Activity of Ubiquitin-dependent Pathway in Response to Oxidative Stress

• DOI: 10.1074/jbc.272.37.23086
• Publication Date: 2002-07-26T14:48:08Z
• Authors: Fu Shang; Xin Gong; Allen Taylor
• Keywords: 0303 health sciences; Ubiquitin-Protein Ligases; Hydrogen Peroxide; Ubiquitin-Activating Enzymes; Epithelium; Ligases; Oxidative Stress; 03 medical and health sciences; Adenosine Triphosphate; Lens, Crystalline; Ubiquitin-Conjugating Enzymes; Animals; Cattle; RNA, Messenger; Oxidation-Reduction; Ubiquitins; Cells, Cultured; Signal Transduction

Relations between the ubiquitin pathway and cellular stress have been noted, but data regarding responses of to oxidative are scanty. This paper documents response this in lens cells. A brief exposure epithelial cells physiologically relevant levels H2O2 induces a transient increase activity ubiquitin-dependent pathway. Ubiquitin conjugation was maximal increased 3.5–9.2-fold over noted untreated by 4 h after removal H2O2. By 24 H2O2, returned level In parallel changes activity, ubiquitin-activating enzyme (E1), as determined thiol ester formation, 2–6.7-fold during recovery from oxidation. Addition exogenous E1 resulted an carrier protein (E2)-ubiquitin esters both H2O2-treated These suggest that is rate-limiting process increases which induced upon oxidation primarily due activity. The oxidation- recovery-induced up-regulation post-synthetic events. Substrate availability E2 activities also appear be related enhancement ubiquitinylation stress. oxidation-induced were associated with intracellular proteolysis, suggesting may play role damaged proteins lens, first line defense against crucial roles maintenance entire organ. Oxidation major its constant light oxidants (1Varma S.D. Chand D. Sharma Y.R. Kuck Jr., J.F. Richards R.D. Curr. Eye Res. 1984; 3: 35-57Crossref PubMed Scopus (278) Google Scholar, 2Spector A. FASEB J. 1995; 9: 1173-1182Crossref (798) 3Taylor Davies K.L.A. Free Radical Biol. Med. 1987; 371-377Crossref (144) 4Taylor Jacques P.F. Dorey C.K. Toxicol. Ind. Health. 1993; 349-371Crossref (50) Scholar). (100–300 μm) has detected using different methods (5Devamanoharan P.S. Ramachandran S. Varma 1991; 10: 831-838Crossref (13) 6Devamanoharan, P. S., Varma, (1995)Ophthalmic , 27, (suppl.), 39–43.Google addition, aqueous humor (the fluid receives nutriture) normal (0.03 mm) or cataract patients (0.08–0.19 various laboratories (7Spector Garner W.H. Exp. 1981; 33: 673-681Crossref (449) 8Giblin F.J. McCready J.P. Kadama T. Reddy V.N. 38: 87-93Crossref (126) 9Ramachandran Morris S.M. Devamanoharan Henei M. 53: 503-506Crossref (62) Oxidation-induced damage includes bulk oxidation, inactivation some key enzymes, DNA breaks, lipid peroxidation (3Taylor 10Blondin Baragi V. Schwartz E. Sadowski J.A. Taylor Radicals 1986; 2: 275-281Crossref (65) 11Blondin Mech. Ageing Dev. 41: 39-46Crossref (32) 12Reddan J.R. Sevilla M.D. Giblin Padgaonkar Dziedzic D.C. Leverenz Misra I.C. Peters J.L. 56: 543-554Crossref (66) 13Spector Kleiman N.J. Huang R.R.C. Wang R.R. 1989; 49: 685-698Crossref (44) 14Spector G.-M. R.-R. Moll H. 12: 163-179Crossref (117) Lens antioxidant systems repair mechanisms ameliorate insult. one putative mechanisms. It participates selective obsolete (15Finley Ozkaynak Varshavsky Cell. 48: 1035-1046Abstract Full Text PDF (634) 16Rechsteiner 66: 615-618Abstract (205) 17Johnson E.S. Bartel B. Seufert W. EMBO 1992; 11: 497-505Crossref (213) 18Huang L.L. Shang F. Nowell T.R. 61: 45-54Crossref 19Shang Biochem. 307: 297-303Crossref (140) 20Spence Sadis Haas A.L. Finley Mol. 15: 1265-1273Crossref 21Bregman D.B. Halaban R. van Gool A.J. Henning K.A. Friedberg E.C. Warren S.L. Proc. Natl. Acad. Sci. U. 1996; 93: 11586-11590Crossref (265) yeast, required withstand (22Cheng L. Watt Piper P.W. Gen. Genet. 1994; 246: 358-362Crossref (71) Scholar) heavy metal toxicity (23Jungmann Reins H.