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• W2162707945 abstract "The mitochondrial uncoupling proteins 2 and 3 (UCP2 and -3) are known to curtail oxidative stress and participate in a wide array of cellular functions, including insulin secretion and the regulation of satiety. However, the molecular control mechanism(s) governing these proteins remains elusive. Here we reveal that UCP2 and UCP3 contain reactive cysteine residues that can be conjugated to glutathione. We further demonstrate that this modification controls UCP2 and UCP3 function. Both reactive oxygen species and glutathionylation were found to activate and deactivate UCP3-dependent increases in non-phosphorylating respiration. We identified both Cys25 and Cys259 as the major glutathionylation sites on UCP3. Additional experiments in thymocytes from wild-type and UCP2 null mice demonstrated that glutathionylation similarly diminishes non-phosphorylating respiration. Our results illustrate that UCP2- and UCP3-mediated state 4 respiration is controlled by reversible glutathionylation. Altogether, these findings advance our understanding of the roles UCP2 and UCP3 play in modulating metabolic efficiency, cell signaling, and oxidative stress processes. The mitochondrial uncoupling proteins 2 and 3 (UCP2 and -3) are known to curtail oxidative stress and participate in a wide array of cellular functions, including insulin secretion and the regulation of satiety. However, the molecular control mechanism(s) governing these proteins remains elusive. Here we reveal that UCP2 and UCP3 contain reactive cysteine residues that can be conjugated to glutathione. We further demonstrate that this modification controls UCP2 and UCP3 function. Both reactive oxygen species and glutathionylation were found to activate and deactivate UCP3-dependent increases in non-phosphorylating respiration. We identified both Cys25 and Cys259 as the major glutathionylation sites on UCP3. Additional experiments in thymocytes from wild-type and UCP2 null mice demonstrated that glutathionylation similarly diminishes non-phosphorylating respiration. Our results illustrate that UCP2- and UCP3-mediated state 4 respiration is controlled by reversible glutathionylation. Altogether, these findings advance our understanding of the roles UCP2 and UCP3 play in modulating metabolic efficiency, cell signaling, and oxidative stress processes. UCP2 and -3 belong to the mitochondrial anion carrier family and are ∼73% homologous to each other, and they are both ∼58% homologous to the highly thermogenic uncoupling protein, UCP1. Whereas UCP3 is expressed in skeletal muscle and brown adipose tissue (BAT) 3The abbreviations used are: BATbrown adipose tissueROSreactive oxygen speciesBSObutathionine sulfoximineOCRoxygen consumption rateBIAMbiotinylated iodoacetamideBioGEEbiotinylated glutathione ethyl esterGRxglutaredoxinNEMN-ethylmaleimidePSSGprotein-glutathioneANOVAanalysis of varianceMPTmitochondrial permeability transition4-HNE4-hydroxynonenal. and to some extent in the heart, UCP2 is found in a wide variety of tissues (1Azzu V. Brand M.D. Trends Biochem. Sci. 2010; 35: 298-307Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). UCP2 and UCP3 have been shown to diminish oxidative stress by lowering the mitochondrial membrane potential (2Nègre-Salvayre A. Hirtz C. Carrera G. Cazenave R. Troly M. Salvayre R. Pénicaud L. Casteilla L. FASEB J. 1997; 11: 809-815Crossref PubMed Scopus (685) Google Scholar, 3Gustafsson H. Söderdahl T. Jönsson G. Bratteng J.O. Forsby A. J. Neurosci. Res. 2004; 77: 285-291Crossref PubMed Scopus (48) Google Scholar). Acute increases in reactive oxygen species (ROS) production increase proton conductance through both UCP2 and UCP3, providing a negative feedback loop to limit further mitochondrial ROS formation (4Echtay K.S. Roussel D. St-Pierre J. Jekabsons M.B. Cadenas S. Stuart J.A. Harper J.A. Roebuck S.J. Morrison A. Pickering S. Clapham J.C. Brand M.D. Nature. 