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• W2038139095 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 IκB 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 ∼300 ARD-containing species in the human proteome. 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 IκB 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 ∼300 ARD-containing species in the human proteome. Post-translational hydroxylation is well established as a modification of collagen and other extracellular proteins but has been considered to be rare in intracellular proteins (1Walsh C.T. Post Translational Modification of Proteins: Expanding Nature's Inventory. Roberts and Co. Publishers, Greenwood Village, CO2006: 331-347Google Scholar). Recently, however, hydroxylations of specific prolyl and asparaginyl residues have been defined as oxygen-regulated signals that determine the stability and activity of the HIF 1The abbreviations used are: HIF, hypoxia-inducible factor; 2OG, 2-oxoglutarate; AP, affinity purification; ARD, ankyrin repeat domain; DMOG, dimethyloxalylglycine; FIH, factor inhibiting HIF; MSE, low/high collision energy MS; UPLC, ultraperformance liquid chromatography; SILAC, stable isotope labeling by amino acids in cell culture; SPA, Sequential Peptide Affinity tag; EGFP, enhanced green fluorescent protein; EV, empty vector; tet, tetracycline; PLGS, ProteinLynx Global Server; IP, immunoprecipitation; DDA, data-directed analysis; siRNA, small interfering RNA; HEK, human embryonic kidney; AR, ankyrin repeat; KH, K homology; SAM, sterile alpha motif. 1The abbreviations used are: HIF, hypoxia-inducible factor; 2OG, 2-oxoglutarate; AP, affinity purification; ARD, ankyrin repeat domain; DMOG, dimethyloxalylglycine; FIH, factor inhibiting HIF; MSE, low/high collision energy MS; UPLC, ultraperformance liquid chromatography; SILAC, stable isotope labeling by amino acids in cell culture; SPA, Sequential Peptide Affinity tag; EGFP, enhanced green fluorescent protein; EV, empty vector; tet, tetracycline; PLGS, ProteinLynx Global Server; IP, immunoprecipitation; DDA, data-directed analysis; siRNA, small interfering RNA; HEK, human embryonic kidney; AR, ankyrin repeat; KH, K homology; SAM, sterile alpha motif. transcriptional complex. Both reactions are catalyzed by members of the 2-oxoglutarate (2OG)-dependent di-oxygenase superfamily: HIF prolyl hydroxylation by PHD (prolyl hydroxylase domain) 1–3 and HIF asparaginyl hydroxylation by FIH (for a review, see Ref. 2Schofield C.J. Ratcliffe P.J. Oxygen sensing by HIF hydroxylases.Nat. Rev. Mol. Cell Biol. 2004; 5: 343-354Crossref PubMed Scopus (1558) Google Scholar). Following the identification of the HIF hydroxylases, searches for alternative (non-HIF) substrates of these enzymes have identified certain IκB and Notch family members and ASB4 (ankyrin repeat and SOCS box protein 4) as substrates of FIH (3Cockman M.E. Lancaster D.E. Stolze I.P. Hewitson K.S. McDonough M.A. Coleman M.L. Coles C.H. Yu X. Hay R.T. Ley S.C. Pugh C.W. Oldham N.J. Masson N. Schofield C.J. Ratcliffe P.J. Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH).Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 14767-14772Crossref PubMed Scopus (220) Google Scholar, 4Coleman M.L. McDonough M.A. Hewitson K.S. Coles C. Mecinovic J. Edelmann M. Cook K.M. Cockman M.E. Lancaster D.E. Kessler B.M. Oldham N.J. Ratcliffe P.J. Schofield C.J. Asparaginyl hydroxylation of the Notch ankyrin repeat domain by factor inhibiting hypoxia-inducible factor.J. Biol. Chem. 2007; 282: 24027-24038Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 5Ferguson III, J.E. Wu Y. Smith K. Charles P. Powers K. Wang H. Patterson C. ASB4 is a hydroxylation substrate of FIH and promotes vascular differentiation via an oxygen-dependent mechanism.Mol. Cell. Biol. 2007; 27: 6407-6419Crossref PubMed Scopus (78) Google Scholar, 6Zheng X. Linke S. Dias J.M. Gradin K. Wallis T.P. Hamilton B.R. Gustafsson M. Ruas J.L. Wilkins S. Bilton R.L. Brismar K. Whitelaw M.L. Pereira T. Gorman J.J. Ericson J. Peet D.J. Lendahl U. Poellinger L. Interaction with factor inhibiting HIF-1 defines an additional mode of cross-coupling between the Notch and hypoxia signaling pathways.