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• W1986618045 abstract "Mitochondrial oxidative phosphorylation provides most cellular energy. As part of this process, cytochrome c oxidase (CcO) pumps protons across the inner mitochondrial membrane, contributing to the generation of the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. During acute inflammation, as in sepsis, aerobic metabolism appears to malfunction and switches to glycolytic energy production. The pro-inflammatory cytokine tumor necrosis factor α (TNFα) has been shown to play a central role in inflammation. We hypothesized that TNFα-triggered cell signaling targets CcO, which is a central enzyme of the aerobic energy metabolism and can be regulated through phosphorylation. Using total bovine and murine hepatocyte homogenates TNFα treatment led to an ∼60% reduction in CcO activity. In contrast, there was no direct effect of TNFα on CcO activity using isolated mitochondria and purified CcO, indicating that a TNFα-triggered intracellular signaling cascade mediates CcO inhibition. CcO isolated after TNFα treatment showed tyrosine phosphorylation on CcO catalytic subunit I and was ∼50 and 70% inhibited at high cytochrome c concentrations in the presence of allosteric activator ADP and inhibitor ATP, respectively. CcO phosphorylation occurs on tyrosine 304 as demonstrated with a phosphoepitope-specific antibody. Furthermore, the mitochondrial membrane potential was decreased in H2.35 cells in response to TNFα. Concomitantly, cellular ATP was more than 35 and 64% reduced in murine hepatocytes and H2.35 cells. We postulate that an important contributor in TNFα-mediated pathologies, such as sepsis, is energy paucity, which parallels the poor tissue oxygen extraction and utilization found in such patients. Mitochondrial oxidative phosphorylation provides most cellular energy. As part of this process, cytochrome c oxidase (CcO) pumps protons across the inner mitochondrial membrane, contributing to the generation of the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. During acute inflammation, as in sepsis, aerobic metabolism appears to malfunction and switches to glycolytic energy production. The pro-inflammatory cytokine tumor necrosis factor α (TNFα) has been shown to play a central role in inflammation. We hypothesized that TNFα-triggered cell signaling targets CcO, which is a central enzyme of the aerobic energy metabolism and can be regulated through phosphorylation. Using total bovine and murine hepatocyte homogenates TNFα treatment led to an ∼60% reduction in CcO activity. In contrast, there was no direct effect of TNFα on CcO activity using isolated mitochondria and purified CcO, indicating that a TNFα-triggered intracellular signaling cascade mediates CcO inhibition. CcO isolated after TNFα treatment showed tyrosine phosphorylation on CcO catalytic subunit I and was ∼50 and 70% inhibited at high cytochrome c concentrations in the presence of allosteric activator ADP and inhibitor ATP, respectively. CcO phosphorylation occurs on tyrosine 304 as demonstrated with a phosphoepitope-specific antibody. Furthermore, the mitochondrial membrane potential was decreased in H2.35 cells in response to TNFα. Concomitantly, cellular ATP was more than 35 and 64% reduced in murine hepatocytes and H2.35 cells. We postulate that an important contributor in TNFα-mediated pathologies, such as sepsis, is energy paucity, which parallels the poor tissue oxygen extraction and utilization found in such patients. Pro-inflammatory cytokine TNFα 3The abbreviations used are: TNF, tumor necrosis factor; CcO, cytochrome c oxidase; ETC, electron transport chain; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide; OxPhos, oxidative phosphorylation; PKA, protein kinase A; PMSF, phenylmethylsulfonyl fluoride; ROS, reactive oxygen species; dH2O, distilled H2O; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HPLC, high pressure liquid chromatography; AKAP, protein kinase A-anchoring protein. 