Complex I within Oxidatively Stressed Bovine Heart Mitochondria Is Glutathionylated on Cys-531 and Cys-704 of the 75-kDa Subunit

• DOI: 10.1074/jbc.m803432200
• Publication Date: 2008-07-09T04:44:59Z
• Authors: Thomas R. Hurd; Raquel Requejo; Aleksandra Filipo...; Stephanie Brown; Tracy A. Prime; Alan J. Robinson; Ian M. Fearnley; Michael P. Murphy
• Keywords: Thiol; Thermus thermophilus; Glutaredoxin; Sulfenic acid

Complex I has reactive thiols on its surface that interact with the mitochondrial glutathione pool and are implicated in oxidative damage many pathologies. However, Cys residues thiol modifications involved not known. Here we investigate complex modification within oxidatively stressed mammalian mitochondria, containing physiological levels of glutaredoxin 2. In mitochondria incubated oxidant diamide, is only glutathionylated 75-kDa subunit. Of 17 subunit, 6 iron-sulfur centers, making them plausible candidates for glutathionylation. Mass spectrometry from bovine heart showed Cys-531 Cys-704 were glutathionylated. The other four non-iron-sulfur center remained as free thiols. glutathionylation also occurred response to relatively mild stress caused by increased superoxide production respiratory chain. Although correlated loss activity, it did increase formation, reversal restore activity. Comparison known structure ortholog Nqo3 Thermus thermophilus suggested I, exposed pool. 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Poland Honzatko Arch. 1995; 319: 1-9Crossref (363) 24Dalle-Donne Gagliano Giustarini 2008; 10: 445-473Crossref (249) was unclear whether intact mitochondria; known, functional significance changes uncertain show intact, subunit discuss implications these stress. ϵ-amino-n-caproic acid Blue Native coelenterazine dodecyl-β-d-maltoside N,N-bis(2 glycine dithiothreitol carbonyl 4-(trifluoromethoxy)phenylhydrazone matrix-assisted time-of-flight tandem mass N-ethylmaleimide glutathione-protein dismutase 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Materials—Rabbit antiserum against Prof. John cross-reacted rat mouse monoclonal antibody (α-GSH) Viro-Gen Corp. Complete protease inhibitor Roche Applied Science. [35S]GSH (942.0 Ci/mmol) PerkinElmer Life Sciences, (DTT) removed prior experiments extraction ethyl acetate. His-tagged Grx2 expressed described biotinylated ester Invitrogen. Rabbit human sarcomeric creatine kinase AbCam enzyme. Preparation Mitochondria, Membranes, I— Bovine prepared (25Cocheme H.M. 283: 1786-1798Abstract (417) Heart preparations stored ice 1–7 h preparation over which time well coupled. membranes disruption blender (26Smith A.L. Methods Enzymol. 1967; 81-86Crossref (466) followed isolation centrifugation (27Walker Buchanan 260: 14-34Crossref (88) solubilization dodecyl β-d-maltoside (DDM, Anatrace, OH) ion-exchange chromatography (28Sharpley M.S. Draghi 45: 241-248Crossref (161) Pooled fractions further purified gel filtration, buffer 0.1% DDM, 1 mm DTT, 10% glycerol -80 °C. Immediately experiments, replaced one lacking DTT Micro Bio-Spin column. Samples without gave results indistinguishable samples DTT. Assays—GSH, protein-glutathione disulfides (PrSSG) assayed using assay adapted 96-well plate reader (29Anderson 1985; 113: 548-555Crossref (2380) 30Scarlett Packer M.A. Porteous C.M. Pharmacol. 1996; 52: 1047-1055Crossref (72) 31Akerboom T.P.M. Sies 1981; 373-382Crossref (1442) Two-dimensional (BN)-PAGE—For BN-PAGE (32Schagger 190-202Crossref (132) 33Schagger von Jagow Anal. 1991; 199: 223-231Crossref (1881) (0.5 mg protein) treated 50 NEM 5 min, pelleted, resuspended 60 μl buffer: 1% 0.75 m (ACA), BisTris-HCl, pH 7.0 4 After incubation 15 suspension clarified an Airfuge™ (Beckman Coulter) p.s.i. (∼100 000 × g) min. Then 3.5 sample buffer, 5% (w/v) Coomassie G 250 (Serva, Germany) 500 ACA, added, resolved 1-mm-thick 5–12% acrylamide gradient 0–20% glycerol, 0.5 (4 °C), overlaid 3.9% stacking same buffer. anode BisTris, 7.0, °C, cathode 0.02% G-250, Tricine, run at4 °C 100 V then overnight 40 Blue. bands excised min room temperature 125 Tris-HCl, SDS, before insertion into wells 3.7% 0.13 6.8, SDS-polyacrylamide gel. SDS-PAGE done either 12.5% linear gel, 5–20% 0–15% sucrose 0.375 8.8. Stacking polymerized around separated electrophoresis 100–120 MiniProtean system (Bio-Rad) 25 Tris, 0.192 glycine, 8.3, running Immunoblotting—Following SDS-PAGE, transferred 0.2-μm nitrocellulose mini Trans-Blot transfer 48 39 0.05% 20% (v/v) methanol, 8.3. blocked PBST (PBS, Tween 20 skimmed milk powder) primary 1–2 (αGSH, 1:1000; αCI75, α-creatine (sarcomeric isoform), 1.25 μg/ml). Blots 1:5000–1:10,000 dilutions appropriate secondary temperature, chemiluminescent reagent (ECL ECL Plus, Amersham Biosciences) according manufacturer's instructions, visualized Super RX photographic film (Fujifilm). To visualize blots ExtrAvidin-peroxidase conjugate (1:1000, Sigma) instead antibody. re-probed stripping 62.5 Tris-HCl (pH 6.8), 2% β-mercaptoethanol 30 washed extensively PBST. Autoradiography—Radiolabeled gels above dried onto filter paper. Gels phosphor screen (GE Healthcare), digitized Typhoon detector Healthcare). Protein Spot Excision Spectrometry—Following razor blade, 0.5-ml tube had pre-washed 50% digested "in-gel" cleavage (34Wilm Shevchenko Houthaeve T. Breit Schweigerer Fotsis Mann Nature. 