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 3368-3373Crossref PubMed Scopus (194) Google Scholar). These intracellular proteins all contain ARDs, and in each case the target asparagine residues lie within the ARD. The ARD is one of the most common amino acid motifs in nature; it is present in over 300 proteins in the human genome (SMART (simple modular architecture research tool) database (7Schultz J. Milpetz F. Bork P. Ponting C.P. SMART, a simple modular architecture research tool: identification of signaling domains.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (2972) Google Scholar)) and conserved in all kingdoms of life (for a review, see Ref. 8Li J. Mahajan A. Tsai M.D. Ankyrin repeat: a unique motif mediating protein-protein interactions.Biochemistry. 2006; 45: 15168-15178Crossref PubMed Scopus (409) Google Scholar). ARDs are composed of a variable number of 33-residue repeats that individually fold into paired antiparallel α-helices linked by a β-hairpin type turn. The hydroxylated asparagine residue is positioned within the hairpin loop that links individual repeats. These findings suggest that asparaginyl hydroxylation might be much more prevalent in intracellular proteins than has been appreciated previously, particularly among ARD-containing proteins. However, this has not been noted in proteomics surveys to date. Furthermore the protein association methods used so far to identify FIH-associated proteins, including yeast two-hybrid screens and affinity purification (AP)-MS technology, have only identified a limited number of ARD-containing proteins as molecules interacting with FIH (3Cockman M.E. Lancaster D.E. Stolze I.P. Hewitson K.S. McDonough M.A. Coleman M.L. Coles C.H. Yu X. Hay R.T. Ley S.C. Pugh C.W. Oldham N.J. Masson N. Schofield C.J. Ratcliffe P.J. Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH).Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 14767-14772Crossref PubMed Scopus (220) Google Scholar, 4Coleman M.L. McDonough M.A. Hewitson K.S. Coles C. Mecinovic J. Edelmann M. Cook K.M. Cockman M.E. Lancaster D.E. Kessler B.M. Oldham N.J. Ratcliffe P.J. Schofield C.J. Asparaginyl hydroxylation of the Notch ankyrin repeat domain by factor inhibiting hypoxia-inducible factor.J. Biol. Chem. 2007; 282: 24027-24038Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 9Rual J.F. Venkatesan K. Hao T. Hirozane-Kishikawa T. Dricot A. Li N. Berriz G.F. Gibbons F.D. Dreze M. Ayivi-Guedehoussou N. Klitgord N. Simon C. Boxem M. Milstein S. Rosenberg J. Goldberg D.S. Zhang L.V. Wong S.L. Franklin G. Li S. Albala J.S. Lim J. Fraughton C. Llamosas E. Cevik S. Bex C. Lamesch P. Sikorski R.S. Vandenhaute J. Zoghbi H.Y. Smolyar A. Bosak S. Sequerra R. Doucette-Stamm L. Cusick M.E. Hill D.E. Roth F.P. Vidal M. Towards a proteome-scale map of the human protein-protein interaction network.Nature. 2005; 437: 1173-1178Crossref PubMed Scopus (2238) Google Scholar, 10Linke S. Hampton-Smith R.J. Peet D.J. Characterization of ankyrin repeat-containing proteins as substrates of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible transcription factor.Methods Enzymol. 2007; 435: 61-85Crossref PubMed Scopus (23) Google Scholar). Although AP-MS can be a powerful method, potentially permitting the identification of protein-protein interactions in a physiological context, the preservation of transient protein associations such as those between enzymes and substrates presents a major challenge to this technology. It was thus possible that important FIH protein-substrate associations had been overlooked. We therefore sought to improve methods for identification of such interactions and for the determination of the extent of FIH-catalyzed hydroxylation in substrate proteins. In analyses of FIH with known HIF, IκB, and Notch receptor substrates we noted that the enzyme-substrate interaction could be stabilized by pretreatment of cells with dimethyloxalylglycine (DMOG; a cell-penetrant inhibitor of 2OG-dependent oxygenases that is metabolized to the 2OG analogue N-oxalylglycine) and defined conditions under which DMOG could be used as a “substrate trapping” agent. Here we describe comparative proteomics screens of untreated cells and cells pre-exposed to DMOG, the use of SILAC to identify preferential DMOG-stabilized interactions with FIH, and the use of alternating low/high collision energy tandem MS to provide simultaneous assignment and quantification of specific sites of FIH-mediated hydroxylation in