Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150407_Ub_Tube_CH.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150407_Ub_Tube_CR.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150407_WCE_CH.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_POX1_H2O2_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/141212_WCE_CR_K63R_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_POX1_Rec_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150526_WCE_H2O2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150407_WCE_CR.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/141212_WCE_CH_K63R_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150127_UB_CR_K63R_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/141212_UB_CH_K63R_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_WCE1_Rec_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150526_POX_H2O2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_WCE1_H2O2_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_POX2_Rec.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150212_WCE_CH_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150127_UB_CR_K63R_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/141212_WCE_CH_K63R_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_POX1_H2O2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/141212_UB_CH_K63R_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_WCE2_Rec_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150212_WCE_CR_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150212_WCE_CH.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_POX2_Rec_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_WCE1_H2O2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_WCE1_H2O2_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_WCE2_H2O2_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/141212_WCE_CH_K63R_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150212_WCE_CH_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150212_WCE_CR_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150526_WCE_Rec.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150526_POX_Rec.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150407_WCE_CR_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/141212_WCE_CR_K63R_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150212_WCE_CH_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150407_WCE_CH_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/141212_UB_CR_K63R_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/141212_WCE_CR_K63R_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150212_WCE_CR.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150212_WCE_CR_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_POX2_H2O2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_POX2_H2O2_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150127_UB_CH_K63R_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/141212_UB_CR_K63R_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150212_WCE_CH_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150212_WCE_CR_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150127_UB_CH_K63R_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/150612_POX1_Rec.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/04/PXD014173/MaxQuant_POXUB_resultsfile_txt.zipprimaryOK200gustavo.silva@duke.eduGustavo SilvaMass SpectrometryShotgun proteomicsNot availableOxidative stressYeastK48 ubiquitinOxidationhttp://www.ebi.ac.uk/pride/archive/projects/PXD014173Cell disruption was performed by glass beads agitation at 4 C in standard buffer 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 25 mM iodoacetamide (IAM), 1x Protease inhibitor cocktail set I (EMD Millipore). Iodoacetamide was added to inhibit a wide class of deubiquitinating enzymes. The extract was centrifuged at 13,000 x g for 30 min at 4 C and protein concentration was determined by Bradford assay (BioRad). Mass spectrometry: Protein denaturation was achieved by 2,2,2-trifluoroethanol (TFE) addition to 100 µg of protein at final concentration of 50 %. Cysteine residues were reduced by 15 mM DTT for 45 min at 55 C and alkylated by 55 mM IAM in the dark at room temperature for 30 min. Samples were diluted with 50 mM Tris-HCl pH 8.0 to reach 5 % of TFE. Trypsin was added to a final concentration of 1:50 w/w enzyme:protein and mixture were incubated for 16 h at 37 C. Reaction was stopped with 1 % (v/v) formic acid (FA), desalted using HyperSep Spin Tip C18 (Thermo), lyophilized and resuspended in 5 % acetonitrile (ACN), 0.1 % FA. Ubiquitinated proteins were isolated from cell lysate by TUBE2 (Tandem Ubiquitin Binding Entities - LifeSensors) affinity purification according to manufacturer’s protocol. Briefly, 1 mg of protein was incubated with TUBE-agarose beads for 2 h at 4 C under agitation. The beads were washed 5 times before elution. To covalently label oxidized proteins, 280 µg of total protein were diluted in 100 mM MES-buffered saline pH 4.7, and EZ-link hydrazide-biotin (Thermo) was added to a final concentration of 5 mM. Samples were incubated at room temperature for one hour. Unreacted biotin-hydrazide was removed and buffer was exchanged by dialysis (Slide-A-Lyzer MINI dialysis devices, 10K MWCO, Thermo) into dialysis buffer (50 mM Tris HCl pH 7.5, 50 mM NaCl) overnight. To isolate biotinylated proteins, dialyzed lysate was incubated with T1 Streptavidin Dynabeads (Invitrogen) for 1 hour at 4C with agitation followed by three washes in TBS-T buffer. For mass spectrometry analysis, samples were eluted in elution buffer (8M urea, 50 mM Tris HCl pH 8.0) at room temperature twice for 20 minutes with agitation. Samples were digested for 3 hours with 2 µg of trypsin/LysC (Promega) at 37C. Samples were diluted with 50 mM Tris HCl pH 8.0 such that [urea] = 0.72 M and were allowed to continue digesting at 37C overnight. Tryptic digestion was halted by adding 1% (v/v) FA. Samples were lyophilized to ~20 µl, re-suspended in 150 µl buffer C (95% H2O, 5% acetonitrile, 0.01% formic acid), and washed using a HyperSep C18 Spin Tip (Thermo). The eluted sample was again lyophilized and resuspended in buffer C.PrideSILACubiquitinylated lysineiodoacetamide derivatized residuemonohydroxylated residueThe output RAW data was processed using the MaxQuant suite {Cox, 2008 #1819} to detect, assign, sequence and quantify the proteins abundance assisted by Andromeda search engine. The data was searched against the yeast Saccharomyces cerevisiae database (Uniprot). Protein identification was performed at 20 ppm tolerance at the FT-mass analyzer and 6 ppm at the IT-MS, with a posterior global false discovery rate at 1 % based on the reverse sequence of the FASTA file (Uniprot). Two trypsin missed cleavages sites were allowed, oxidation of methionine was searched as variable post-translational modification and cysteine carbamidomethylation as fixed. Only proteins with more than one peptide were considered for further analysis and for the core dataset (oxidation/ubiquitinated/expression), a given protein had to be present in all biological replicates.ProteomicsGustavo SilvaLTQ Orbitrap EliteDepartment of Biology, Duke University, USAPARTIALSaccharomyces Cerevisiae (baker's Yeast)gustavo.silva@duke.edu31482721 Manohar S, Jacob S, Wang J, Wiechecki KA, Koh HWL, Simões V, Choi H, Vogel C, Silva GM. Polyubiquitin Chains Linked by Lysine Residue 48 (K48) Selectively Target Oxidized Proteins In Vivo. Antioxid Redox Signal. 2019 31(15):1133-1149 10.1089/ars.2019.7826Duke UniversityUnited States<b><i>Aims:</i></b> Ubiquitin is a highly conserved protein modifier that heavily accumulates during the oxidative stress response. Here, we investigated the role of the ubiquitination system, particularly at the linkage level, in the degradation of oxidized proteins. The function of ubiquitin in the removal of oxidized proteins remains elusive because of the wide range of potential targets and different roles that polyubiquitin chains play. Therefore, we describe in detail the dynamics of the K48 ubiquitin response as the canonical signal for protein degradation. We identified ubiquitin targets and defined the relationship between