MassIVEapplication/xmlftp://massive.ucsd.edu/v01/MSV000080695/primaryOK2000000Cormac Taylarhttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=a374f7b3732c4ae7bfd32f94eaa34d52cormac.taylor@ucd.ieMassIVE52MOD:00425 - "A protein modification that effectively replaces one hydrogen atom with a hydroxyl group."ProteomicsQ ExactiveHomo Sapiens (ncbitaxon:9606)Principal Investigator. SBI (System Biology Ireland), Conway Institute, UCDThe asparagine hydroxylase, factor inhibiting HIF (FIH), confers oxygen-dependence upon the hypoxia-inducible factor (HIF), a master regulator of the cellular adaptive response to hypoxia. Studies investigating whether asparagine hydroxylation is a general regulatory oxygen-dependent modification have identified multiple non-HIF targets for FIH. However, the functional consequences of this outside of the HIF pathway remain unclear. Here, we demonstrate that the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) is a substrate for hydroxylation by FIH on N22. Mutation of N22 leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. Furthermore, in cultured cells, overexpression of N22A mutant OTUB1 impairs cellular metabolic processes when compared to wild type. Based on these data, we hypothesize that OTUB1 is a target for functional hydroxylation by FIH. Additionally, we propose that our results provide new insight into the regulation of cellular energy metabolism during hypoxic stress and the potential for targeting hydroxylases for therapeutic benefit.FIH Regulates Cellular Metabolism through Hydroxylation of the Deubiquitinase OTUB1.Scholz Carsten C CC, Rodriguez Javier J, Pickel Christina C, Burr Stephen S, Fabrizio Jacqueline-Alba JA, Nolan Karen A KA, Spielmann Patrick P, Cavadas Miguel A S MA, Crifo Bianca B, Halligan Doug N DN, Nathan James A JA, Peet Daniel J DJ, Wenger Roland H RH, Von Kriegsheim Alex A, Cummins Eoin P EP, Taylor Cormac T CTprojections, thiol-dependent ubiquitinyl hydrolase activity, Metabolisms, ovary tumor, A4, Bioenergetics, NSHPT, protein, thiol-dependent ubiquitin-specific protease activity, binding site, Oxygen Deficiencies, Mutations, protein polypeptide chains, oxigeno, 2, Expenditure, E948, protein aggregate, response to intermittent hypoxia, DL-Asparagine, OVARY NEOPL, ubiquitin hydrolase activity, asparagina, High Mobility Protein 20, asparagine, N, L-Asparagine, O, Oxygen-16, Ubiquitin-related 1, EIG8, proteins, ubiquitin C-terminal hydrolase, ASN, Asn, NEOPL OVARIAN, Oxygen, APF-1, familial isolated, [OO], papilla, intermediary metabolism, response to sustained hypoxia, thiol-dependent deubiquitinase, ubiquitin C-terminal hydrolase activity, Deubiquitinating, ubiquitinyl hydrolase activity, Cancer of Ovary, anatomical protrusion, familial, lamina, flanges, CEP52, Sauerstoff, shelf, 2310046M24Rik, regulator, Hydroxylations, region, Ubiq, autosomal recessive, Ovary Neoplasm, ovarian tumour, Ubiquitin, Disauerstoff, dioxygen, positional polypeptide feature, FIH1, Deubiquitinases, shelves, Human Ubiquitin, Hypoxemia, Ovary Neoplasms, A830014H24Rik, 2-amino-3-carbamoylpropanoic acid, projection, response to lowered oxygen tension, ridge, ovary neoplasm, Energy Metabolisms, DMDA, spine, Aldehyde, OTB1, HYPOC1, Anoxemia, Dioxygen, OTU1, region or site annotation, lamellae, ubiquitinyl hydrolase 1 activity, Neoplasms, N22, Deficiencies, Hasp, E-948, Gene, ovary tumour, tumor of ovary, neoplasm of ovary, ATP Dependent Proteolysis Factor 1, 8O, Deubiquitinating Enzyme, Ubiquitin carboxyl extension protein 80, protein-containing complex, autosomal dominant, GPRC2A, process of organ, deubiquitylase, TYPE, Human, protrusion, Oxygen Deficiency, DAGA4, lamella, OVARIAN NEOPL, ovarian tumors, hydroxylation, deubiquitinase activity, dioxygene, polypeptide chain, Ovarian, Metabolism, response to hypoxic stress, Gene Products, HMG-20, ubiquitin-like protein