Asparagine Hydroxylation is a Reversible Post-translational Modification
2020; Elsevier BV; Volume: 19; Issue: 11 Linguagem: Inglês
10.1074/mcp.ra120.002189
ISSN1535-9484
AutoresJavier Rodríguez, Cameron D. Haydinger, Daniel J. Peet, Lan K. Nguyen, Alex von Kriegsheim,
Tópico(s)Advanced Proteomics Techniques and Applications
ResumoAmino acid hydroxylation is a common post-translational modification, which generally regulates protein interactions or adds a functional group that can be further modified. Such hydroxylation is currently considered irreversible, necessitating the degradation and re-synthesis of the entire protein to reset the modification. Here we present evidence that the cellular machinery can reverse FIH-mediated asparagine hydroxylation on intact proteins. These data suggest that asparagine hydroxylation is a flexible and dynamic post-translational modification akin to modifications involved in regulating signaling networks, such as phosphorylation, methylation and ubiquitylation. Amino acid hydroxylation is a common post-translational modification, which generally regulates protein interactions or adds a functional group that can be further modified. Such hydroxylation is currently considered irreversible, necessitating the degradation and re-synthesis of the entire protein to reset the modification. Here we present evidence that the cellular machinery can reverse FIH-mediated asparagine hydroxylation on intact proteins. These data suggest that asparagine hydroxylation is a flexible and dynamic post-translational modification akin to modifications involved in regulating signaling networks, such as phosphorylation, methylation and ubiquitylation. Post-translational modifications (PTMs) are chemical alterations of amino acids or proteins that increase the complexity of the proteome and allow the cell to modify protein function in a dynamic or sustained manner (1Walsh C.T. Garneau-Tsodikova S. Gatto Jr, G.J. Protein posttranslational modifications: the chemistry of proteome diversifications.Angew. Chem. Int. Ed. Engl. 2005; 44: 7342-7372Crossref PubMed Scopus (1025) Google Scholar, 2Hunter T. Tyrosine phosphorylation: thirty years and counting.Curr. Opin. Cell Biol. 2009; 21: 140-146Crossref PubMed Scopus (486) Google Scholar, 3Ciechanover A. Proteolysis: from the lysosome to ubiquitin and the proteasome.Nat. Rev. Mol. Cell Biol. 2005; 6: 79-87Crossref PubMed Scopus (814) Google Scholar, 4Duan G. Walther D. The roles of post-translational modifications in the context of protein interaction networks.PLoS Comput. Biol. 2015; 11: e10040491Crossref Scopus (234) Google Scholar). Some of these modifications are irreversible, such as the cleavage of a polypeptide chain resulting in altered activity of the protein. Nevertheless, most PTMs are reversible and comprise the dynamic addition or elimination of functional groups that range from large oligosaccharide chains to a few atoms, such as glycosylation, phosphorylation, acetylation, methylation, or carboxylation. The reversibility of these PTMs is frequently achieved by the action of pairs of enzyme classes with opposing functions, one of which catalyzes the forward reaction and another the reverse reaction (5Tonks N.K. Protein tyrosine phosphatases: from genes, to function, to disease.Nat. Rev. Mol. Cell Biol. 2006; 7: 833-846Crossref PubMed Scopus (1254) Google Scholar, 6Trewick S.C. McLaughlin P.J. Allshire R.C. Methylation: lost in hydroxylation?.EMBO Rep. 2005; 6: 315-320Crossref PubMed Scopus (158) Google Scholar, 7Gray S.G. Teh B.T. Histone acetylation/deacetylation and cancer: an "open" and "shut" case?.Curr. Mol. Med. 2001; 1: 401-429Crossref PubMed Scopus (83) Google Scholar). In cases where one of the reactions is thermodynamically unfavorable, the reaction may not be straightforwardly reversible and therefore includes intermediate products. An example would be the formation and dissolution of cysteine bonds, where the formation of the bond can include the oxidation of the sulfur of cysteine to sulfenic acid, which then forms a disulfide bond by reacting with another cysteine (8Rehder D.S. Borges C.R. Cysteine sulfenic acid as an intermediate in disulfide bond formation and nonenzymatic protein folding.Biochemistry. 