2002; 415: 96-99Crossref PubMed Scopus (1150) Google Scholar, 5Echtay K.S. Murphy M.P. Smith R.A. Talbot D.A. Brand M.D. J. Biol. Chem. 2002; 277: 47129-47135Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Furthermore, UCP2 and UCP3 have also been implicated in many physiologic functions, suggesting that the common underlying mechanism may be ROS-mediated cell signaling. brown adipose tissue reactive oxygen species butathionine sulfoximine oxygen consumption rate biotinylated iodoacetamide biotinylated glutathione ethyl ester glutaredoxin N-ethylmaleimide protein-glutathione analysis of variance mitochondrial permeability transition 4-hydroxynonenal. Previous work has reported that UCP3 protects against insulin resistance and obesity (6Harper M.E. Green K. Brand M.D. Annu. Rev. Nutr. 2008; 28: 13-33Crossref PubMed Scopus (91) Google Scholar, 7Choi C.S. Fillmore J.J. Kim J.K. Liu Z.X. Kim S. Collier E.F. Kulkarni A. Distefano A. Hwang Y.J. Kahn M. Chen Y. Yu C. Moore I.K. Reznick R.M. Higashimori T. Shulman G.I. J. Clin. Invest. 2007; 117: 1995-2003Crossref PubMed Scopus (148) Google Scholar, 8Costford S.R. Crawford S.A. Dent R. McPherson R. Harper M.E. Diabetologia. 2009; 52: 2405-2415Crossref PubMed Scopus (21) Google Scholar, 9Costford S.R. Chaudhry S.N. Crawford S.A. Salkhordeh M. Harper M.E. Am. J. Physiol. Endocrinol. Metab. 2008; 295: E1018-E1024Crossref PubMed Scopus (56) Google Scholar), whereas UCP2 has been implicated a broad range of functions, including hormone secretion from the pancreas, immune cell function, and feeding behavior (10Alves-Guerra M.C. Rousset S. Pecqueur C. Mallat Z. Blanc J. Tedgui A. Bouillaud F. Cassard-Doulcier A.M. Ricquier D. Miroux B. J. Biol. Chem. 2003; 278: 42307-42312Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 11Parton L.E. Ye C.P. Coppari R. Enriori P.J. Choi B. Zhang C.Y. Xu C. Vianna C.R. Balthasar N. Lee C.E. Elmquist J.K. Cowley M.A. Lowell B.B. Nature. 2007; 449: 228-232Crossref PubMed Scopus (542) Google Scholar, 12Pi J. Bai Y. Daniel K.W. Liu D. Lyght O. Edelstein D. Brownlee M. Corkey B.E. Collins S. Endocrinology. 2009; 150: 3040-3048Crossref PubMed Scopus (145) Google Scholar, 13Derdak Z. Mark N.M. Beldi G. Robson S.C. Wands J.R. Baffy G. Cancer Res. 2008; 68: 2813-2819Crossref PubMed Scopus (186) Google Scholar). For UCP2, most of these functions are linked to ROS level buffering (14Andrews Z.B. Liu Z.W. Walllingford N. Erion D.M. Borok E. Friedman J.M. Tschöp M.H. Shanabrough M. Cline G. Shulman G.I. Coppola A. Gao X.B. Horvath T.L. Diano S. Nature. 2008; 454: 846-851Crossref PubMed Scopus (577) Google Scholar, 15Pecqueur C. Alves-Guerra M.C. Gelly C. Levi-Meyrueis C. Couplan E. Collins S. Ricquier D. Bouillaud F. Miroux B. J. Biol. Chem. 2001; 276: 8705-8712Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 16Diao J. Allister E.M. Koshkin V. Lee S.C. Bhattacharjee A. Tang C. Giacca A. Chan C.B. Wheeler M.B. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12057-12062Crossref PubMed Scopus (59) Google Scholar). Indeed, UCP2 null mice on various genetic backgrounds exhibit oxidative stress in many tissue types and a decrease in the circulating glutathione (GSH)/glutathione disulfide ratio (2Nègre-Salvayre A. Hirtz C. Carrera G. Cazenave R. Troly M. Salvayre R. Pénicaud L. Casteilla L. FASEB J. 1997; 11: 809-815Crossref PubMed Scopus (685) Google Scholar, 12Pi J. Bai Y. Daniel K.W. Liu D. Lyght O. Edelstein D. Brownlee M. Corkey B.E. Collins S. Endocrinology. 