modifier, MAM, SCG3, HHC, ubiquitin-specific protease activity, positional, Anoxemia., ligand, Energy, tumour of ovary, ridges, dependence, Expenditures, Asparagin, UBP, 40S ribosomal protein S27a, Enzyme, ubiquitin, HHC1, site, protein tagging activity, laminae, tumour of the ovary, molecular oxygen, data, Oxygen 16, AI850305, ovarian neoplasm, ovary neoplasm (disease), protein complex, tumor of the ovary, anatomical process, Proteins, Ovary, hypoparathyroidism familial isolated, Deubiquitinase, oxygen, LGMD2C, Enzymes, Anoxia, Ubiquitin A-52 residue ribosomal protein fusion product 1, native protein, natural protein, hypoparathyroidism, Ubiquitin-related 2, Protein, Neoplasm, CAR, oxygene, ATP-Dependent Proteolysis Factor 1, OXYGEN MOLECULE, flange, organ process, familial isolated hypoparathyroidism, Energy Expenditure, covalent modifier, O2, FHH, DMDA1, PCAR1, INSDC_feature:misc_binding, Energy Expenditures, 4-diamino-4-oxobutanoic acid, Ovarian Neoplasms, ovarian tumor, Bioenergetic, UCH2, Protein Gene Products, Gene Proteins, processes, process, FIH, deubiquitinating enzyme, Deficiency, binding_or_interaction_site, 60S ribosomal protein L40, ovarian tumours, protein tag, familial isolated 1, neoplasm of the ovary, deubiquitinase, SCARMD2, processus, Ubiquitin-related, regulation, cellular metabolism, E 948projections, Oxidases, thiol-dependent ubiquitinyl hydrolase activity, Metabolisms, ovary tumor, A4, Bioenergetics, NSHPT, protein, thiol-dependent ubiquitin-specific protease activity, Oxygen Deficiencies, Mutations, protein polypeptide chains, Monooxygenase, oxigeno, 2, Expenditure, E948, protein aggregate, response to intermittent hypoxia, Mixed Function., DL-Asparagine, OVARY NEOPL, ubiquitin hydrolase activity, asparagina, High Mobility Protein 20, asparagine, Function Oxygenase, N, L-Asparagine, O, Oxygen-16, Ubiquitin-related 1, EIG8, proteins, ubiquitin C-terminal hydrolase, ASN, Asn, NEOPL OVARIAN, Oxygen, APF-1, familial isolated, [OO], papilla, intermediary metabolism, response to sustained hypoxia, Function Oxidase, thiol-dependent deubiquitinase, ubiquitin C-terminal hydrolase activity, Deubiquitinating, ubiquitinyl hydrolase activity, Cancer of Ovary, anatomical protrusion, familial, lamina, flanges, Cultured, CEP52, results, Hydroxylase, Sauerstoff, shelf, 2310046M24Rik, regulator, Hydroxylations, Ubiq, autosomal recessive, Ovary Neoplasm, ovarian tumour, Ubiquitin, Disauerstoff, dioxygen, Mixed Function, Oxidase, FIH1, Deubiquitinases, shelves, Human Ubiquitin, Hypoxemia, Ovary Neoplasms, A830014H24Rik, 2-amino-3-carbamoylpropanoic acid, projection, response to lowered oxygen tension, ridge, ovary neoplasm, Energy Metabolisms, DMDA, spine, Aldehyde, Cultured Cells, OTB1, HYPOC1, Anoxemia, Dioxygen, OTU1, lamellae, ubiquitinyl hydrolase 1 activity, Neoplasms, N22, Deficiencies, Hasp, E-948, Gene, ovary tumour, tumor of ovary, neoplasm of ovary, ATP Dependent Proteolysis Factor 1, 8O, Deubiquitinating Enzyme, Ubiquitin carboxyl extension protein 80, protein-containing complex, autosomal dominant, GPRC2A, process of organ, deubiquitylase, TYPE, Human, protrusion, Oxygen Deficiency, DAGA4, lamella, OVARIAN NEOPL, ovarian tumors, hydroxylation, deubiquitinase activity, dioxygene, polypeptide chain, Ovarian, Metabolism, response to hypoxic stress, Gene Products, HMG-20, ubiquitin-like protein modifier, MAM, SCG3, HHC, Oxygenases, ubiquitin-specific protease activity, ligand, Mixed Function Oxidase, Energy, tumour of ovary, ridges, dependence, Expenditures, Asparagin, UBP, 40S ribosomal protein S27a, Enzyme, Mixed Function Oxidases, ubiquitin, HHC1, protein tagging activity, laminae, tumour of the ovary, molecular oxygen, data, Oxygen 16, AI850305, ovarian neoplasm, ovary neoplasm (disease), protein complex, tumor of the ovary, anatomical process, Proteins, Ovary, hypoparathyroidism familial