target proteins. In total, the work identified 12 ARD-containing proteins that associate with FIH in a DMOG-enhanced manner. Detailed MS-based characterization of three of these proteins, Rabankyrin-5, RNase L, and Tankyrase-2, confirmed that all are FIH substrates and revealed the presence of multiple hydroxylation sites that are differentially hydroxylated by FIH, including at least eight sites on Tankyrase-2. The findings indicate that asparaginyl hydroxylation is a common post-translational modification, at least among ARD-containing proteins, and identify these proteins as the largest class of protein hydroxylation targets known to date. Human embryonic kidney (HEK) 293 cells stably expressing SPA-tagged FIH (Sequential Peptide Affinity tag; 3× FLAG epitope tag, tobacco etch virus protease site, and calmodulin binding peptide (11Zeghouf M. Li J. Butland G. Borkowska A. Canadien V. Richards D. Beattie B. Emili A. Greenblatt J.F. Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes.J. Proteome Res. 2004; 3: 463-468Crossref PubMed Scopus (150) Google Scholar)) were used in proteomics screens for FIH-co-precipitating proteins. An expression construct for stable expression of SPA-tagged fusion proteins (pcDNA3/NSPA) was created by inserting an N-terminal SPA tag (custom synthesis; GenScript Corp.) into pcDNA3 (Invitrogen) via BamHI/EcoRI sites into which full-length FIH (or EGFP control) cDNA generated by PCR was subcloned. Sequence-verified constructs were transfected into 293 cells, and stable clones were identified by selection in G418 (1 mg/ml). Clones expressing the lowest levels of FLAG-transgene were used in this study. For the screen, SPA-FIH and control cells (SPA-EGFP) were cultured in the presence or absence of 1 mm DMOG for 16 h. Cells were harvested in IP+ buffer (3Cockman M.E. Lancaster D.E. Stolze I.P. Hewitson K.S. McDonough M.A. Coleman M.L. Coles C.H. Yu X. Hay R.T. Ley S.C. Pugh C.W. Oldham N.J. Masson N. Schofield C.J. Ratcliffe P.J. Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH).Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 14767-14772Crossref PubMed Scopus (220) Google Scholar), and SPA-tagged complexes were immunopurified with FLAG affinity gel (EZview™, Sigma) prior to elution (500 mm NH4OH (pH 11), 0.5 mm EDTA), dilution in Laemmli buffer, and SDS-PAGE analysis. Co-precipitating species that demonstrated DMOG-inducible capture upon Coomassie Blue or silver staining were excised and digested with trypsin. Peptides were analyzed by Tandem MS on a Q-Tof Premier™ instrument (Waters). This approach led to the identification of RIPK4 (four unique peptides; Mascot score, 70) and RNase L (four unique peptides; Mascot score, 152). For the SILAC screen, U2OS cells expressing FLAG-FIH (tet-FIH) or empty vector (tet-EV) under the control of a doxycycline-inducible promoter were used (3Cockman M.E. Lancaster D.E. Stolze I.P. Hewitson K.S. McDonough M.A. Coleman M.L. Coles C.H. Yu X. Hay R.T. Ley S.C. Pugh C.W. Oldham N.J. Masson N. Schofield C.J. Ratcliffe P.J. Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH).Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 14767-14772Crossref PubMed Scopus (220) Google Scholar). Two isotopically distinct populations of tet-FIH cells were created by serial passage in arginine- and lysine-deficient Dulbecco’s modified Eagle’s medium containing 10% dialyzed fetal bovine serum supplemented with either normal (“light”) isotopic abundance (0.68 μm) l-lysine and (0.54 μm) l-arginine or with heavy isotopic forms of l-lysine (U-13C6; Lys6) and l-arginine (U-13C6,15N4; Arg10) at identical concentrations (SILAC Protein ID and Quantitation Media kit; Invitrogen). FIH was induced in both populations after six cell doublings by addition of doxycycline (0.5 μg/ml; 18 h), whereas only the heavy population was exposed to DMOG (1 mm; 16 h). Cells were harvested in IP+ buffer, quantitated, and normalized for total protein content. Efficient incorporation of the heavy label was confirmed by digesting 20 μg of methanol/chloroform-precipitated cell lysate with trypsin and analysis by tandem MS; >99% of peptides assigned by Mascot carried a mass label (data not shown). FIH complexes were immunopurified from heavy and light lysates by FLAG affinity gel. The affinity gel was washed, pooled, and eluted before desalting and digestion