2009; 150: 3040-3048Crossref PubMed Scopus (145) Google Scholar). Increased expression of UCP2 in cancer cells is associated with the acquisition of drug-resistant phenotypes, a phenomenon related to the ROS-quenching function of UCP2 (17Mailloux R.J. Adjeitey C.N. Harper M.E. PLoS One. 2010; 5: e13289Crossref PubMed Scopus (80) Google Scholar, 18Harper M.E. Antoniou A. Villalobos-Menuey E. Russo A. Trauger R. Vendemelio M. George A. Bartholomew R. Carlo D. Shaikh A. Kupperman J. Newell E.W. Bespalov I.A. Wallace S.S. Liu Y. Rogers J.R. Gibbs G.L. Leahy J.L. Camley R.E. Melamede R. Newell M.K. FASEB J. 2002; 16: 1550-1557Crossref PubMed Scopus (155) Google Scholar). The absence of UCP3 in skeletal muscle increases oxidative damage and perturbs skeletal muscle metabolism (19Vidal-Puig A.J. Grujic D. Zhang C.Y. Hagen T. Boss O. Ido Y. Szczepanik A. Wade J. Mootha V. Cortright R. Muoio D.M. Lowell B.B. J. Biol. Chem. 2000; 275: 16258-16266Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). Increased UCP3 expression in muscle augments fatty acid metabolism and also curtails ROS production during fatty acid oxidation (20Bezaire V. Spriet L.L. Campbell S. Sabet N. Gerrits M. Bonen A. Harper M.E. FASEB J. 2005; 19: 977-979Crossref PubMed Scopus (113) Google Scholar, 21Anderson E.J. Yamazaki H. Neufer P.D. J. Biol. Chem. 2007; 282: 31257-31266Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Remarkably, although they have been linked to a plethora of cellular processes, the molecular control of UCP2 and UCP3 has remained elusive. Reactive cysteine residues of cellular proteins are known sites of regulation by conjugation to GSH, a process referred to as glutathionylation. This is especially relevant for mitochondria because a large fraction of the mitochondrial proteome contains exposed thiols that can be covalently modified by GSH. We were intrigued by the fact that UCP2 and UCP3 proteins contain several cysteine residues located in predicted membrane-spanning domains and in a loop region in the mitochondrial matrix (22Boss O. Samec S. Paoloni-Giacobino A. Rossier C. Dulloo A. Seydoux J. Muzzin P. Giacobino J.P. FEBS. Lett. 1997; 408: 39-42Crossref PubMed Scopus (998) Google Scholar). Here, we provide the first evidence that implicates reversible glutathionylation in the regulation of UCP2 and UCP3 function. We also show that glutathionylation and ROS-induced deglutathionylation work in tandem to turn UCP2 and UCP3 off and on, respectively. This novel mechanism for UCP2 and UCP3 control provides new insight into the putative role of these proteins for cellular ROS buffering. These results also improved our understanding of the function of these proteins in various physiological processes. Thymocytes were isolated from UCP2 null (knock-out (KO)) and wild-type (WT) mice as described previously (23Krauss S. Buttgereit F. Brand M.D. Biochim. Biophys. Acta. 1999; 1412: 129-138Crossref PubMed Scopus (35) Google Scholar). The thymus was immediately removed and placed in ice-cold glucose-free Dulbecco's modified Eagle's medium (DMEM) containing 5 mm glutamine, 1 mm pyruvate, 2% FBS and buffered with HEPES, pH 7.4 (Glc-free DMEM) and then pressed through a metal tea strainer. Cells were washed twice with Glc-free DMEM and counted for oxygen consumption rate determinations. Primary myoblasts were isolated from WT and UCP3 null (KO) mice as described (24Rando T.A. Blau H.M. J. Cell Biol. 1994; 125: 1275-1287Crossref PubMed Scopus (804) Google Scholar). Primary myoblasts were maintained on Matrigel-coated 60-mm2 plates in DMEM containing 5 mm dextrose, 1 mm pyruvate, 4 mm glutamine, 10% (v/v) fetal bovine serum (FBS), and 1% antimycotics antibiotics (A.A.). For experiments, primary myoblasts were grown to 70% and then differentiated for up to 7 days in DMEM containing 5 mm dextrose, 1 mm pyruvate, 4 mm glutamine, 2% FBS, and 1% A. A. Cells were then