-A. Schobert C. Jentsch Nature. 361: 369-371Crossref (235) plays regulation diverse processes, including signal transduction (24Hicke Riezman 84: 277-287Abstract (671) 25Madura K. Science. 265: 1454-1458Crossref (133) 26Palombella V.J. Rando O.J. Goldberg Maniatis 78: 773-785Abstract (1922) 27Chen Z.J. Parent 853-862Abstract (871) Scholar), cell cycle control (28Finley Ciechanover 37: 43-55Abstract (360) 29Goebl M.G. Yochem McGrath Byers 1988; 241: 1331-1335Crossref (323) differentiation (30Hondermarck Sy Bradshaw R.A. Arfin Biophys. Commun. 189: 280-288Crossref (31) malignant transformation (31Scheffner Huibregtse J.M. Howley P.M. 91: 8797-8801Crossref (236) 32Scheffner Nuber 373: 81-83Crossref (756) apoptosis (33Muller L.M. BioEssays. 17: 677-684Crossref hallmark covalent attachment form ubiquitin-protein conjugates termed ubiquitinylation. best known selectively targeting for degradation, such calmodulin, histones H2A H2B, actin, membrane receptors serves regulatory function without them cytosolic degradation (34Jennissen H.P. Eur. 231: 1-30Crossref (112) Previous studies showed culture inhibited 1 mm (19Shang Recent work laboratory indicated mild enhanced cultured lenses (35Shang Gong X. Palmer H.J. 1997; 64: 21-23Crossref (79) However, no information steps available. Multiple enzymes involved process, activated (E1) 1The abbreviations used are: E1, enzyme; E2, protein; E3, ligase; BLEC, bovine cells; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; HMW, high molecular weight; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate. via formation bond E1. then passed (E2), linked bond. either directly substrates ligase (E3). Several E2s E3s identified. diversity responsible substrate specificity work, we effect on studied We investigated Trizma (Tris-base), dithiothreitol, creatine phosphate, phosphokinase, ATP, 2-deoxyglucose, Coomassie Blue R-250, chloramine T obtained Sigma. Acrylamide,N,N′-methylene-bisacrylamide,N,N,N′,N′-tetramethylenediamine, 2-mercaptoethanol, sodium dodecyl sulfate, glycine, mass standards purchased Bio-Rad. Hexokinase Worthington, magnesium chloride Fisher. Na125I 125I-protein NEN Life Science Products. Anti-ubiquitin antibody anti-E1 produced New Zealand White rabbits injection SDS-denatured conjugated γ-globulin synthetic peptides ovalbumin. Bovine (BLEC) Dulbecco's modified Eagle's medium containing 10% (v/v) fetal serum, penicillin (100 units/ml), streptomycin μg/ml), amphotericin (250 ng/ml). maintained at 37 °C 95% air 5% CO2. Exposure performed serum- phenol red-free 0.1 mmH2O2 30 min. collected immediately H2O2-free allow recover Control treated exactly exposed except not included medium. viability H2O2was monitored exclusion trypan blue 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide staining. reduced glutathione described previously ATP bioluminescent somatic assay kit (Sigma) according manufacturer's instructions. BLEC harvested min 1, 2, homogenized 50 mmTris-HCl, pH 7.6, mixture inhibitors proteinase isopeptidase (5 EDTA, 10 μm hemin, 4-(2-aminoethyl) benzene sulfonyl fluoride, E-64, 2 μg/ml aprotinin, mmiodoacetamide). Following SDS-PAGE separation transfer nitrocellulose, blots probed affinity purified polyclonal followed incubation ubiquitin, conjugates, autoradiography quantified image analysis. Tris, mmDTT, 7.6. supernatant ability catalyze endogenous exogenous125I-labeled ubiquitin. done final volume μl, Tris-HCl, 5 MgCl2, AMP-PNP (36Johnston N.L. Cohen R.E. Biochemistry. 30: 7514-7522Crossref (34) μg 125I-ubiquitin (≈106 cpm), aldehyde, μl (10 mg protein/ml). reaction started addition supernatant. 