with trypsin. Protein(s) binding in a DMOG-inducible manner was assigned on the basis of an increased ratio of heavy to light peptides as determined by Mascot or ProteinLynx Global Server (PLGS; Waters). A control (FLAG) immunoprecipitation (IP) was performed in parallel on tet-EV cells that were passaged in light medium and exposed to doxycycline (0.5 μg/ml; 18 h) and DMOG (1 mm; 16 h). This IP provided a list of contaminants that was subtracted from the FIH screen to define specific FIH interactors. Material was subjected to nano-ultraperformance liquid chromatography tandem mass spectrometry analysis (nano-UPLC-MSE or -MS/MS) using a 75-μm-inner diameter × 25-cm C18 nanoAcquity™ UPLC™ column (1.7-μm particle size; Waters) and a 90-min gradient of 2–45% solvent B (solvent A: 99.9% H2O, 0.1% formic acid; solvent B: 99.9% acetonitrile, 0.1% formic acid) on a Waters nanoAcquity UPLC system (final flow rate, 250 nl/min; 7000 p.s.i.) coupled to Q-TOF Premier tandem mass spectrometer (Waters). Data were acquired in high definition low/high collision energy MS (MSE) mode (low collision energy, 4 eV; high collision energy ramping from 15 to 40 eV, switching every 1.5 s). Alternatively MS analysis was performed in data-directed analysis (DDA) mode (MS to MS/MS switching at precursor ion counts greater than 10 and MS/MS collision energy dependent on precursor ion mass and charge state). All raw MS data were processed with PLGS software (version 2.2.5) including deisotoping. For MSE data MS/MS spectra were reconstructed by combining all masses with identical retention times. The mass accuracy of the raw data was corrected using Glu-fibrinopeptide (200 fmol/μl; 700 nl/min flow rate; 785.8426 Da [M + 2H]2+) that was infused into the mass spectrometer as a lock mass during sample analysis. MS, MSE, and MS/MS data were calibrated at intervals of 30 s. A UniProtKB/Swiss-Prot database (release 55; June 17, 2008; number of human sequence entries, 19,804) was used for database searches of each run with the following parameters: peptide tolerance, 15 ppm; fragment tolerance, 0.015 Da; trypsin missed cleavages, 1; variable modifications, carbamidomethylation and Met/Pro/Asn/Lys/Asp oxidation. Assignments of asparaginyl hydroxylations that were detected by PLGS were evaluated and verified upon manual inspection. In every case, peptides containing hydroxyasparagine were uniquely assigned to one protein. Each MS/MS spectrum was processed for deisotoping and deconvolution using MaxEnt3 (MassLynx 4.1), and all assignments are documented by an MS/MS spectrum included in this study. For the analysis of MSE-derived SILAC data, PLGS (version 2.2.5) was used to search against the UniProtKB/Swiss-Prot database (release 55) with the following parameters: peptide tolerance, 15 ppm; fragment tolerance, 0.015 Da; carbamidomethylation as a fixed modification; and [13C]Lys (+6 Da) and [13C,15N]Arg (+10 Da) as variable modifications. DDA-derived MS/MS spectra (peak lists) were searched against the UniProtKB/Swiss-Prot database using either PLGS as described or alternatively using Mascot version 2.2 (Matrix Science) with the following parameters: peptide tolerance, 0.2 Da; 13C = 1; fragment tolerance, 0.1 Da; missed cleavages, 2; instrument type, ESI-Q-TOF; variable modifications, carbamidomethylation, methionine/asparagine oxidation, and for SILAC data label Lys +6 Da and Arg +10 Da. All database searches were restricted to human species because of the complexity of the searches when combined with multiple modifications. The interpretation and presentation of MS/MS data were performed according to published guidelines (12Taylor G.K. Goodlett D.R. Rules governing protein identification by mass spectrometry.Rapid Commun. Mass Spectrom. 