exposed to the superoxide-producing quinone menadione (0–20 μm), butathionine sulfoximine (BSO; 0–100 μm), or diamide (0–500 μm). Differentiated myotubes were then treated accordingly for experimentation. Muscle mitochondria were isolated as described previously (25Seifert E.L. Bézaire V. Estey C. Harper M.E. J. Biol. Chem. 2008; 283: 25124-25131Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Skeletal muscle from forelimbs, hind limbs, and the pectoral region were pooled, cleaned, and placed in basic medium (140 mm KCl, 20 mm HEPES, 5 mm MgCl2, 1 mm EGTA, pH 7.0). Following mincing, tissue was placed in homogenizing medium (basic medium supplemented with 1 mm ATP and 1% BSA (w/v)) and one unit of protease (subtilisin A) and homogenized using a glass/Teflon Potter-Elvehjem tissue grinder. Mitochondria were then isolated as described (25Seifert E.L. Bézaire V. Estey C. Harper M.E. J. Biol. Chem. 2008; 283: 25124-25131Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). BAT mitochondria were isolated using a standard protocol, as described (26Shabalina I.G. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 2004; 279: 38236-38248Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Interscapular BAT was dissected from 2–3 mice/genotype, cleaned, and pooled in a 250 mm sucrose solution. The tissue was minced and then homogenized by hand using a glass/Teflon Potter-Elvehjem tissue grinder in a 250 mm sucrose solution containing 0.2% BSA. Mitochondria were isolated as described (26Shabalina I.G. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 2004; 279: 38236-38248Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Protein concentration was determined by Bradford assay using BSA as the standard. Mitochondrial preparations were kept on ice, and all experiments were conducted within 4 h following completion of the isolation procedure. Experiments in skeletal muscle mitochondria were conducted in incubation medium containing 120 mm KCl, 1 mm EGTA, 5 mm KH2PO4, 5 mm MgCl2, and 5 mm HEPES, pH 7.4, supplemented with 0.3% defatted BSA. Experiments in BAT mitochondria were conducted in incubation medium containing 125 mm sucrose, 20 mm Tris-HCl, 2 mm MgCl2, 4 mm KH2PO4, 1 mm EDTA, pH 7.2. An aliquot of mitochondria from BAT and skeletal muscle was frozen at −20 °C for immunoblot analysis. Oxygen consumption was measured in isolated mitochondria (0.5 mg/ml) or thymocytes at 37 °C using a Clark-type oxygen electrode (Hansatech, Norfolk, UK) and incubated in the appropriate incubation medium assumed to contain 406 nmol of oxygen/ml at 37 °C. Mitochondria were preincubated in the absence or presence of 2 μm diamide for skeletal muscle mitochondria and 100 μm diamide for BAT mitochondria with 1 μg/ml of oligomycin for 10 min at 37 °C before oxygen consumption measurements. 10 mm pyruvate and 5 mm malate were the substrates. For determinations in thymocytes, 20 × 106 cells were incubated in DMEM containing 5 mm glucose, 4 mm glutamine, and 1 mm pyruvate for 15 min at 37 °C containing oligomycin (1 μg/ml) and in the presence or absence of diamide (100 μm). Following incubations, cells were then placed in the Clark-type electrode chamber for oxygen consumption measurements. An aliquot of thymocytes was kept for immunoblot analysis of UCP2 levels. Membrane potential (Δψm) was determined fluorimetrically, using safranin O dye (5 μm; excitation 485 nm, emission 580 nm) in isolated mouse mitochondria (0.5 mg/ml) incubated in the requisite incubation medium at 37 °C (27Silva J.P. Shabalina I.G. Dufour E. Petrovic N. Backlund E.C. Hultenby K. Wibom R. Nedergaard J. Cannon B. Larsson N.G. EMBO J. 2005; 24: 4061-4070Crossref PubMed Scopus (94) Google Scholar). Prior to assessments, mitochondria were preincubated for 10 min in diamide (0–200 