3The abbreviations used are: TNF, tumor necrosis factor; CcO, cytochrome c oxidase; ETC, electron transport chain; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide; OxPhos, oxidative phosphorylation; PKA, protein kinase A; PMSF, phenylmethylsulfonyl fluoride; ROS, reactive oxygen species; dH2O, distilled H2O; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HPLC, high pressure liquid chromatography; AKAP, protein kinase A-anchoring protein. exerts a wide range of inflammatory, immune-modulatory, and metabolic effects. TNFα is associated with various diseases such as sepsis, atherosclerosis, and hepatic failure and initiates its biological effects by binding to high affinity cell surface receptors (TNF receptor types 1 and 2) (1Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (2963) Google Scholar). Receptor ligation in different types of cells is associated with an increased production of reactive oxygen species (ROS). It is well documented that TNFα increases ROS in the mitochondria and that it does so in the cytoplasm in a NADPH-dependent fashion. Mitochondria are considered an early target in TNFα-induced cytotoxicity, because they appear swollen with a reduction in cristae membrane structure in the early course of endotoxinemia and sepsis (2Brealey D. Brand M. Hargreaves I. Heales S. Land J. Smolenski R. Davies N.A. Cooper C.E. Singer M. Lancet. 2002; 360: 219-223Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, 3Schulze-Osthoff K. Bakker A.C. Vanhaesebroeck B. Beyaert R. Jacob W.A. Fiers W. J. Biol. Chem. 1992; 267: 5317-5323Abstract Full Text PDF PubMed Google Scholar). It was suggested that early mitochondrial dysfunction and inhibition of the oxidative phosphorylation (OxPhos) system play a pivotal role in impaired O2 utilization during inflammatory processes (2Brealey D. Brand M. Hargreaves I. Heales S. Land J. Smolenski R. Davies N.A. Cooper C.E. Singer M. Lancet. 2002; 360: 219-223Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, 4Singer M. Crit. Care Med. 2005; 33: S539-S542Crossref PubMed Scopus (24) Google Scholar). During endotoxinemia and sepsis, glycolytic ATP production was increased (5Berg S. Sappington P.L. Guzik L.J. Delude R.L. Fink M.P. Crit. Care Med. 2003; 31: 1203-1212Crossref PubMed Scopus (32) Google Scholar). Apparently, OxPhos was inhibited, although increased tissue oxygen levels suggest cellular availability of O2 (6Rosser D.M. Stidwill R.P. Jacobson D. Singer M. J. Appl. Physiol. 1995; 79: 1878-1882Crossref PubMed Scopus (85) Google Scholar), indicating a decrease of oxygen utilization (7Kreymann G. Grosser S. Buggisch P. Gottschall C. Matthaei S. Greten H. Crit. Care Med. 1993; 21: 1012-1019Crossref PubMed Scopus (193) Google Scholar, 8Boneh A. Cell Mol. Life Sci. 2006; 63: 1236-1248Crossref PubMed Scopus (24) Google Scholar).The formation of the cellular energy carrier ATP is the result of both anaerobic and aerobic processes. Anaerobic ATP generation is catalyzed by phosphoglycerate kinase and pyruvate kinase, and GTP is produced by succinyl coenzyme A synthetase. However, ∼95% of cellular energy is generated through the aerobic pathway, the mitochondrial OxPhos process that includes an elaborate electron transport chain (ETC), in which O2 functions as the terminal electron acceptor and is reduced to water. The flow of electrons from NADH and FADH2 through the ETC is coupled to the pumping of protons across the mitochondrial inner membrane, which creates a transmembrane electrochemical potential (ΔΨm). Through this process, a proton-motive force is generated that is utilized by ATP synthase to produce ATP from ADP and phosphate. As a byproduct of the OxPhos process, an estimated 1-2% of cellular O2 is converted into superoxide anions (O2⋅¯) (9Richter C. Park J.W. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6465-6467Crossref PubMed Scopus (1477) Google Scholar). OxPhos is regulated at various sites by numerous factors, including the ATP/ADP ratio (10Napiwotzki J. Shinzawa-Itoh K. Yoshikawa S. Kadenbach B. Biol. Chem. 1997; 378: 1013-1021Crossref PubMed Scopus (91) Google Scholar), PO2, and the amount of ROS and nitric oxide (11Palacios-Callender M. Quintero M. Hollis V.S. Springett R.J. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7630-7635Crossref PubMed Scopus (158) Google Scholar, 12Brunori M. Giuffre A. Sarti P. J. Inorg. Biochem. 