2005; 193420Crossref PubMed Scopus (90) Google Scholar). In addition, individual MS/MS spectra for peptides with a Mascot Mowse score lower than 40 (Expect <0.015) were inspected manually and included in the statistics only if a series of at least four continuous y or b ions was observed. For the analysis of MSE data using PLGS, ARD proteins were included when detected with a score above 20 and/or their probability to be present in the mixture was >50% as calculated by the software. Three other ARD proteins were included with a probability of less than 50% and a protein score of below 20 because the heavy versus light peptide ratios indicated that they were DMOG-inducible (Table I). Protein identification was also based on the assignment of at least two peptides with the exception of Notch2, which was shown previously to be an FIH substrate (Ref. 4Coleman M.L. McDonough M.A. Hewitson K.S. Coles C. Mecinovic J. Edelmann M. Cook K.M. Cockman M.E. Lancaster D.E. Kessler B.M. Oldham N.J. Ratcliffe P.J. Schofield C.J. Asparaginyl hydroxylation of the Notch ankyrin repeat domain by factor inhibiting hypoxia-inducible factor.J. Biol. Chem. 2007; 282: 24027-24038Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar; see supplemental Fig. S5 for MS/MS assignment).Table IIdentification of FIH-interacting ARD protein candidates using a SILAC-based proteomics screenProteinAccession no. (Swiss-Prot)Protein scoreaMascot (DDA), PLGS (MSE).MS/MS modeSequence coveragePeptides totalHeavybDerived from sample treated with DMOG.LightcDerived from untreated sample.%Factor inhibiting HIFdBait protein used as internal standard for an equal ratio of heavy to light peptides.Q9NWT63652DDA771296366Rabankyrin-5eFIH interaction of this protein confirmed in this study.Q9P2R3491DDA12990138MSE2518182Ankyrin repeat and KH domain-1Q8IWZ390DDA2651Tankyrase-2eFIH interaction of this protein confirmed in this study.Q9H2K251DDA233041MSE101293Ankyrin repeat domain-52Q8NB4637MSE111165Notch2fShown previously to be on FIH substrate (4).Q0472122DDA1110Ankyrin repeat domain-27Q96NW421MSE7642Ankyrin repeat and SAM domain-1Q9262521MSE4321Ankyrin repeat domain-60gIncluding peptide/protein probabilities of <50% as calculated by PLGS.Q9BZ1916MSE3012102Ankyrin repeat domain-35gIncluding peptide/protein probabilities of <50% as calculated by PLGS.Q8N28313MSE1923158IκBεgIncluding peptide/protein probabilities of <50% as calculated by PLGS.Q1416411MSE8651a Mascot (DDA), PLGS (MSE).b Derived from sample treated with DMOG.c Derived from untreated sample.d Bait protein used as internal standard for an equal ratio of heavy to light peptides.e FIH interaction of this protein confirmed in this study.f Shown previously to be on FIH substrate (4Coleman M.L. McDonough M.A. Hewitson K.S. Coles C. Mecinovic J. Edelmann M. Cook K.M. Cockman M.E. Lancaster D.E. Kessler B.M. Oldham N.J. Ratcliffe P.J. Schofield C.J. Asparaginyl hydroxylation of the Notch ankyrin repeat domain by factor inhibiting hypoxia-inducible factor.J. Biol. Chem. 2007; 282: 24027-24038Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar).g Including peptide/protein probabilities of <50% as calculated by PLGS. Open table in a new tab To assess whether FIH interaction with a detected protein was inducible by DMOG, the ratios of peptides with incorporated stable amino acids (Lys6/Arg10 for samples that included DMOG treatment) versus unlabeled peptides (samples without DMOG treatment) were examined. In cases where peptide assignments were matching to more than one protein, the corresponding MS/MS spectra were assigned manually. As an internal control, heavy and light tryptic peptides derived from FIH were evaluated for equal mixing of both sample sets (Table I). The local “in-house” Mascot server used for this study is supported and maintained by the Computational Biology Research Group at the University of Oxford. Whole cell extracts were prepared in IP+ buffer with 400 μg of extract as input. FIH pulldowns used FLAG affinity gel, whereas endogenous IPs used 2 μg of anti-ARD antibody sourced from the following: anti-Tankyrase (Clone 19A449, Abcam), anti-RNase L (2E9, Abcam), and anti-Rabankyrin-5 (13Schnatwinkel C. Christoforidis S. Lindsay M.R. Uttenweiler-Joseph S. Wilm M. Parton R.G. Zerial M. The Rab5 effector Rabankyrin-5 regulates and coordinates different endocytic mechanisms.PLoS Biol. 2004; 2: E261Crossref PubMed Scopus (160) Google Scholar) or species/isotype-matched control IgG (all supplied by Abcam). Immunoblotting was performed using the same panel of antibodies, including FLAG-horseradish peroxidase (Sigma) and anti-FIH antibody, which was raised in the host laboratory and described previously (14Stolze I.P. Tian Y.M. Appelhoff R.J. Turley H. Wykoff C.C. Gleadle J.M. Ratcliffe P.J. Genetic analysis of the role of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible factor (HIF) in regulating HIF transcriptional target genes.J. Biol. Chem. 