2010; 49: 7748-7755Crossref PubMed Scopus (109) Google Scholar, 9Oka O.B. Bulleid N.J. Forming disulfides in the endoplasmic reticulum.Biochim. Biophys. Acta. 2013; 1833: 2425-2429Crossref PubMed Scopus (80) Google Scholar). Oxidation or more precisely hydroxylation of proteins on residues other than cysteine was recognized as a PTM in the 1960s, when the enzymatic hydroxylation of proline and lysine was identified as taking place during collagen synthesis (10Kivirikko K.I. Prockop D.J. Hydroxylation of proline in synthetic polypeptides with purified protocollagen hydroxylase.J. Biol. Chem. 1967; 242: 4007-4012Abstract Full Text PDF PubMed Google Scholar, 11Hutton Jr., J.J. Kaplan A. Udenfriend S. Conversion of the amino acid sequence gly-pro-pro in protein to gly-pro-hyp by collagen proline hydroxylase.Arch. Biochem. Biophys. 1967; 121: 384-391Crossref PubMed Scopus (28) Google Scholar). Subsequently, the molecular mechanism of hydroxylation catalyzed by the evolutionarily conserved family of the 2-Oxoglutarate dependent dioxygenases (2OG-ox) was described (12Ploumakis A. Coleman M.L. OH, the places you'll go! Hydroxylation, gene expression, and cancer.Mol. Cell. 2015; 58: 729-741Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Loenarz C. Schofield C.J. Physiological and biochemical aspects of hydroxylations and demethylations catalyzed by human 2-oxoglutarate oxygenases.Trends Biochem. Sci. 2011; 36: 7-18Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). The 2OG-ox are a family of proteins composed of over 60 enzymes of which the so-called HIF-hydroxylases form a subgroup consisting of four hydroxylases: 3 prolyl hydroxylases (PHD1,2 and 3) and an asparaginyl hydroxylase "factor inhibiting HIF" (FIH). HIF-hydroxylases act as sensors of the oxygen levels within the cells. Under normoxic conditions prolyl hydroxylases (PHD1, 2 and 3) catalyze the specific hydroxylation of two proline residues in the alpha subunit of HIF1, the master regulator of the hypoxic response. Once hydroxylated, HIF1α is bound by the von Hippel-Lindau ubiquitin ligase (VHL) complex, which promotes the ubiquitination and rapid proteasomal degradation of HIF1α, resulting in the ablation of the protein under normoxic conditions (14Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. von Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.Science. 2001; 292: 468-472Crossref PubMed Scopus (4434) Google Scholar, 15Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr, W.G. HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing.Science. 2001; 292: 464-468Crossref PubMed Scopus (3874) Google Scholar, 16Yu F. White S.B. Zhao Q. Lee F.S. HIF-1alpha binding to VHL is regulated by stimulus-sensitive proline hydroxylation.Proc. Natl. Acad. Sci. U S A. 2001; 98: 9630-9635Crossref PubMed Scopus (644) Google Scholar, 17Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.Nature. 1999; 399: 271-275Crossref PubMed Scopus (4118) Google Scholar, 18Appelhoff R.J. Tian Y.M. Raval R.R. Turley H. Harris A.L. Pugh C.W. Ratcliffe P.J. Gleadle J.M. Differential function of the prolyl hydroxylases PHD1, PHD2, and PHD3 in the regulation of hypoxia-inducible factor.J. Biol. Chem. 2004; 279: 38458-38465Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar). FIH, the other component of the subfamily, catalyzes an asparagine hydroxylation in the C terminus of HIF1α. Upon hydroxylation of this residue, the interaction with co-factors required for the formation of the active transcription factor complex is impeded, resulting in the down-regulation of HIF-driven transcription in the presence of oxygen (19Mahon P.C. Hirota K. Semenza G.L. FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity.Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1119) Google Scholar, 20Lando D. Peet D.J. Whelan D.A. Gorman J.J. Whitelaw M.L. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch.Science. 2002; 295: 858-861Crossref PubMed Scopus (1271) Google Scholar). Over the past few years, it has been argued that HIF1α is not the only protein that is hydroxylated by HIF-hydroxylases and several additional substrates, particularly of FIH, have been postulated and validated (21Cockman M.E. Webb J.D. Kramer H.B. Kessler B.M. Ratcliffe P.J. Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.Mol. Cell. Proteomics. 2009; 8: 535-546Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In a similar manner to that observed in the context of HIFα, hydroxylation of these other substrates by FIH alters the physicochemical properties of the hydroxylated domains. These changes can induce or destroy protein-protein interactions that ultimately control substrate activity, folding or localization (22Scholz C.C. Rodriguez J. Pickel C. Burr S. Fabrizio J.A. Nolan K.A. Spielmann P. Cavadas M.A. Crifo B. Halligan D.N. Nathan J.A. Peet D.J. Wenger R.H. Von Kriegsheim A. Cummins E.P. Taylor C.T. FIH regulates cellular metabolism through hydroxylation of the deubiquitinase OTUB1.PLos Biol. 2016; 14: e10023471Crossref Scopus (60) Google Scholar, 23Rodriguez J. Pilkington R. Garcia Munoz A. Nguyen L.K. Rauch N. Kennedy S. Monsefi N. Herrero A. Taylor C.T. von Kriegsheim A. Substrate-trapped interactors of PHD3 and FIH cluster in distinct signaling pathways.Cell Rep. 2016; 14: 2745-2760Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 24Kiriakidis S. Henze A.T. Kruszynska-Ziaja I. Skobridis K. Theodorou V. Paleolog E.M. Mazzone M. Factor-inhibiting HIF-1 (FIH-1) is required for human vascular endothelial cell survival.FASEB J. 2015; 29: 2814-2827Crossref PubMed Scopus (22) Google Scholar, 25Karttunen S. Duffield M. Scrimgeour N.R. Squires L. Lim W.L. Dallas M.L. Scragg J.L. Chicher J. Dave K.A. Whitelaw M.L. Peers C. Gorman J.J. Gleadle J.M. Rychkov G.Y. Peet D.J. Oxygen-dependent hydroxylation by FIH regulates the TRPV3 ion channel.J. Cell Sci. 2015; 128: 225-231Crossref PubMed Scopus (32) Google Scholar, 26Janke K. Brockmeier U. Kuhlmann K. Eisenacher M. Nolde J. Meyer H.E. Mairbaurl H. Metzen E. Factor inhibiting HIF-1 (FIH-1) modulates protein interactions of apoptosis-stimulating p53 binding protein 2 (ASPP2).J. Cell Sci. 2013; 126: 2629-2640Crossref PubMed Scopus (43) Google Scholar). Presently, it has been suggested that the hydroxylation mediated by HIF-hydroxylases is an irreversible process, and that the hydroxylation can only be reset by the degradation and new synthesis of a nonhydroxylated protein (27Gorres K.L. Raines R.T. Prolyl 4-hydroxylase.Crit. Rev. Biochem. Mol. Biol. 2010; 45: 106-124Crossref PubMed Scopus (406) Google Scholar, 28Schofield C.J. Ratcliffe P.J. Oxygen sensing by HIF hydroxylases.Nat. Rev. Mol. Cell Biol. 2004; 5: 343-354Crossref PubMed Scopus (1611) Google Scholar, 29Chan D.A. Sutphin P.D. Yen S.E. Giaccia A.J. Coordinate regulation of the oxygen-dependent degradation domains of hypoxia-inducible factor 1 alpha.Mol. Cell Biol. 2005; 25: 6415-6426Crossref PubMed Scopus (191) Google Scholar). A mass spectrometric study monitoring two FIH-mediated hydroxylation sites of Rabankyrin-5 substantiated this, as no evidence of dehydroxylation was found under the investigated conditions (30Singleton R.S. Trudgian D.C. Fischer R. Kessler B.M. Ratcliffe P.J. Cockman M.E. Quantitative mass spectrometry reveals dynamics of factor-inhibiting hypoxia-inducible factor-catalyzed hydroxylation.J. Biol. Chem. 2011; 286: 33784-33794Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Nevertheless, some authors have proposed the existence of a dehydroxylation mechanism (25Karttunen S. Duffield M. Scrimgeour N.R. Squires L. Lim W.L. Dallas M.L. Scragg J.L. Chicher J. Dave K.A. Whitelaw M.L. Peers C. Gorman J.J. Gleadle J.M. Rychkov G.Y. Peet D.J. Oxygen-dependent hydroxylation by FIH regulates the TRPV3 ion channel.J. Cell Sci. 2015; 128: 225-231Crossref PubMed Scopus (32) Google Scholar, 31Giaccia A. Siim B.G. Johnson R.S. HIF-1 as a target for drug development.Nat. Rev. Drug Discov. 