protein ubiquitination and oxidation during the stress response. <b><i>Results:</i></b> Combining oxidized protein isolation, linkage-specific ubiquitination screens, and quantitative proteomics, we found that K48 ubiquitin accumulated at both the early and late phases of the stress response. We further showed that a fraction of oxidized proteins are conjugated with K48 ubiquitin. We identified ∼750 ubiquitinated proteins and ∼400 oxidized proteins that were modified during oxidative stress, and around half of which contain both modifications. These proteins were highly abundant and function in translation and energy metabolism. <b><i>Innovation and Conclusion:</i></b> Our work showed for the first time that K48 ubiquitin modifies a large fraction of oxidized proteins, demonstrating that oxidized proteins can be targeted by the ubiquitin/proteasome system. We suggest that oxidized proteins that rapidly accumulate during stress are subsequently ubiquitinated and degraded during the late phase of the response. This delay between oxidation and ubiquitination may be necessary for reprogramming protein dynamics, restoring proteostasis, and resuming cell growth.Polyubiquitin Chains Linked by Lysine Residue 48 (K48) Selectively Target Oxidized Proteins <i>In Vivo</i>.Manohar Sandhya S, Jacob Samson S, Wang Jade J, Wiechecki Keira A KA, Koh Hiromi W L HWL, Simões Vanessa V, Choi Hyungwon H, Vogel Christine C, Silva Gustavo M GMsodium salt, Water, perfluoroethylene, ammonium formate, 13C-labeled, PLIP, cadmium salt, MeCN, TFE, BOUND WATER, magnesium formate, Basodexan, monosomy 2q32, Apaf-1, oxidane, zinc salt, Strepavidin, Type 2A-interacting protein, WATER, l(2)k09905, imperforate anus with hand, HOH, 5730420M11Rik, dmTAF[[II]]230, protein polypeptide chains, sal1, hydrogen chloride, 5-[(3aS, cobalt(II) formate dihydrate, Biotin, CH3-C#N, B1, HCL-C, 1, Urea, 2, Analysis, Ubiquitinated, HTATIP, nup154, DmelCG8428, SET, Spin, C79325, TFIID TAF250, hac-1, Analyses, cel, phosphatase 2A inhibitor I2PP2A, weight-weight percentage, cis-Hexahydro-2-oxo-1H-thieno(3, Denaturations, ITF-2b, proteins, foot, DmelCG4299, AI047805, salt, set, SPIN, Wasserstoffchlorid, decreased, C1 4B, HSAL1, cPLA2, h, scientific observation, SEF-2, BTKAP1, anatomical tube, ACN, 6-anhydro-alpha-L-galactopyranosyl-(1->3)-beta-D-galactopyranan, D1, Acn, sample, monosomy 2q32-q33, Half Cystine, F2C=CF2, TIP, GTFII-I, nickel salt, 6030441I21Rik, sal-1, dTAF[[II]]230, Rombellin, Zinc Cysteinate, cobalt (+2) salt, anon-53Fa, TAF200, Ethene, l(2)SH0173, BAP135, tip, ANAC091, Spectrum Analysis, Expanded, (3aS-(3aalpha, Putative MAPK-activating protein PM10, H2O, common salt, tetrafluoro-, sodium (4:1:1) salt, Dm1, magnesium salt, [4)-3, SATB2-associated syndrome, Sepharose C1, Del(2)(q32), Chlorwasserstoff, BAP-135, TIP60, leukemic reticuloendotheliosis, Dialysis, CG6829, Townes-Brocks syndrome, dark/hac-1/dapaf-1, 1H-Thieno(3, perfluoroethene, 2q32q33 microdeletion syndrome, HLA-DR-associated protein II, DI-2, I-2Dm, formate, Spectrometry, cromium (+3), inhibitor of granzyme A-activated DNase, GtfII-I, lysed material, study protocol, beta Trypsin, w/w, I-2PP1, Speed, TAF-IBETA, l(2)W5, Taf250, duct, 5730422P05Rik, ammonium (4:1) salt, TAF-Ibeta, methyl cyanide, chlorane, lead (+2) salt, tlp, TIP41, D-(+)-biotin, Biokur, TAF230, Deacura, nickel formate dihydrate, lead formate, TBS1, chlorure d'hydrogene, aluminum salt, Medebiotin, Carbamide, Deubiquitinating Enzyme, protein-containing complex, Chalk, CG7826, ESA1, agua, C2F4, SATB2 syndrome, LEMD5, SEF2-1, renal-ear-anal-radial syndrome, CG7835, Gene Products, CG42273, Half-Cystine, Dialyses, Carmol, renal-Ear-anal-radial syndrome, Gelfert, ZNF794, Biotin Hermes, dTAF[[II]]250, Acinus, 2310061K05Rik, Hydrogen chloride, thallium (+1) salt, cell, L Cysteine, homopolymer, beta-Trypsin, rubidium salt, Fluoroplast, 2pp2a, methanoic acid, study assay, CG10574, Sal-1, acinusL, with hand, dTAF250, 4-d]imidazole-4-valeric acid, Enzyme, DmelCG4579, ms(2)zk, hairy cell leukemia, 2PP2A, acinusS, Kochsalz, dSET, dSet, tetrafluoroethene, Ptc1, ASP-I2, PTHB1, WBSCR6, measuring, dark/dapaf-1/hac-1, mass