μm) and then treated with 1 μg/ml oligomycin to induce state 4 respiration conditions. The substrates used were 5 mm pyruvate and 3 mm malate for BAT and 5 mm succinate for skeletal muscle. The XF24 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA) was employed to determine the mitochondrial bioenergetic characteristics of primary myotubes exposed to menadione, diamide, and/or BSO. Primary myoblasts were seeded at 50,000 cells/ml in Matrigel-coated 24-well Seahorse XF24 culture plates. Upon differentiation and reaching confluence, the cells were exposed to menadione (0–20 μm) or BSO (0–100 μm) for 24 h and then incubated for 30 min at 37 °C in HCO3-free DMEM containing 5–10 mm glucose, 4 mm l-glutamine, and 1 mm pyruvate. Measurement of oxygen consumption rates (OCRs) was performed for three measurement intervals to assess basal metabolic rate (one measurement interval consists of a 2-min mixing, 2-min incubation, and 2-min measurement step). State 4 respiration, maximal metabolic rate, and extramitochondrial O2 consumption were ascertained by injecting oligomycin (1 μg/ml), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (1 μm), and antimycin A (1 μm). The impact of glutathionylation on state 4 respiration was determined by exposing cells to 100 μm diamide for 15 min prior to the injection of oligomcycin. OCR was normalized to total cellular protein/well using the Bradford assay. Reactive cysteines and glutathionylation sites were tested by BIAM (Invitrogen) and BioGEE (Invitrogen) labeling (28Reynaert N.L. van der Vliet A. Guala A.S. McGovern T. Hristova M. Pantano C. Heintz N.H. Heim J. Ho Y.S. Matthews D.E. Wouters E.F. Janssen-Heininger Y.M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13086-13091Crossref PubMed Scopus (355) Google Scholar, 29Marino S.M. Li Y. Fomenko D.E. Agisheva N. Cerny R.L. Gladyshev V.N. Biochemistry. 2010; 49: 7709-7721Crossref PubMed Scopus (31) Google Scholar). Briefly, partially purified UCP3 was diluted to 4 mg/ml in sucrose buffer (250 mm sucrose, Tris-HCl, pH 7) containing 100 μm BIAM or BioGEE and incubated for 30 min at room temperature in the dark under constant agitation. For control experiments, the samples were preincubated for 30 min at room temperature in 10 mm dithiothreitol (DTT) or 5 mm N-ethylmaleimide (NEM). Samples were then passed through a PD-10 Sephadex G25 desalting column (GE Healthcare) and eluted by centrifugation at 1000 rpm for 1 min (30Clavreul N. Adachi T. Pimental D.R. Ido Y. Schöneich C. Cohen R.A. FASEB J. 2006; 20: 518-520Crossref PubMed Scopus (116) Google Scholar). BIAM and BioGEE-labeled proteins were then pulled out of solution with streptavidin-coated beads overnight at 4 °C. Beads were then collected, and the supernatant fraction was placed in an ice-cold minitube containing Laemmli buffer. Beads were separated from the BIAM and BioGEE-labeled proteins using sucrose media containing 5 m urea. Upon removal of the deproteinated beads, the sample was placed in an ice-cold minitube containing Laemmli buffer constituting the pull-down fraction. Samples were electrophoresed under non-reducing conditions, and then UCP3 was detected by immunoblot using anti-UCP3 antibody (Abcam). The glutathionylation of UCP3 was tested with glutaredoxin 1 (GRx1; Sigma). For GRx1, experiments were performed as described previously (31Applegate M.A. Humphries K.M. Szweda L.I. Biochemistry. 