2005; 99: 324-336Crossref PubMed Scopus (105) Google Scholar), as well as hormonal influences (13Arnold S. Goglia F. Kadenbach B. Eur. J. Biochem. 1998; 252: 325-330Crossref PubMed Scopus (172) Google Scholar).Cytochrome c oxidase (CcO, complex IV) is the terminal enzyme of the electron transport chain. CcO accepts electrons from cytochrome c and transfers them to molecular oxygen, which is reduced to water. At the same time protons are pumped across the inner mitochondrial membrane leading to the generation of the mitochondrial membrane potential ΔΨm. CcO exhibits the three characteristic regulatory means known to act on key metabolic enzymes: isoform expression, allosteric control, and reversible phosphorylation. There are three liver and heart type isoform pairs of subunits VIa, VIIa, and VIII (reviewed in Ref. 14Hüttemann M. Lee I. Liu J. Grossman L.I. FEBS J. 2007; 274: 5737-5748Crossref PubMed Scopus (51) Google Scholar) in addition to a lung-specific isoform of CcO subunit IV, a testes-specific isoform of subunit VIb, and a third isoform of subunit VIII (15Hüttemann M. Kadenbach B. Grossman L.I. Gene (Amst.). 2001; 267: 111-123Crossref PubMed Scopus (143) Google Scholar, 16Hüttemann M. Jaradat S. Grossman L.I. Mol. Reprod. Dev. 2003; 66: 8-16Crossref PubMed Scopus (95) Google Scholar, 17Hüttemann M. Schmidt T.R. Grossman L.I. Gene (Amst.). 2003; 312: 95-102Crossref PubMed Scopus (47) Google Scholar); CcO is allosterically regulated through adenine nucleotides and thyroid hormone T2 (reviewed in Ref. 18Ludwig B. Bender E. Arnold S. Hüttemann M. Lee I. Kadenbach B. Chembiochem. 2001; 2: 392-403Crossref PubMed Scopus (185) Google Scholar); and there is clear evidence that CcO can be phosphorylated (19Bender E. Kadenbach B. FEBS Lett. 2000; 466: 130-134Crossref PubMed Scopus (149) Google Scholar, 20Miyazaki T. Neff L. Tanaka S. Horne W.C. Baron R. J. Cell Biol. 2003; 160: 709-718Crossref PubMed Scopus (182) Google Scholar, 21Steenaart N.A. Shore G.C. FEBS Lett. 1997; 415: 294-298Crossref PubMed Scopus (73) Google Scholar, 22Prabu S.K. Anandatheerthavarada H.K. Raza H. Srinivasan S. Spear J.F. Avadhani N.G. J. Biol. Chem. 2006; 281: 2061-2070Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). We have recently shown that CcO is targeted for cAMP-dependent phosphorylation on tyrosine 304 of catalytic subunit I in vivo, which leads to strong enzyme inhibition (23Lee I. Salomon A.R. Ficarro S. Mathes I. Lottspeich F. Grossman L.I. Hüttemann M. J. Biol. Chem. 2005; 280: 6094-6100Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). In addition the substrate of CcO, cytochrome c, can be phosphorylated in vivo on tyrosine 97, which also leads to an inhibition of the reaction with CcO (24Lee I. Salomon A.R. Yu K. Doan J.W. Grossman L.I. Hüttemann M. Biochemistry. 2006; 45: 9121-9128Crossref PubMed Scopus (78) Google Scholar). The complex regulation of CcO suggests an important role for CcO in the overall regulation of aerobic energy production. This hypothesis is supported by several recent studies suggesting that metabolic flux in the ETC is tightly coupled to CcO under physiological conditions, which assigns the role of the rate-limiting step to CcO (25Villani G. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1166-1171Crossref PubMed Scopus (174) Google Scholar, 26Villani G. Greco M. Papa S. Attardi G. J. Biol. Chem. 1998; 273: 31829-31836Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 27Kunz W.S. Kudin A. Vielhaber S. Elger C.E. Attardi G. Villani G. J. Biol. Chem. 2000; 275: 27741-27745Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 28Acin-Perez R. Bayona-Bafaluy M.P. Bueno M. Machicado C. Fernandez-Silva P. Perez-Martos A. Montoya J. Lopez-Perez M.J. Sancho J. Enriquez J.A. Hum. Mol. Genet. 