percentage, 1-(14)C-labeled, dApaf-1, BcDNA:LD21772, Proteins, Expanded PTFE, Roche, Deubiquitinase, BG:DS00004.13, chloridohydrogen, potassium formate, Mischung, copper (+2) salt, acetonitrile, buffer, ME2, deafness, Cell, mKIAA0670, bnch, dTAF230, l(2)k02511, 6aR)-2-oxohexahydro-1H-thieno[3, APAF1, native protein, Temperatures, Natriumchlorid, I-2PP2A, Hac-1, carbonyldiamide, C18, cupric formate, Dm I-2, 4-d]imidazol-4-yl]pentanoic acid, Ptch, TAF[[II]]250/230, NaCl, aluminum formate, Perfluoraethylen, MES, dJ142L7.2, Mass Spectrum Analyses, Taf[[II]]250, AU020952, Politef, lithium formate, Gabunat, underdeveloped, GORE TEX, CG4579, Dark/Dapaf-1/HAC1, Townes-Brocks syndrome 1, ethanenitrile, (1->4)-3, 4)imidazole-4-valeric acid, mes, ME-IV, plan specification, Gene Proteins, Protein Denaturations, PTFE, bHLHb19, [OH2], hydrochloric acid, MITF-2B, MITF-2A, Sta, STA, Hac-1/Dark, DIWS, Nup32D, IPP2A2, 2-tetrafluoroethylene, Meth, NCMe, vitamin B7, DmelCG6829, Sepharose C1 4B, classic hairy cell leukaemia, zinc formate, lead salt, Hydrogenchlorid, protein, GORE-TEX, extracted material, A230106A15Rik, Teflon, halite, ammonium tetraformate, BANF, dJ69E11.3, Min, Williams-Beuren syndrome chromosomal region 6 protein, 6aR)-Hexahydro-2-oxo-1H-thieno[3, Apaf1, Tetrafluoraethylen, protein aggregate, monosomy 2q32q33, present in fewer numbers in organism, ARK, DmelCG42273, Mass Spectrum Analysis, Sepharose, TAF-I, E927b, 2610036I19Rik, (3aS, 2610510L13Rik, hypoplasia, biotina, min, arc, mAPC, EDMD, biotine, SEF2, BIOTIN, copper, ark, T1, imperforate, cloruro sodico, IGAAD, Fluon, reaction, fSAP152, chromosome 2q32-q33 deletion syndrome, lithium salt, Hermes, DmelCG10574, Ice, Diws1t, dihydridooxygen, MKS, dApaf1, biotinum, dihydrogen oxide, buffer., ammonium (2:1) salt, Deubiquitinating, phapii, Medobiotin, Tube, rock salt, wide/broad, D-Apaf-1, ZC2HC5, 4B, aqua, Dmel_CG7826, StF-IT-1, HTATIP1, TAFII-250, classic hairy cell leukemia, TAF250/230, ur, 4S, 4-d)imidazole-4-pentanoic acid, Spectroscopy, with imperforate anus and hypoplastic thumbs, glass, any method, TUBE, carbamide, TAFII250, tetrafluoroethylene, HCL, SRF-Phox1-interacting protein, Biotin Ratiopharm, BBS13, Goretex, 4-d)imidazoline-4-valeric acid, nickel (+2) salt, Dmel_CG7835, Ptc, D-Biotin, Crystal, Mnb, MNB, l(2R)W5, and Ear anomalies, ammonium salt, Biotine Roche, Tetrafluoraethen, znf794, Deubiquitinases, HCl, E2-2, cobaltous formate, hydrogen hydroxide, Del(2)(q32q33), L-Cysteine, CG4299, hac1, sall1a, AW124434, CG17603, natrii chloridum, TAF[[II]], acqua, Townes syndrome, dapaf-1S, wide, H2NC(O)NH2, apaf-1, dapaf-1L, WBS, SR3-5, Acetamide, copper salt, Sepharose 4B, CG8428, cis-(+)-Tetrahydro-2-oxothieno[3, Wasser, i2pp2a, 1728, 4-d]imidazol-4-yl)pentanoic acid, d230, Hydrazide, Cysteine Hydrochloride, Dapaf-1/HAC-1, Biotine, Psychomotor agitation, cesium salt, Gene, dTAFII250, broad, Spectrum Analyses, ACINUS, Vitamin H, foot and ear anomalies, EfW1, TFII-I, PHAPII, Buffer, formic acid, Bruton tyrosine kinase-associated protein 135, Dark/Hac-1/dApaf1, Karbamid, method, Hac1, potassium salt, Dark/Hac-1/dApaf-1, template-activating factor I, polypeptide chain, reduced, dmTAF1, glass syndrome, Taf230, subnumerary, 14C-labeled, Arabidopsis NAC domain containing protein 91, method used in an experiment, Polytef, 4beta, Harnstoff, Tina, Mass, E2.2, zk, IB291, ITF2, tiny, AA673430, 6aalpha))-, cloruro de hidrogeno, Mass Spectroscopy, Pro-Mega, TAF250, Taf200, l(2)10432, Glass, Coenzyme R, ITF-2, ipp2a2, Tcf-4, dapaf-1, chlorure de sodium, Taf1p, dapaf, dark, eau, decreased number, calcium formate, lysate, DARK, taf-ibeta, HEL-S-89, (+)-cis-Hexahydro-2-oxo-1H-thieno[3, Agarose, FEP, Tarflen, cromium (+3) salt, BTK-associated protein 135, AA553322, l(2)01501, TAF, dArk, Dapaf-1, Renal dialysis, 6-An-alpha-L-Galp-(1->3)-beta-D-Galp-(1->]n, 2-iodo-, sodium formate, sensorineural, small, 2q32-q33 microdeletion syndrome, DYRK1, TAF[[II]]250, nickel formate, 2q32q33 microdeletion syndromes, 3H-labeled, protein complex, sensorineural deafness