20 min, stopped × Laemmli buffer. After boiling 100 separated 15% SDS-PAGE. For negative control, experiment replaced 4.5 units hexokinase 12 2-deoxyglucose. drying gel, it film 2–4 days. densitometry autoradiogram. activating conjugating (E2s) their property (37Ciechanover Elias Heller Hershko Chem. 1982; 257: 2537-2542Abstract 38Hershko 1983; 258: 8206-8214Abstract 39Haas A.R. Baboshina O. Williams 270: 9407-9412Abstract (119) of125I-ubiquitin buffer (50 4% SDS, 8 m urea, 6.8). standing room temperature gels. gels drying. determining density bands disappeared autoradiogram reduction β-mercaptoethanol. about 120 kDa subunit 110 kDa, 8.5 kDa). masses E2-ubiquitin ∼8.5 higher than corresponding (38Hershko concentrations assays saturating 15–30-fold unlabeled (40Jahngen J.H. Blondin Eisenhauer 261: 13760-13767Abstract Thus, competition the125I-ubiquitin negligible. Total RNA isolated Trizol reagent (41Chomczynski BioTechniques. 532-535PubMed RNAs electrophoretically 1% agarose 2% formaldehyde transferred Hybond-N+ (Amersham Corp.). cDNA human was32P-labeled Megaprime labeling Prehybridization hybridization carried out SSC (150 chloride, 15 citrate, 7.0), Denhardt's (0.02% Ficoll, 0.02% polyvinylpyrrolidone, serum albumin), 50% formamide, salmon sperm 42 °C. nonspecifically bound radiolabels removed two washes 0.2 SSC, 0.2% SDS 60 mRNA scanning normalized 28 S rRNA. contrast prior study physiological (0.1 peroxide. To assess (GSH), indicator redox status (42Giblin Reddan Schrimscher 1990; 50: 795-804Crossref (73) treatment. GSH decreased 46% treatment treatment, pretreatment remained thereafter. rapid restitution confirms active system restore protective apparatus permanently altered exposure. indicate H2O2need less time since still lower Levels another (43Schraufstatter I.U. Hinshaw Hyslop P.A. Spragg R.G. Cochrane C.G. Clin. Invest. 77: 1312-1320Crossref (436) 44Schraufstatter Sklar L.A. 83: 4908-4912Crossref (438) 45Brunk U.T. Zhang Dalen Ollinger 19: 813-822Crossref (103) (Table I). stress, ∼50%. One hour recovery, ∼30% observed But found total together staining data, more survived can H2O2.Table ILevels H2O2exposure recoveryTreatmentsATP nmol/106 (mean ± S.E.,n= 4)Control10.53 1.1530 exposure5.64 1.531-h recovery7.58 1.144-h recovery11.04 2.2324-h recovery11.89 2.09 Open table new tab varied 16 >200 (Fig. 1). More had (>200 When weight (HMW) 20–40% compared HMW-ubiquitin Upon 10–20% 1,lanes 3–5). low conjugate (45 kDa) time-dependent manner transiently decreases conjugates. possible oxidatively challenged alteration test possibility incubating 125I-labeled (an analog supports does support degradation). As shown Fig. (in ATP-dependent fashion, shown) sizes de novo formed >112 kDa. 1) novoubiquitinylation when 2) point lane 2), 30–280% 2). extent varies experiment, trend constant. Accordingly, report range three independent experiments hereafter II). Reasons discrepancy increasedde clear time. Although appeared even stressed (≈50 vastly exceed K (0.5 reticulocyte (46Haas Rose I.A. 10329-10337Abstract decrease involve temporary Both supplied assay.Table IIRanges with/without recoveryNo additional E1With E1No H2O2H2O22-aCells min.H2O2and Rec2-bCells allowed h.No h.Ubiquitin conjugates11.3–3.83.5–9.23.8–7.53.8–9.26.1–17E111.4–3.12–6.710–1810–1810–18E210.8–1.92.1–6.46.4–173.9–148.8–23The autograms. densities each these moieties designated unit. Densities H2O2-exposed recovered (Rec) given relative unit summarized five (no E1) (with experiments.2-a Cells min.2-b h. experiments. exposure, there further 2,lanes (250–820% cells) 5). 3 B). up-regulated during, from, determine if H2O2-induced E2s, extracts 47Haas Warms J.V.B. 2543-2548Abstract based process; thus, reflect (39Haas Consistent compare Table II), 40–210% H2O2(Fig. 3, A–C, significant A, lanes 3–5 100–570% B, 6 Unlike remain unchanged like 110–540% andB). 22 24.5 30.5 respectively. Since requires paralleled plausible oxidation/recovery-induced caused examine possibility, added assay. 540–1600% unstressed C, versus 1; preparations substantial 3; E2-thiol exo