola P, Chang GW, Clifford SC, Maher ER, Pugh CW, Ratcliffe PJ, and Maxwell PH
- Subjects
- Animals, Basic Helix-Loop-Helix Transcription Factors, COS Cells, Cysteine Endopeptidases metabolism, DNA-Binding Proteins chemistry, Hypoxia-Inducible Factor 1, Hypoxia-Inducible Factor 1, alpha Subunit, Immunoblotting, Multienzyme Complexes metabolism, Mutagenesis, Site-Directed, Mutation, Missense, Nuclear Proteins chemistry, Oxygen metabolism, Plasmids metabolism, Precipitin Tests, Proteasome Endopeptidase Complex, Protein Binding, Protein Biosynthesis, Protein Structure, Tertiary, Proteins chemistry, Proteins genetics, Proteins physiology, Rats, Reticulocytes metabolism, Substrate Specificity, Time Factors, Transfection, Von Hippel-Lindau Tumor Suppressor Protein, DNA-Binding Proteins metabolism, Ligases, Nuclear Proteins metabolism, Proteins metabolism, Trans-Activators, Transcription Factors, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases, Ubiquitins metabolism
- Abstract
The von Hippel-Lindau tumor suppressor protein (pVHL) has emerged as a key factor in cellular responses to oxygen availability, being required for the oxygen-dependent proteolysis of alpha subunits of hypoxia inducible factor-1 (HIF). Mutations in VHL cause a hereditary cancer syndrome associated with dysregulated angiogenesis, and up-regulation of hypoxia inducible genes. Here we investigate the mechanisms underlying these processes and show that extracts from VHL-deficient renal carcinoma cells have a defect in HIF-alpha ubiquitylation activity which is complemented by exogenous pVHL. This defect was specific for HIF-alpha among a range of substrates tested. Furthermore, HIF-alpha subunits were the only pVHL-associated proteasomal substrates identified by comparison of metabolically labeled anti-pVHL immunoprecipitates from proteosomally inhibited cells and normal cells. Analysis of pVHL/HIF-alpha interactions defined short sequences of conserved residues within the internal transactivation domains of HIF-alpha molecules sufficient for recognition by pVHL. In contrast, while full-length pVHL and the p19 variant interact with HIF-alpha, the association was abrogated by further N-terminal and C-terminal truncations. The interaction was also disrupted by tumor-associated mutations in the beta-domain of pVHL and loss of interaction was associated with defective HIF-alpha ubiquitylation and regulation, defining a mechanism by which these mutations generate a constitutively hypoxic pattern of gene expression promoting angiogenesis. The findings indicate that pVHL regulates HIF-alpha proteolysis by acting as the recognition component of a ubiquitin ligase complex, and support a model in which its beta domain interacts with short recognition sequences in HIF-alpha subunits.
- Published
- 2000
- Full Text
- View/download PDF
30. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.
- Author
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Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, Wykoff CC, Pugh CW, Maher ER, and Ratcliffe PJ
- Subjects
- Cell Hypoxia, Cobalt pharmacology, Cysteine Endopeptidases metabolism, Gene Expression Regulation, HeLa Cells, Humans, Hypoxia-Inducible Factor 1, Hypoxia-Inducible Factor 1, alpha Subunit, Iron Chelating Agents pharmacology, Multienzyme Complexes metabolism, Neovascularization, Pathologic genetics, Neovascularization, Pathologic metabolism, Proteasome Endopeptidase Complex, Protein Binding drug effects, Response Elements, Transfection, Tumor Cells, Cultured, Von Hippel-Lindau Tumor Suppressor Protein, von Hippel-Lindau Disease genetics, von Hippel-Lindau Disease metabolism, von Hippel-Lindau Disease pathology, DNA-Binding Proteins metabolism, Genes, Tumor Suppressor, Ligases, Nuclear Proteins metabolism, Oxygen metabolism, Proteins metabolism, Transcription Factors, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases
- Abstract
Hypoxia-inducible factor-1 (HIF-1) has a key role in cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, angiogenesis and apoptosis. The alpha subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. Cobaltous ions or iron chelators mimic hypoxia, indicating that the stimuli may interact through effects on a ferroprotein oxygen sensor. Here we demonstrate a critical role for the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL in HIF-1 regulation. In VHL-defective cells, HIF alpha-subunits are constitutively stabilized and HIF-1 is activated. Re-expression of pVHL restored oxygen-dependent instability. pVHL and HIF alpha-subunits co-immunoprecipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding complex. In cells exposed to iron chelation or cobaltous ions, HIF-1 is dissociated from pVHL. These findings indicate that the interaction between HIF-1 and pVHL is iron dependent, and that it is necessary for the oxygen-dependent degradation of HIF alpha-subunits. Thus, constitutive HIF-1 activation may underlie the angiogenic phenotype of VHL-associated tumours. The pVHL/HIF-1 interaction provides a new focus for understanding cellular oxygen sensing.
- Published
- 1999
- Full Text
- View/download PDF
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