with imperforate anus and hypoplastic thumbs, igaad, strontium formate, rear syndrome, l(3)84Ab, Biotin Gelfert, apaf1, strontium salt, 5-(2-oxohexahydro-1H-thieno[3, 4]imidazole-4-valeric acid, Enzymes, with imperforate anus and thumb anomalies, [HCl], TBS, Denaturation, cis-Tetrahydro-2-oxothieno(3, anus, 4]imidazoline-4-valeric acid, biotin, natural protein, p230, water, Protein, I2PP2A, AI551343, Hydrochloride, TFIID, table salt, Dyrk1, 25/11, Dark, Dark/Apaf-I, ACETONITRILE, cyanomethane, Biodermatin, Mass Spectrum, Biotin-Ratiopharm, AW550900, SAS, tbs, nup32D, TAF[[II]]230, POC12, uree, urea, CC1, Tripcellim, hexahydro-2-oxo-, TAF[II]250, dApaf-1/DARK/HAC-1, chromic formate, sample population, UREA, l(2)10403, Protein Gene Products, Trypure, dSET/TAF-Ibeta, 2610030F17Rik, DmelCG17603, concentration, Townes-Brocks-branchiootorenal-like syndrome, calcium salt, TCV-interacting protein, AA407739, SCH, dAPAF-1, TAF1, sodium chlorideUbiquitin, Protein Gene Products, Gene Proteins, protein polyubiquitinylation, Protein., polyubiquitin, Protein, Gene Products, Proteins, Gene, protein polyubiquitylation, proteins, poly-Bru, cox4, posttranslational modification, 2-amino-4-(methylsulfanyl)butanoic acid, Cysteine Hydrochloride, Raw, determination, False, nucleocapsid, Brewer's, L Isomer, Gene, baker's yeast, protein, protein-containing complex, Saccharomyces italicus, peptide, CG10664, Polypeptides, protein polypeptide chains, L-Isomer Methionine, Methionine, ClvPrd, peptido, polypeptide chain, posterior, IV, Gene Products, L-Methionine, Del(8)44H, Half-Cystine, DmelCG10664, protein aggregate, Pedameth, Svc, present in organism., Search Engines, proportion, posttranslational amino acid modification, Coproporphyrinogenase, F, peptides, COXIV, M, L Cysteine, beta-Trypsin, proteins, Baker, analyzer, posttranslational protein modification, Saccharomyces uvarum var. melibiosus, COXA_DROME, S. cerevisiae, S cerevisiae, Candida robusta, 2-amino-4-(methylthio)butanoic acid, Cox4, 10^[-6], Saccharomyces capensis, ppm, COX, Baker's, peptidos, 1.3.3.3, Mycoderma cerevisiae, Half Cystine, brewer's yeast, metionina, COX-VA, Racemethionine, posterior end of organism, ratio, data, Data Set, Saccharomyces cerevisiae 'var. diastaticus', DL-Methionine, Zinc Cysteinate, protein complex, Proteins, 2-Amino-4-(methylthio)butyric acid, Search, L-Isomer, Peptide, Engine, Baker's Yeasts, polypeptide, native protein, natural protein, Saccharomyces diastaticus, VA, AL024441, Protein, chemical analysis, sequence, core, IV-1, Methionin, methionine, Hmet, COX IV, fixed, Data Base, Met, Saccharomyces oviformis, Yeast, Col4a-1, PTM, Va, Coprogen oxidase, COX IV-1, tend, proportionality, Tripcellim, rate, L-Cysteine, beta Trypsin, primary structure of sequence macromolecule, post-translational amino acid modification, Protein Gene Products, Gene Proteins, Trypure, CG14724, post-translational modification, quotient, alpha-amino-gamma-methylmercaptobutyric acid, Peptid, DmelCG14724, Polypeptide, assay, Baker's Yeast, variable, Liquimeth, Brewer's Yeast, Cox4a, Baker Yeastmulticellular organismal catabolic process, single-organism catabolic process, protein translation, advanced, Plays, Peptidomics, polyubiquitin, Metabolisms, number, Gene, protein breakdown, Bioenergetics, Protein Homeostasis, ATP Dependent Proteolysis Factor 1, protein, Ubiquitin carboxyl extension protein 80, protein-containing complex, cellular protein breakdown, presence, body system, temporal, Human, protein polypeptide chains, polypeptide chain, protein polyubiquitinylation, protein amino acid oxidation, Metabolism, responsivity, Gene Products, HMG-20, system, DmF2, ubiquitin-like protein modifier, Expenditure, protein aggregate, Fs(3)Hor, Ubiquitinated, protein degradation, Toy, lod, reactivity, DmelCG2684, Ubiquitylation, anatomical systems, pheromone catabolism, High Mobility Protein 20, Playthings, growth of cell, catabolism, non-developmental growth of a unicellular organism, Ubiquitin-related 1, Energy, Homeostases, proteins, protein polyubiquitylation, metabolic process resulting in cell growth, number