2008; 47: 473-478Crossref PubMed Scopus (106) Google Scholar) with some modifications. Purified UCP3 was incubated in sucrose buffer for 15 min at 37 °C under constant agitation and in the dark with 0.5 mm GSH in the absence or presence of 10 units of GRx1. The samples were then incubated in 200 μm BioGEE for 30 min at room temperature under constant agitation and in the dark. The reaction was quenched with 5 mm NEM and passed through a PD-10 Sephadex G25 desalting column. The amount of UCP3 was accessed by immunoblot using anti-UCP3 antibody (Abcam). Kidneys from WT and UCP2 null (KO) mice were excised, flash-frozen, and stored at −80 °C. On the day of experiments, kidneys were placed in 10 ml of Trizma (Tris base)-buffered sucrose solution (125 mm sucrose, 25 mm Trizma, 2 mm MgCl2, 1 mm EGTA, pH 7.2) and homogenized on ice using a glass/Teflon Potter-Elvehjem tissue grinder. The homogenate was then centrifuged at 150 × g for 5 min at 4 °C to remove any particulates and whole cells, and the supernatant was then tested for protein content. Protein was diluted to 4 mg/ml in sucrose buffer and incubated for 30 min in the dark at room temperature under constant agitation with 100 μm BioGEE. Reactions were quenched by the addition of NEM. 15 μg of total protein was then loaded in a 6–15% linear gradient SDS-gel and then electrophoresed under standard conditions. UCP2 biotinylated with BioGEE was detected by probing blots for 1 h at room temperature in the dark using an anti-biotin HRP-conjugated antibody (1:4000 dilution in 5% nonfat skim milk; Abcam). The electrophoretic mobility of UCP2 in the biotin blots was confirmed by probing for UCP2 on the same gel with anti-UCP2 antibody. The assay was optimized by loading various amounts of protein (5–60 μg) and by varying antibody dilution (1:500 to 1:4000) and incubation time (30 min to 24 h). To test whether UCP3 was glutathionylated, we employed mitochondria isolated from the skeletal muscle of WT and UCP3 null (KO) mice. Mitochondria were diluted to 4 mg/ml in sucrose buffer and incubated in BioGEE. Reactions were stopped by the addition of NEM. Protein was then loaded in a 6–15% linear gradient SDS-gel and then electrophoresed under standard conditions. Detection of biotinylation was performed using anti-biotin HRP-conjugated antibody (1:4000 dilution in 5% nonfat skim milk; Abcam). The electrophoretic mobility of UCP3 in the biotin blots was confirmed by probing for these proteins on the same gel with anti-UCP3 (1:1000 dilution, 24-h incubation at 4 °C; Abcam) antibody. CMV plasmid constructs containing the full-length human UCP3 were purchased from Origene Technologies (Rockville, MD). For the site-directed mutagenesis, suitable primers (from Sigma; 5′-gcaggcacagcagccgcttttgctgacctcgt-3′ and 3′-cgtccgtgtcgtcggcgaaaacgactggagca-5′ for UCP3C25A, 5′-ttggagccggcttcgctgccacagtggtgg-3′ and 3′-aacctcggccgaagcgacggtgtcaccacc-5′ for UCP3C230A, and 5′-tacttcagccccctcgacgctatgataaagatggtggc-3′ and 3′-atgaagtcgggggagctgcgatactatttctaccaccg-5′ for UCP3C259A, respectively) were used to change codons from cysteine to alanine. Primers were designed using the QuikChange II mutagenesis primer design application (Agilent Technologies). Mutants were generated using the QuikChange II site-directed mutagenesis kit (Agilent Technologies). Plasmids carrying the appropriate point mutation were confirmed using BLAST and Expasy. Transient transfections were carried out with Lipofectamine 2000 according to the manufacturer's instructions. Briefly, HEK293 cells were seeded at 90% confluence in Matrigel-coated 60-mm dishes in antibiotic/antimycotic-free low glucose DMEM containing 10% FBS. Cells were treated with plasmid-Lipofectamine complex in antibiotic/antimycotic-free DMEM containing 10% FBS and incubated for ∼36 h in the cell incubator. The cell monolayer was washed once with PBS and then incubated for 1 h at 37 °C in serum and antimycotic/antibiotic-free DMEM containing 1 mm BioGEE. The cell monolayer was then quickly washed twice, and the cells were lysed on ice with precipitation buffer containing 25 mm NEM. BioGEE-labeled proteins were isolated as described above. UCP3 was detected