379: 466-469Crossref (1502) do this, slice pressure liquid grade water (100 μl) min; acetonitrile, 100% acetonitrile (20 10 pieces completely SpeedVac 37 ∼ rehydrated 3–7 CaCl2 12.5 ng/μl trypsin (Roche Science) sequencing endoproteinase Asp-N Peptides extracted 4% formic (ARISTAR grade, Merck), 60% (Romil). All digests examined MALDI-TOF-TOF spectrometer (model 4700 Proteomics Analyzer, Biosystems) α-cyano-hydroxy-trans-cinnamic matrix. For digests, instrument calibrated autolysis products (m/z 2163.057 2273.160) calcium related matrix ion 1060.048). digestions, Pseudomonas fragi product 1723.743) two lysis 830.404 1329.753). Peptide sequences obtained MS spectrometer. masses peptide fragmentation data compared National Center Biotechnology Information nr 20080103 base entries MASCOT (35Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref (6661) parameters follows: tolerance ± ppm, MS/MS 0.8 Da, allow missed variable modification. generally acquired automatically, spectra interpreted automatically Mascot MS-MS web-based tool When investigated, peaks MALDI-TOF identified manually Data Explorer (Applied Biosystems). Identifications subsequently supported analyzed calculator feature software digest capillary reverse phase chromatography, portion MALDI-TOF-TOF. ROS Assays—To measure (1 protein/ml) first KPi (50 KPi, 8, EGTA, μm DTPA, neocuproine) pelleted (14,000 g 7 min), twice ml Membranes (0.2 (CLZ) luminometer (Berthold Auto-LumatPlus LB 953) additions indicated figure legends, chemiluminescence recorded s every 5-min 36Lucas Solano 206: 273-277Crossref (71) Chemiluminescence stable duration incubation, average recorded. Superoxide measured methods CLZ dihydroethidine (37Zhao Joseph Fales Sokoloski E.A. Levine R.L. Vasquez-Vivar Kalyanaraman 102: 5727-5732Crossref (472) 38Zhao Kalivendi Nithipatikom 34: 1359-1368Crossref (638) measurements, μg ±1 diamide 120 KCl, HEPES, EDTA, 7.2, supplemented CLZ, succinate 8 μg/ml rotenone glutamate/malate substrates. above. assay, dihydroethidine. Succinate (10 mm) ±2.5 antimycin Triton X-100 added later salmon sperm DNA. fluorescence intercalated DNA 3-ml stirred cuvette Shimadzu RF-5301 fluorimeter (λex 520 nm, λem 590 nm). Control amount sufficient maximize ethidium bromide presence quench fluorescence. This assesses 2-hydroxyethidium ethidium, whereas 2-hydroethidium specific superoxide, pathways formation under conditions General effect activities, KCl (120 7.2 7.5) centrifugation. 20–40 protein/ml rotenone-sensitive (ϵ340 = 6.22 mm-1·cm-1) 7.4, 300 nm antimycin, NADH, decylubiquinone phosphatidylcholine mg/ml) (39James A.M. Wei Y.-H. Pang C.-Y. 318: 401-407Crossref (143) rate typically ∼90% uninhibited rate. II/III succinate, KCN pre-equilibration cytochrome c (ϵ550 21 mm-1·cm-1). background negligible. Respiration rates Clark-type electrode (Rank Brothers, Bottisham, Cambridge, UK). Rat (1–2 chamber 0, 0.5, diamide. respiration measured. 2–3 (FCCP; μm) uncoupled concentration determined bicinchoninic serum albumin standard (40Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. 150: 76-85Crossref (18348) Multiple Sequence Alignments Comparative Modeling Subunit—Orthologous UniProt resource BLASTP (41Altschul S.F. Madden T.L. Schaffer A.A. Z. Miller W. Lipman Nucleic Acids Res. 1997; 3389-3402Crossref (58760) Sequences aligned ClustalW (42Thompson J.D. Higgins D.G. Gibson T.J. 1994; 22: 4673-4680Crossref (54899) weight gap penalties preferential placement gaps loop regions, rather than elements. A set nonredundant selected diverse organisms determine extent residue conservation members family, including thermophilus, Deinococcus radiodurans, Reclinomonas americana, Bos taurus, Drosophila melanogaster, Caenorhabditis elegans, Neurospora crassa, Arabidopsis thaliana, Phytophthora infestans, Acanthamoeba castellani, Dictyostelium discoideum. more closely taurus set, mammals, Gallus gallus, Xenopus laevis, Danio rerio. alignment identify location structure. comparative modeling program MODELLER (43Sali Blundell 1993; 234: 779-815Crossref (10292) calculate model orthologous those pairwise sequence alignments. PyMOL analyze structures local environment residues. Subunit Is Glutathionylated Oxidatively Stressed Mitochondria—To induce stress, converted GSSG led protein-mixed thiol-disulfide (Fig. 1A). proteins, lysed alkylating prevent transglutathionylation, nonreducing probed 1B, αGSH). Diamide treatment number reversed reductant Reprobing co-migrated glutathionyla