of, NTef2, Expenditures, cellular protein catabolism, protein catabolism, Proteostases, APF-1, 40S ribosomal protein S27a, Puppets, cellular protein degradation, ubiquitin, has or lacks parts of type, cellular growth, Play, 26S proteasome, ubiquitination, protein tagging activity, Puppet, incidence, accumulated, protein anabolism, protein biosynthetic process, degradation, protein complex, Proteins, Protein Homeostases, late, function, extra or missing physical or functional parts, CEP52, Fs(3)Sz11, mereological quality, cell expansion, count in organism, Ubiquitin A-52 residue ribosomal protein fusion product 1, native protein, natural protein, Playthings and Play, Ubiquitin-related 2, non-developmental cell growth, Protein, precocious, protein formation, Plaything, protein biosynthesis, ATP-Dependent Proteolysis Factor 1, connected anatomical system, metabolism resulting in cell growth., Ubiq, poly-, Energy Expenditure, Ubiquitin, proteasome, covalent modifier, pheromone catabolic process, breakdown, Homeostasis, Human Ubiquitin, Toys, Energy Expenditures, Lds, early, Bioenergetic, Protein Gene Products, organ system, Gene Proteins, Energy Metabolisms, multicellular organismal protein catabolic process, 60S ribosomal protein L40, Eukaryota protein, protein synthesis, protein tag, Horka, cellular protein catabolic process, cardinality, CG2684, Fs(3)Horka, Ubiquitin-related, response, quantitative, presence or absence in organismDNA Oxidative, multicellular organismal catabolic process, single-organism catabolic process, protein translation, polyubiquitin, Metabolisms, Mbp1, Nitrative Stress, protein breakdown, Bioenergetics, protein, Damage, Long Term, protein polypeptide chains, Oxidative DNA, Oxidative, Roles, protein polyubiquitinylation, protein ubiquitylation, responsivity, Nitro-Oxidative Stress, Concepts, Nitro-Oxidative Stresses, Expenditure, protein aggregate, Oxidative Injury, Fs(3)Hor, myd, Ubiquitinated, Effect, DNA Damage, protein degradation, Oxidative Injuries, DmelCG2684, Ubiquitylation, High Mobility Protein 20, catabolism, non-developmental growth of a unicellular organism, Oxidative Cleavage, Ubiquitin-related 1, Mbp-1, Homeostases, proteins, metabolic process resulting in cell growth, W, Oxidative DNA Damages, NTef2, Anti-oxidative, APF-1, Oxidative Stress Injuries, Oxidative Stresses, cellular protein degradation, Role Concepts, cellular growth, 26S proteasome, ubiquitination, Long-Term Effects, accumulated, protein anabolism, protein biosynthetic process, wide/broad, gyltl1b-b, Longterm Effect, late, CEP52, results, Fs(3)Sz11, Role Concept, Oxidative and Nitrosative Stress, MDDGA6, mKIAA0609, non-developmental cell growth, Role, precocious, protein formation, DNA Oxidative Damages, metabolism resulting in cell growth., Ubiq, Ubiquitin, fg, proteasome, breakdown, gyltl1b, mdc1d, Human Ubiquitin, expanded, early, Nitro-Oxidative, organ system, MDC1D, wide, Energy Metabolisms, multicellular organismal protein catabolic process, enr, protein synthesis, enlarged, Horka, CG2684, Fs(3)Horka, Oxidative Stress Injury, disease characteristic, DNA, time, Oxidative Damage, big, Oxidative Stress, advanced, Peptidomics, Effects, Antioxidative, number, Antioxidative Stress, Gene, Protein Homeostasis, ATP Dependent Proteolysis Factor 1, Ubiquitin carboxyl extension protein 80, broad, protein-containing complex, body system, cellular protein breakdown, presence, froggy, Gyltl1a, Human, Stresses, period, large, Oxidative Nitrative, polypeptide chain, Stress Injury, Metabolism, Gene Products, Oxidative Cleavages, HMG-20, system, DmF2, ubiquitin-like protein modifier, qualifier, Oxidative Damages, lod, reactivity, Injury, anatomical systems, pheromone catabolism, growth of cell, Longterm, MDDGB6, Energy, protein polyubiquitylation, Long-Term, Expenditures, cellular protein catabolism, protein catabolism, Proteostases, protein ubiquitinylation, BPFD#36, 40S ribosomal protein S27a, Anti oxidative Stress, Oxidative DNA Damage, Antioxidative Stresses, great, ubiquitin, Long-Term Effect, protein tagging activity, modifier, degradation, protein