by immunoblot as described above. Primary WT myotubes were treated for 1 h with serum-free LG DMEM containing 1 mm BioGEE. Monolayers were then washed once with serum-free DMEM and then treated with serum-free DMEM containing 0–100 μm H2O2 for 15 min. Cells were then immediately washed with PBS and lysed with precipitation buffer containing 25 mm NEM. BioGEE-labeled proteins were extracted as described above, and the presence of UCP3 was tested by immunoblot. Protein content was tested by a BCA assay. Cells exposed to BioGEE and H2O2 as described above were also tested for ROS levels. Immediately following H2O2 exposure, WT cells were incubated for 30 min at 37 °C in differentiating media containing 20 μm dichlorofluorescein diacetate. Fluorescence was determined as described (17Mailloux R.J. Adjeitey C.N. Harper M.E. PLoS One. 2010; 5: e13289Crossref PubMed Scopus (80) Google Scholar). Primary WT and UCP3 null (KO) myotubes were treated with 100 μm diamide for 30 min and then isolated by trypsinization. Mitochondria were isolated by sonication and differential centrifugation as described (32Mailloux R.J. Harper M.E. FASEB J. 2010; 24: 2495-2506Crossref PubMed Scopus (55) Google Scholar) with some modifications. 50 mm NEM was included in the mitochondrial isolation buffer. Mitochondria were permeabilized with 1% (w/v) digitonin on ice for 30 min, and the specific activity of complex I was then determined using 1.5 mm NADH and 0.5 mg/ml mitochondrial protein. NADH consumption was monitored as described (32Mailloux R.J. Harper M.E. FASEB J. 2010; 24: 2495-2506Crossref PubMed Scopus (55) Google Scholar). Mitochondrial protein concentration was determined using the Bradford assay. Primary myotubes from WT mice were treated with diamide (0–500 μm) for 1 h in serum-free media and then lysed in a radioimmune precipitation buffer containing 50 mm NEM (to quench diamide reaction). Protein-glutathione (PSSG) adducts were detected using anti-PSSG antibody (Virogen). PSSG adducts were detected as described (33Hill B.G. Higdon A.N. Dranka B.P. Darley-Usmar V.M. Biochim. Biophys. Acta. 2010; 1797: 285-295Crossref PubMed Scopus (71) Google Scholar). Primary myotubes were exposed to BSO (0–100 μm) for 24 h and then trypsinized and washed with PBS, and then total GSH levels were tested using a GSH detection kit provided by Sigma. WT and KO primary myotubes were seeded, grown, and differentiated in Matrigel-coated 96-well plates. Upon full differentiation, cells were treated for 24 h with differentiating medium containing 0–40 μm menadione or for 30 min with serum-free DMEM containing 0–500 μm diamide. Following the exposure, cell monolayers were washed, probed with propidium iodide, and then tested for amount of cell death as described (32Mailloux R.J. Harper M.E. FASEB J. 2010; 24: 2495-2506Crossref PubMed Scopus (55) Google Scholar). Immunoblots were carried out as described previously (32Mailloux R.J. Harper M.E. FASEB J. 2010; 24: 2495-2506Crossref PubMed Scopus (55) Google Scholar). The membranes were blocked and probed for 3–24 h at 4 °C with primary antibodies directed against UCP3 (1:1000 dilution for cell lysate, 1:500 dilution of detection following BioGEE pull-down from cell lysate, 1:2500 dilution for purified UCP3; Abcam), UCP2 (1:2000, N-19; Santa Cruz Biotechnology, Inc.), UCP1 (1:10,000; Sigma), protein-glutathione adducts (1:500; Virogen), troponin T (1:1000; Abcam), and SDH (1:2000; Santa Cruz Biotechnology, Inc.). SDH and troponin T served as the loading standards. Membranes were then incubated for 1 h at room temperature with the requisite horseradish peroxidase-conjugated secondary antibodies (anti-rabbit, anti-mouse, or anti-goat 1:2000; Santa Cruz Biotechnology, Inc.). Blots were incubated in enhanced chemiluminescent