2004; 279: 42719-42725Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Where necessary, secondary detection used Trueblot™ horseradish peroxidase-conjugated antibody (eBioscience). Plasmids encoding full-length Tankyrase-2 (pFLAG/TNKS2 (15Sbodio J.I. Lodish H.F. Chi N.W. Tankyrase-2 oligomerizes with tankyrase-1 and binds to both TRF1 (telomere-repeat-binding factor 1) and IRAP (insulin-responsive aminopeptidase).Biochem. J. 2002; 361: 451-459Crossref PubMed Scopus (107) Google Scholar)), RNase L (pcDNA3/RNase L-GFP (16Le Roy F. Laskowska A. Silhol M. Salehzada T. Bisbal C. Characterization of RNABP, an RNA binding protein that associates with RNase L.J. Interferon Cytokine Res. 2000; 20: 635-644Crossref PubMed Scopus (8) Google Scholar)), and Rabankyrin-5 (pEYFP/Rabankyrin-5 (13Schnatwinkel C. Christoforidis S. Lindsay M.R. Uttenweiler-Joseph S. Wilm M. Parton R.G. Zerial M. The Rab5 effector Rabankyrin-5 regulates and coordinates different endocytic mechanisms.PLoS Biol. 2004; 2: E261Crossref PubMed Scopus (160) Google Scholar)) were expressed transiently in 293T cells using FuGENE 6™ transfection reagent (Roche Applied Science). FIH levels were modulated by co-transfection of pcDNA3/FIH in a 1:5 ratio with plasmid encoding the relevant ARD protein or by knockdown of endogenous FIH with prevalidated siRNA duplexes using Oligofectamine reagent (Invitrogen). Plasmids and siRNA sequences have been described previously (14Stolze I.P. Tian Y.M. Appelhoff R.J. Turley H. Wykoff C.C. Gleadle J.M. Ratcliffe P.J. Genetic analysis of the role of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible factor (HIF) in regulating HIF transcriptional target genes.J. Biol. Chem. 2004; 279: 42719-42725Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Cells were lysed in IP+ buffer (3Cockman M.E. Lancaster D.E. Stolze I.P. Hewitson K.S. McDonough M.A. Coleman M.L. Coles C.H. Yu X. Hay R.T. Ley S.C. Pugh C.W. Oldham N.J. Masson N. Schofield C.J. Ratcliffe P.J. Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH).Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 14767-14772Crossref PubMed Scopus (220) Google Scholar), and ARD substrate was immunopurified using either anti-green fluorescent protein antibody (Clone 3E1, Cancer Research UK) coupled to protein A-agarose (Millipore) or FLAG affinity gel. Samples were eluted in ammonium hydroxide and either resolved by SDS-PAGE or desalted by methanol/chloroform precipitation prior to tryptic digestion as described previously (17Xu D. Suenaga N. Edelmann M.J. Fridman R. Muschel R.J. Kessler B.M. Novel MMP-9 substrates in cancer cells revealed by a label-free quantitative proteomics approach.Mol. Cell. Proteomics. 2008; 7: 2215-2228Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). DMOG is a cell-penetrating precursor of N-oxalylglycine, a 2OG analogue that competitively inhibits many 2OG-dependent oxygenases including FIH (Fig. 1A). To pursue the possibility that exposure of cells to this compound might stabilize FIH-substrate interactions sufficiently to permit the identification of novel substrates of FIH we first performed co-immunoprecipitation experiments using cell lines stably expressing SPA-tagged FIH or control SPA-EGFP. HEK293 cells were transfected with pcDNA3 encoding FIH with an N-terminal SPA tag that had been shown not to interfere with FIH enzymatic activity. Transfectants expressing modestly elevated levels of SPA-FIH, when compared with endogenous FIH, were selected for this study. Cells were exposed to DMOG following which SPA-FIH-associated proteins were purified from cell extracts and resolved by SDS-PAGE. These experiments demonstrated that exposure to DMOG (1 mm for a period of 16 h) was sufficient to enhance the capture of FIH-associated species as revealed by Coomassie Blue staining (Fig. 1B). The two FIH-associated species that were defined in this way were excised, digested with trypsin, and analyzed by LC-MS/MS. This revealed the species to be ankyrin repeat and FYVE domain-containing protein 1 (Rabankyrin-5) and receptor-interacting serine/threonine-protein kinase 4 (RIPK4), both ARD-containing p" @default.
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• W2038139095 date "2009-03-01" @default.
• W2038139095 modified "2023-09-27" @default.
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