2003; 2: 803-811Crossref PubMed Scopus (531) Google Scholar), but this notion is only founded on the assumption that PTMs should be generally reversible. Some data have emerged that implicitly suggest that asparagine hydroxylation may be a reversible PTM after all. First, in contrast to proline hydroxylation of HIF, FIH-mediated asparagine hydroxylation does not lead to the rapid decrease of protein half-life. This suggests that FIH-substrates are longer-lived proteins and that the cell would therefore benefit from a mechanism of resetting the level of hydroxylation in a more dynamic, nondestructive manner (21Cockman M.E. Webb J.D. Kramer H.B. Kessler B.M. Ratcliffe P.J. Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.Mol. Cell. Proteomics. 2009; 8: 535-546Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). One such example is transient receptor potential vanilloid 3 (TRPV3), an ion channel that is hydroxylated by FIH. Hydroxylation on an asparagine residue reduces TRPV3-mediated current, whereas hypoxia, FIH inhibition or mutation of the asparagine residue potentiates it without affecting protein stability. Intriguingly, the increases in current through the channel are observable in less than one hour of hypoxia or FIH inhibition. This rapid response indicates that there has to be a very rapid turnover of the protein or that the hydroxylation can be reversed without destruction and re-synthesis of TRPV3 (25Karttunen S. Duffield M. Scrimgeour N.R. Squires L. Lim W.L. Dallas M.L. Scragg J.L. Chicher J. Dave K.A. Whitelaw M.L. Peers C. Gorman J.J. Gleadle J.M. Rychkov G.Y. Peet D.J. Oxygen-dependent hydroxylation by FIH regulates the TRPV3 ion channel.J. Cell Sci. 2015; 128: 225-231Crossref PubMed Scopus (32) Google Scholar). Moreover, indirect evidence from mathematical modeling indicates that signaling networks require reversibility of asparagine hydroxylation. Nguyen et al. published a comprehensive ODE-based mathematical model of the immediate HIF network (32Nguyen L.K. Cavadas M.A. Scholz C.C. Fitzpatrick S.F. Bruning U. Cummins E.P. Tambuwala M.M. Manresa M.C. Kholodenko B.N. Taylor C.T. Cheong A. A dynamic model of the hypoxia-inducible factor 1alpha (HIF-1alpha) network.J. Cell Sci. 2013; 126: 1454-1463Crossref PubMed Scopus (86) Google Scholar). Intriguingly, the mathematical model assumes irreversibility of both proline hydroxylations but includes an undefined reaction that leads to the dehydroxylation of the N-terminal asparagine residue (Fig. 1A). The reversibility of the asparagine hydroxylation was required for the model to reproduce the experimental data, which showed the transient induction of HIF protein levels and transcriptional output. Upon removal of the unspecified dehydroxylation reaction, the model predicts that HIF levels and activity would increase in a sustained manner, which is at odds with the experimental observations (Fig. 1B–1E). Overall, although these data give initial foundation to the hypothesis that asparagine hydroxylation is reversible, no direct evidence of reversibility has been produced. Notably, amino acid methylation was initially thought to be a static modification (33Ramchandani S. Bhattacharya S.K. Cervoni N. Szyf M. DNA methylation is a reversible biological signal.Proc. Natl. Acad. Sci. U S A. 1999; 96: 6107-6112Crossref PubMed Scopus (269) Google Scholar) but was subsequently demonstrated to be reversed through the action of 2OG-ox. With this in mind, we set out to examine the potential reversibility of asparagine hydroxylation by using quantitative MS. All reagents were purchased from Sigma, Gillingham, UK unless otherwise stated. HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mm glutamine (Invitrogen, Carlsbad, CA) and 10% fetal calf serum (Invitrogen). Plasmids were transfected with LipofectAMINE 2000 (Invitrogen) according to the vendor's instructions. SILAC media was generated by custom made DMEM medium lacking l-arginine and l-lysine (Thermo Fisher Scientific).This media was supplemented l-Arginine-13C 15N (R10) and l-Lysine-13C 15N (K8) (Cambridge Isotope Laboratories, Inc., Leicester, UK) but not with FCS to prevent incorporation of 12C-Lysine or Arginine. All antibodies were from commercial sources: anti-FLAG M2 peroxidase was obtained from Sigma Aldrich (F4042, 1:1,000 dilution), anti-HIF1α was from BD Biosciences (610958 1:1,000 dilution), anti-GFP and anti-myc were from Cell Signaling (D5.1-9B11, 1:2,000 dilution,) anti-tubulin