complex, Proteins, Protein Homeostases, function, Anti-oxidative Stresses, Qualifier, Concept, cell expansion, Oxidative Nitrative Stress, count in organism, Ubiquitin A-52 residue ribosomal protein fusion product 1, native protein, natural protein, Ubiquitin-related 2, Oxidative Nitrative Stresses, Protein, Long Term Effects, protein biosynthesis, ATP-Dependent Proteolysis Factor 1, connected anatomical system, disease qualifier, poly-, Energy Expenditure, covalent modifier, pheromone catabolic process, Anti-oxidative Stress, Homeostasis, DNA Oxidative Damage, Energy Expenditures, Cleavage, Lds, Modifier, Bioenergetic, Longterm Effects, Protein Gene Products, Gene Proteins, 60S ribosomal protein L40, protein tag, Stress, cellular protein catabolic process, Ubiquitin-related, response, quantitative, Nitro Oxidative Stress, presence or absence in organismUbiquitin, covalent modifier, High Mobility Protein 20, Protein., Proteins, Human Ubiquitin, Ubiquitin-related 1, Gene, ATP Dependent Proteolysis Factor 1, proteins, Ubiquitin carboxyl extension protein 80, CEP52, Human, Protein Gene Products, APF-1, Gene Proteins, 40S ribosomal protein S27a, Ubiquitin A-52 residue ribosomal protein fusion product 1, 60S ribosomal protein L40, protein tag, Ubiquitin-related 2, ubiquitin, Protein, Gene Products, HMG-20, Ubiquitin-related, ATP-Dependent Proteolysis Factor 1, ubiquitin-like protein modifier, protein tagging activity, UbiqfalseK48 ubiquitin selectively targets oxidized proteins in vivoUbiquitin is a highly conserved eukaryotic protein responsible for regulating a variety of functions when conjugated to target proteins. Ubiquitin is essential, accumulates during the stress response, and is the canonical signal for protein degradation by the proteasome. Still, the function of ubiquitin in response to oxidative stress, particularly in the removal of oxidized proteins remains elusive because of the number of potential targets and different roles that polyubiquitin chains can play. Since polyubiquitin chains linked by K48 ubiquitin are the most abundant and canonical signal for protein degradation, we investigated the roles of K48 ubiquitin in the degradation of oxidized proteins. Combining protein oxidation, linkage-specific ubiquitination screens, and quantitative proteomics, we found K48 ubiquitin accumulated in a bimodal fashion, at both the early and late phase of response. We further showed that a fraction of oxidized proteins are conjugated with K48 ubiquitin in vivo. Using quantitative proteomics we identified ~750 ubiquitinated proteins and ~400 oxidized proteins that were differentially modified during the stress response, and around half the proteins were both oxidized and ubiquitinated. These proteins were highly abundant and function in translation and energy metabolism. Finally, we showed that the ubiquitination system is required for degradation of oxidized proteins, suggesting that oxidized proteins that rapidly accumulate during stress are ubiquitinated, and degraded during the later recovery phase. This temporal disconnect may be necessary for reprogramming protein dynamics, restoring proteostasis, and resuming cell growth.2021-04-302019-06-07PXD01417344664499175582580388043330295358511145120678110806124619624619728421880903206375811363350906836351524260275838538791187947398007NCBITaxon:12805723071114301555714683514526236491508472746707364113546138858035823290313888691568895399784475187043511232946928123977133103430436548135833925198183659219813143205965581914936329572637371922226938124781866132838828139558434513586722294914081691804541619341390363453058611204162326442104117061274432108595614963674283166694369455972945281132081003923751463308793694478420896431744712704459741194669400667145458793299352932010198126469041066114575512165994048339251216597216595160621213312831282128033952321832172920435315228112454349825747874980197525555362312951053948824261914548140479NCBITaxon:21575515343989903712059687623193516309800588845581817808173630943179698936160488566140985956641624251505907118702118705320496306386323555432046632226186633129945