substrate for visualization (ECL kit, Thermo Scientific). Unless otherwise stated, all data are expressed as mean ± S.D. Statistical analysis was performed using Statview software (SAS Institute Inc.) except for Student's t tests, which were performed in Microsoft Excel. Comparison of control with treated groups was performed with Student's t test, and for comparison of multiple treatments in the same group with a control, one-way ANOVA with a post hoc Tukey's test was used. As a first approach, we determined if partially purified UCP3 protein could be covalently modified with the two agents BIAM and BioGEE in vitro. BIAM is routinely used to test the presence of reactive cysteines on proteins, whereas BioGEE, a cell-permeable glutathione molecule attached to biotin, is used to test if proteins can be glutathionylated. We found that UCP3 was readily recovered following BIAM treatment and streptavidin pull-down (Fig. 1a). By maintaining the cysteines in a reduced state or by blocking the cysteines with NEM, the yield of UCP3 in the pull-down was significantly diminished, confirming the presence of reactive cysteines in this protein (Fig. 1a). Similar observations were made with BioGEE (Fig. 1a). The level of recoverable UCP3 following BioGEE treatment was decreased substantially by the reducing and blocking agents DTT and NEM, revealing the requirement of cysteines for the conjugation of GSH to UCP3 (Fig. 1a). Glutathionylation of proteins has been shown to be mediated by GRx, a thiol-rich enzyme involved in antioxidative defense (34Gallogly M.M. Starke D.W. Mieyal J.J. Antioxid. Redox Signal. 2009; 11: 1059-1081Crossref PubMed Scopus (177) Google Scholar). Incubation of UCP3 with GRx1 and GSH prior to BioGEE treatment and pull-down with streptavidin significantly diminished the recovery of UCP3 (Fig. 1b). In contrast, incubations performed in the absence of GRx1 increased UCP3 recovery following BioGEE treatment and streptavidin pull-down. These data indicate that the glutathionylation of UCP3 is enzymatically mediated in vitro by the GSH exchanger, GRx. We next tested whether BioGEE could covalently modify UCP3 in mitochondria from skeletal muscle. Mitochondria were treated with BioGEE and then tested for biotin content by immunoblot using anti-biotin antibodies. Immunoblotting for BioGEE-modified mitochondrial proteins revealed bands at ∼34 kDa, which corresponds to the electrophoretic mobility of UCP3 (Fig. 1c). Moreover, these immunoreactive bands were only detected in mitochondria from WT mice. Furthermore, a slight shift in the electrophoretic mobility of UCP3 in the BioGEE-treated WT mitochondria was observed. This is most likely due to the covalent modification of this protein with BioGEE (Fig. 1c). Detection of BioGEE-modified proteins with anti-biotin antibodies also revealed a number of other immunoreactive bands at a range of molecular weights indicating that a number of mitochondrial proteins can be modified by GSH in skeletal muscle mitochondria. This is not surprising because the mitochondrial proteome is known to be very susceptible to glutathionylation (35Hurd T.R. Costa N.J. Dahm C.C. Beer S.M. Brown S.E. Filipovska A. Murphy M.P. Antioxid. Redox Signal. 2005; 7: 999-1010Crossref PubMed Scopus (167) Google Scholar). To confirm that UCP3 is glutathionylated and to identify the" @default.
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• W2162707945 date "2011-06-01" @default.
• W2162707945 modified "2023-10-06" @default.
• W2162707945 title "Glutathionylation Acts as a Control Switch for Uncoupling Proteins UCP2 and UCP3" @default.
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• W2162707945 doi "https://doi.org/10.1074/jbc.m111.240242" @default.
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