was purchased from Santa Cruz (sc-8035, 1:1,000 dilution) and anti-V5 was obtained from Invitrogen (R96025, 1:5000 dilution). DMOG was obtained from Cayman Chemical (71210), DFO and cycloheximide were purchased from SIGMA (D9533- C4859). For immunoprecipitation anti-Flag-M2 beads (Sigma Aldrich), anti-myc beads (Cell Signaling-9B11) and anti-GFP (GFP-Trap Magnetic Agarose-Chromotek, Munich, Germany) were used. GFP-HIF1A was a gift from Alex Chong (Aston University), FLAG-TNKS2 from Addgene (#34691). Cells were lysed in ice-cold lysis buffer (1% Triton X-100, 20 mm Tris-HCl (pH 7.5), 150 mm NaCl), supplemented with protease (5 μg/ml leupeptin, 2,2 μg/ml aprotinin), phosphatase inhibitors (20 mm β-glycerophosphate). Lysates were cleared of debris by centrifugation at 20,000 × g for 10 min in a benchtop centrifuge (4 °C). Total lysates were fractionated by SDS-PAGE and transferred onto nitrocellulose filters. Immunocomplexes were visualized by enhanced chemiluminescence detection (GE Healthcare) with horseradish peroxidase–conjugated secondary antibodies (Bio-Rad Laboratories). Thioredoxin-6 histidine (Trx-6H) tagged human HIF-2α CAD (774-874) and maltose binding protein (MBP) tagged human FIH were expressed in BL21(DE3) E. coli as previously described (34Lando D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor.Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1219) Google Scholar). Protein expression was induced with 0.5 mm IPTG. CAD was induced for 8 h at 30 °C and purified using a HisTrap HP column (GE Healthcare, Sydney, Australia). FIH was induced for 16 h at 16 °C and purified using a MPBTrap HP column (GE Healthcare). Proteins were exchanged into 150 mm NaCl, 20 mm Tris-HCl pH 8 using PD-10 desalting columns (GE Healthcare). Ten μm CAD was hydroxylated by incubation with 10 μm FIH, 112.5 mm NaCl, 65 mm Tris-HCl, 4 mm sodium ascorbate, 500 μm DTT, 30 μm FeSO4 and 40 μm 2-oxoglutarate for 2.5 h at 37 °C, and repurified using a HisTrap HP column (GE Healthcare). FIH −/− HeLa cells (35Chen D.Y. Fabrizio J.A. Wilkins S.E. Dave K.A. Gorman J.J. Gleadle J.M. Fleming S.B. Peet D.J. Mercer A.A. Ankyrin repeat proteins of Orf virus influence the cellular hypoxia response pathway.J. Virol. 2017; 91 (1): e0143016Crossref Scopus (12) Google Scholar) were lysed in ice-cold lysis buffer (1% Triton X-100, 20 mm Tris-HCl (pH 7.5), 150 mm NaCl), supplemented with HALT EDTA-free protease inhibitors). Protein concentration was ∼0.5 mg/ml. 1 μg of hydroxylated CAD was incubated under shaking at 37 °C with 1 ml of FIH −/− HeLa lysate for 3 h (or 0 h for control). Precipitated with Ni-NTA-agarose beads (Qiagen) and digested with Trypsin and GluC and processed as described. Mass spectrometry was done using a Lumos Fusion (Thermo Fisher Scientific, Bremen, Germany) mass spectrometer coupled to an RLS-nano uHPLC (Thermo Fisher Scientific). Peptides were separated by a 40-min linear gradient from 5–30% Acetonitrile, 0.05% Acetic acid. Mass range was 646.5-653.5. XIC were generated with a width of (646.6980-646.7140)/(652.0300-653.0440) for the for nonhydroxylated/hydroxylated peptide. Peptide elution times were calibrated using hydroxylated/nonhydroxylated standards. HEK293T cells were transfected with transfected with the different plasmids indicated. The different treatments were done 24 h post-transfection. To prevent re-hydroxylation, all lysis and wash buffers were supplemented with 1 mm NOG, a pan-hydroxylase inhibitor. Post treatment, the cells were lysed in lysis buffer (1% Triton X-100, 150 mm NaCl, 20 mm Tris–HCl pH 7.5, 1 mm EDTA pH 7.5). Immunoprecipitation, washing, and digest was performed on a KingFisher Duo robotic station (Thermo Fisher Scientific). 5 µl of magnetic antibody bead slurry, anti-Flag-M2 beads (Sigma Aldrich), anti-myc beads (Cell signaling-9B11) and anti-GFP (GFP-Trap Magnetic Agarose-Chromotek) respectively, was dilute in 100 µl of lysis buffer and loaded in row H of a 96 deep-well plate. 