isolated, Deubiquitinase, oxygen, Cell, LGMD2C, Enzymes, Anoxia, Ubiquitin A-52 residue ribosomal protein fusion product 1, native protein, natural protein, hypoparathyroidism, Ubiquitin-related 2, Protein, Neoplasm, Oxygenase, CAR, Mixed, oxygene, ATP-Dependent Proteolysis Factor 1, OXYGEN MOLECULE, flange, organ process, familial isolated hypoparathyroidism, Energy Expenditure, covalent modifier, O2, FHH, DMDA1, PCAR1, Energy Expenditures, Mixed Function Oxygenase, Monooxygenases, 4-diamino-4-oxobutanoic acid, Ovarian Neoplasms, ovarian tumor, Bioenergetic, UCH2, Protein Gene Products, Gene Proteins, processes, process, FIH, deubiquitinating enzyme, Deficiency, 60S ribosomal protein L40, ovarian tumours, protein tag, familial isolated 1, neoplasm of the ovary, Cultured Cell, deubiquitinase, SCARMD2, processus, Ubiquitin-related, regulation, cellular metabolism, E 948, HydroxylasesDeubiquitinating, ubiquitinyl hydrolase activity, OTU1, thiol-dependent ubiquitinyl hydrolase activity, ubiquitinyl hydrolase 1 activity, familial, hypoparathyroidism familial isolated, Deubiquitinase, NSHPT, Deubiquitinating Enzyme, autosomal dominant, GPRC2A, deubiquitylase, thiol-dependent ubiquitin-specific protease activity, Enzymes, hydroxylation, deubiquitinase activity, hypoparathyroidism, 2310046M24Rik, CAR, Hydroxylations, autosomal recessive, familial isolated hypoparathyroidism, AI850305., HHC, ubiquitin-specific protease activity, ubiquitin hydrolase activity, FHH, PCAR1, FIH1, Deubiquitinases, EIG8, A830014H24Rik, ubiquitin C-terminal hydrolase, UCH2, UBP, familial isolated, FIH, deubiquitinating enzyme, Enzyme, familial isolated 1, intermediary metabolism, deubiquitinase, HHC1, OTB1, HYPOC1, cellular metabolism, thiol-dependent deubiquitinase, ubiquitin C-terminal hydrolase activityDeubiquitinating, ubiquitinyl hydrolase activity, OTU1, thiol-dependent ubiquitinyl hydrolase activity, ubiquitinyl hydrolase 1 activity, familial, hypoparathyroidism familial isolated, Deubiquitinase, NSHPT, Deubiquitinating Enzyme, autosomal dominant, GPRC2A, deubiquitylase, thiol-dependent ubiquitin-specific protease activity, Enzymes, hydroxylation, deubiquitinase activity, hypoparathyroidism, 2310046M24Rik, CAR, Hydroxylations, autosomal recessive, familial isolated hypoparathyroidism, AI850305., HHC, ubiquitin-specific protease activity, ubiquitin hydrolase activity, FHH, PCAR1, FIH1, Deubiquitinases, EIG8, A830014H24Rik, ubiquitin C-terminal hydrolase, UCH2, UBP, familial isolated, FIH, deubiquitinating enzyme, Enzyme, familial isolated 1, intermediary metabolism, deubiquitinase, HHC1, OTB1, HYPOC1, cellular metabolism, thiol-dependent deubiquitinase, ubiquitin C-terminal hydrolase activity0PXD002103trueFIH regulates cellular metabolism through hydroxylation of the deubiquitinase OTUB1.The asparagine hydroxylase, factor inhibiting HIF (FIH) confers oxygen-dependence upon the hypoxia-inducible factor (HIF), a master regulator of the cellular adaptive response to hypoxia. Studies investigating whether asparagine hydroxylation is a general regulatory oxygen-dependent modification have identified multiple non-HIF targets for FIH. However the functional consequences of this outside of the HIF pathway remain unclear. Here, we demonstrate that the deubiquitinase ovarian tumor domain containing, ubiquitin aldehyde binding protein 1 (OTUB1) is a substrate for hydroxylation by endogenous FIH on N22. Mutation of N22 leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. Furthermore, mutant OTUB1 (lacking the hydroxylation site) impairs cellular metabolic processes when compared to wild type. Based on these data, we hypothesize that OTUB1 is a target for functional hydroxylation by FIH, and propose that this provides new insight into the regulation of cellular energy metabolism during hypoxia.Tue Mar 28 20:40:00 BST 2017MSV00008069526752685