658606884535121612956717136044100160235870026420369456941016763410210761277351279334281461002935786174633132529111870021576418096997457744491566667547604452467675663366411363367102398709NCBITaxon:4055932582359092788396526498217498216347256569156934982116544872056971089448663661410NCBITaxon:49328715568910245980810005894526808732311561263730391141554219325552187268723872487298727123598755529547624223141463055180066556484198822299767982510216998231174673897641041051155744689444474153NCBITaxon:5055796016776744685960065349118226370824812991145784586326547876569835566369838192011164034624136810893161001428099612NCBITaxon:548681NCBITaxon:582054641430774794640986099586522831966272905876689605960615729513722187594467087562270861711013354833090349741387834306154319474703342499859961588655974649987039202111072878272154078211908570752061894804189853502471587796271902528674636096469644375939146247274612874601134506253452659197221963852638588596NCBITaxon:61575052110517237263419610526419407722774694163481123869NCBITaxon:27036125061747679274596153840052573131368626979699034694171344346360347515363296440421018510976063314961612862879669968583558595968912801726071018568729411009118696141633316780781186941436183NCBITaxon:6191118698840231719118691443906NCBITaxon:1029821000712571189694836419080218787870384766455347095145122578656870861822607046183260705260707NCBITaxon:780NCBITaxon:1502451516658457702921158612184520928574707856100761921263854504457383379192875NCBITaxon:10239NCBITaxon:71428291268063130957044830064175158510555241590841228233446900815025754122810430085210977970671356225833136185509924NCBITaxon:5693826883064052779617946995833424090615101515NCBITaxon:631240674482428130767070876138370828167728503515819901195195337024924053608881756261583558333760212671928532177357267119350184645177217743737111049056371215348112153239576769014726647091571256274579031133355085962859634792491308126387121203511082332648373153214693NCBITaxon:961531858614126220029813144937608333411149703965583332101841385691006581237561275045426662551519788749200375036009412934971530094114903524725175014012573983649908515151438992NCBITaxon:11552374539883747445446415259311792866432359839062887051580119459932716011497861603293338922853422999810894535936457428412754135122511775058NCBITaxon:1040750277916797181673871349158910306502780291595412629158515849327159570210022612876891355889309451464792031242164133280382803519284343985808946210803481793921182590287889295027906901749342725632273218903829286757221247190400727NCBITaxon:5796537457114311233521140206411981087135547714245072295333555714154293157546224213921390114319338293550833292307411292211484896160106772526499975744727580240550717629936658121405357622941281607839576320667240054029654318431832246596205755499229575757592845933079725518113549706671436733115955644421432138184922311240607210969762261111795752981818431921949633347986626528104660104421454747962678253567097477317513565050NCBITaxon:13136076993894246887955201488751979596214114082241408223421972890922853164351867631854315206494089414424232NCBITaxon:17732140921157789297932447042075591011774990674990731298991311227323166317018736745101161035899151328426265872103609833413926961130798133679535889316407103401530333733015819130641399253068839103372123368199404458644981153571147161224911131316634305312017554993512993850573176669642688451513458421572678728623862111510416079129831539222746891198351125522856253232660469136167633631689704132888188551167610807723264248618140843439376520965731116234273068808637721666898626NCBITaxon:2697049339862410312NCBITaxon:138305959997586448643319729724629176343863725673786391233435105231100905141405449317998686554055348654514712454667605681042986357124679151641884019373345087711982159739757429719133734275779850762554702563181313686571303443623951655865886223864946454634076NCBITaxon:1107128835649184588NCBITaxon:592011087439145943267671111678107740844087977895349679440812269003803943512814595361235178616243230399662356128695978540964097954186942003089544243277869762791292499545115574840905180426430409286924093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