500 µl of lysate was loaded into row G, 300 µl of lysis buffer were loaded into rows E and F. 300 µl of Wash buffer (150 mm NaCl, 20 mm Tris–HCl pH 7.5, 1 mm EDTA pH 7.5) were loaded into rows B-D. Row A contained the 100 µl of digest buffer (2 M Urea, 50 mm Tris–HCl pH 7.5, 1 mm DTT, 5 µg/ml porcine trypsin (Promega, Southampton, UK) 5 µg/ml GluC (Promega)). The robot picked up beads in row H, transported them to row G and released and mixed them for 2 h. Beads where picked up and released subsequently into rows F to B with 1 min mixing in between. The washed beads were then transported into row A and digested a 27 °C for 30 min under mixing. Beads where then removed and digest continued for 8 h at 37 °C. After iodoacetamide modification and acidification of the samples, the peptide mixtures were desalted using homemade C18 tips. The desalted and lyophilized peptides were resuspended in 0.1% TFA and subjected to mass spectrometric analysis by reversed-phase nano-LC–MS/MS 5 µl of the resuspended peptides were analysed by reversed-phase nano-LC–MS/MS using a nano-Ultimate 3000 liquid chromatography system and a QExactive plus or Lumos Fusion mass spectrometer (both Thermo Fisher Scientific). Flow-rates were 400 nl/min. Peptides were loaded onto an self-packed analytical column (uChrom, nanoLCMS Solutions, Oroville, CA; 1.6, 0.075 mm × 25 cm) using a 67-min gradient Buffer A, 2% acetonitrile 0.5% Acetic Acid, Buffer B, 80% acetonitrile, 0.5% Acetic Acid; (0–16 min: 2% buffer B, 16–56 min: 3–35% buffer B, 56–62 min: 99% buffer B; 62–67 min 2% buffer B. The QExactive was operated in top-12, data-dependent mode with a 30-s dynamic exclusion range. Full-scan spectra recording in the Orbitrap was in the range of m/z 350 to m/z 1,650 (resolution: 70,000; AGC: 3e6 ions). MS2 was performed with an isolation window of 1.4, AGC 5e4, HCD collision energy of 26, Scan range from 140 to 200 ms maximum injection time. The Lumos was operated in data-dependent mode with a 10-s dynamic exclusion range. Full-scan spectra recording in the Orbitrap was in the range of m/z 350 to m/z 1400 (resolution: 240,000; AGC: 7.5e5 ions). MS2 was performed in the ion trap, isolation window 0.7, AGC 2e4, HCD collision energy of 28, rapid scan rate, Scan range 145–1,450 m/z, 50 ms maximum injection time and an overall cycle time of 1 s. To calculate the relative molar ionization efficiency we devised a method that assumes a peptide exists predominantly in two molecular states, hydroxylated and unmodified. By determining the relative abundance of either species with respect to a reference intensity we estimated the relative ionization efficiency for either unmodified or hydroxylated peptides. To determine the relative ionization efficiencies of unmodified peptides, we generated a list of TNKS2/HIF1a/TRPV3 peptides, respectively, detectable in light and heavy labeled samples, excluded M-containing peptides (and N-containing peptides in the case of TNKS2) and summed up the intensities in the heavy channel of latter time point. We then divided the intensity of the unmodified containing heavy peptide by this sum to generate the relative intensity of the nonhydroxylated peptide over a reference intensity. Rel.IonizationEfficiencyN803=IntensityH(N803)∑HifpeptidenHifpeptideIntensityH in the case where FIH expression induced stochiometric hydroxylation (such as N803 of HIF1a), we summed up the intensities of the same peptides in the light channel of the untreated sample (at time point 0) and divided the intensity of the hydroxylated peptide by this sum The ratio of the respective relative intensities equals the ratio of the molar ionization intensities. Rel.IonizationEfficiencyN803Rel.IonizationEfficiencyNox803=MolarIonizationEfficiencyN803MolarIonizationEfficiencyNox803 If the hydroxylation appeared not to be stochiometric, such as in TRPV3, we included an additional step. We calculated the ratios of the nonhydroxylated peptide in heavy conditions. This gave us the ratio of the relative ionization efficiency of the unmodified peptide. We repeated this step for the light labeled, unmodified peptide at time point 0. The difference in the relative ionization efficiency of the unmodified peptides allows calculating the difference of occupancy between the DMOG treated sample and the control allowing us to calculate the % of occupancy of the unmodified peptide. Knowing this occupancy then allows to calculate the occupancy of the hydroxylated peptide and the relative ionization intensity by multiplying the occupancy with the ratio of the light labeled hydroxylated peptide over the sum of peptides. These calculations generated a ratio of molecular ionization efficiencies of unmodified/hydroxylated peptide. We used this to transform the ratio modified/unmodified as calculate by MaxQuant into molar ratios. The molar ratios were converted into estimated occupancies by dividing the ratio by itself +1; Occupancy=RatioRatio+1 using the Perseus software (36Tyanova S. Temu T. Sinitcyn P. Carlson A. Hein M.Y. Geiger T. Mann M. Cox J. The Perseus computational platform for comprehensive analysis of (prote)omics data.Nat. Methods. 2016; 13: 731-740Crossref PubMed Scopus (3530) Google Scholar). In cases where we were unable to calculate the ratio of molar ionization efficiency, we estimated it to be 1. Overall, 150 biological replicate samples were analyzed. 8 for the determination of DMOG efficiency (two controls), 48 and 48 for the TNKS2 DMOG and TNKS2 DMOG/CHX experiments (24 controls in each sample set), 20 for the TNKS2 DFO experiment (10 controls), 6 for the TRPV3 DMOG experiment (2 controls), 16 for the HIF1a DMOG experiment (4 controls) and 4 samples for the HIF2a in vitro dihydroxylation experiment. Samples sequences for MS analysis were randomized using the excel =RAND() command. Statistical tests were performed using Graphpad Prism 8 (multiple t tests), distribution was assumed to be normal. Protein intensities and hydroxylation occupancies are shown with error bars representing standard error of mean (S.E.). The MS raw data were analyzed by the MaxQuant and Andromeda (1.6.10.43) software package (37Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment.J. Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3450) Google Scholar) using the pre-selected conditions for analysis (specific proteases, 2 missed cleavages, 7 amino acids minimum length). Protease was set to trypsin or trypsin+GluC, for the TNKS2/TRPV3 or HIF1a pulldowns, respectively. Carbamylation (C) was selected as fixed modification. Variable modifications were N-terminal acetylation (protein), oxidation (MPN) and heavy labeled amino acids (K8R10). FDR was set to 0.01. MS/MS spectra were searched against the human Uniprot database (09/2019 UP000005640_9606.fasta; 20,656 entries) and a the MaxQuant contaminant database (246 entries) with a mass accuracy of 4.5 ppm (for MS) and 20 ppm or 0.5 Da (MS/MS OT or IT). Peak matching was selected and was limited to within a 0.7 min elution window with a mass accuracy of 4.5 ppm. To in silico predict the effects that putative dehydroxylation may have on dynamic behavior of the HIF-1α signaling pathway, we modified a well-calibrated mathematical model of the HIF pathway that we developed previously (32Nguyen L.K. Cavadas M.A. Scholz C.C. Fitzpatrick S.F. Bruning U. Cummins E.P. Tambuwala M.M. Manresa M.C. Kholodenko B.N. Taylor C.T. Cheong A. A dynamic model of the hypoxia-inducible factor 1alpha (HIF-1alpha) network.J. Cell Sci. 2013; 126: 1454-1463Crossref PubMed Scopus (86) Google Scholar), by removing the steps representing asparagine dehydroxylation in this model (Fig. 1A). This was done by setting the kinetic parameters describing the rate of these reactions to null in the model's ordinary differential equations. Simulations of the intact and the adjusted models under various conditions (i.e. hydroxylase inhibition by DMOG and JNK, and 1 and 3% hypoxia, Fig. 1B–1E) show that removal of the asparagine dehydroxylation steps failed to reproduce the experimental patterns of HIF-1α expression. There are several analytical techniques that can be applied to the quantitation of changes in PTMs, such as the use of modification-specific antibodies or the monitoring of shifts in the apparent molecular weight in PAGE (38Snell C.E. Turley H. McIntyre A. Li D. Masiero M. Schofield C.J. Gatter K.C. Harris A.L. Pezzella F. Proline-hydroxylated hypoxia-inducible factor 1alpha (HIF-1alpha) upregulat
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