2003; 12: 329-339Crossref PubMed Scopus (66) Google Scholar).The effect of TNFα on the ETC and in particular on CcO has not been studied. We hypothesized that TNFα mediates an early inhibitory effect on the OxPhos system and specifically inhibits CcO, because oxygen utilization is impaired in conditions involving TNFα. We predicted that CcO inhibition in turn leads to a decrease in ΔΨm and, consequently, reduced ATP levels. We here investigated the effect of TNFα on mammalian liver CcO in cell homogenates, isolated mitochondria, and purified CcO. We found that CcO activity was consistently reduced in bovine and murine liver cell homogenates in response to TNFα, whereas CcO activity of isolated mitochondria and isolated CcO in response to TNFα treatment did not change. This indicates that an intact cellular infrastructure is required for signal transduction from the cell surface, triggered by TNFα receptors, to the mitochondria and subsequently to CcO as a terminal target of this pathway. In addition, we show that TNFα treatment leads to reduced ΔΨm and ATP levels. We propose a model in which TNFα-mediated CcO inhibition leads to tissue dysoxia, which suppresses aerobic ATP production and causes a shift to the glycolytic pathway.EXPERIMENTAL PROCEDURESIsolation of Bovine and Murine Mitochondria—The chemicals were purchased from Sigma unless otherwise stated. Fresh C57BL/6 mouse liver tissue (2 g) was chopped into small pieces using scissors, whereas frozen cow liver tissue (200 g) was passed through a meat grinder, leaving most cells intact. The samples were supplemented with five volumes of buffer A (250 mm sucrose, 20 mm Tris-Cl (pH 7.4), 2 mm EDTA, 10 mm KF, 2 mm EGTA, 1 mm PMSF) and incubated for 5 min in the absence or presence of 20 ng/ml TNFα at room temperature. Mitochondria were isolated under conditions that preserve the physiological phosphorylation status as previously described (23Lee I. Salomon A.R. Ficarro S. Mathes I. Lottspeich F. Grossman L.I. Hüttemann M. J. Biol. Chem. 2005; 280: 6094-6100Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 24Lee I. Salomon A.R. Yu K. Doan J.W. Grossman L.I. Hüttemann M. Biochemistry. 2006; 45: 9121-9128Crossref PubMed Scopus (78) Google Scholar). Briefly, using 200 g of tissue, mitochondria were isolated at 4 °C, and buffer A was supplemented with unspecific tyrosine phosphatase inhibitor sodium vanadate (1 mm) for all subsequent steps. The samples were further homogenized with a commercial blender using a 5-fold volume of buffer A. After centrifugation (650 × g, 10 min), the supernatant was collected through cheesecloth. The pellet was homogenized and centrifuged to increase mitochondrial yield. Combined supernatants were centrifuged (16,300 × g, 20 min), and mitochondria were resuspended in 200 ml of buffer A using a Teflon homogenizer (150 rpm, four strokes), and buffer A was added to a volume of 2.5 L and centrifuged at low speed (370 × g for 5 min) to remove contaminants. The supernatant was centrifuged (16,300 × g, 20 min) to collect mitochondria, which were washed one more time by resuspension and centrifugation as described above. The mitochondrial pellet was resuspended in buffer A and adjusted to a final protein concentration of 20 mg/ml.Isolation of CcO—CcO isolation starting from purified cow liver mitochondria was performed as previously described (23Lee I. Salomon A.R. Ficarro S. Mathes I. Lottspeich F. Grossman L.I. Hüttemann M. J. Biol. Chem. 2005; 280: 6094-6100Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Per 1 ml of isolated mitochondria, 250 μl of buffer B (1 m KH2PO4, pH 7.4, 10 mm KF, 2 mm EGTA, 1 mm sodium vanadate) were added. Mitochondrial proteins were solubilized by dropwise addition of 1 ml of 20% Triton X-114/1 g of mitochondrial protein under stirring. The membrane fraction was collected by centrifugation for 30 min at 195,000 × g and resuspended with a Teflon glass homogenizer after addition of 100 ml of buffer C (200 mm KH2PO4, pH 7.4, 10 mm KF, 2 mm EGTA, 1 mm sodium vanadate). The sample was centrifuged for 25 min at 195,000 × g, and the pellet was resuspended in 100 ml of buffer D (200 mm KH2PO4, pH 7.4, 5% Triton X-100, 10 mm KF, 2 mm EGTA, and 1 mm sodium vanadate) as above, followed by centrifugation (20 min at 195,000 × g). The CcO-containing supernatant was collected, and CcO was further extracted from the pellet by repeating the previous step two times. The supernatants were combined and diluted with 3 volumes of dH2O. The sample was applied to 300 ml of DEAE Sephacel (fast flow; GE Healthcare) equilibrated with buffer E (50 mm KH2PO4, pH 7.4, 0.1% Triton X-100). The column was washed with 800 ml of buffer E, and the proteins were eluted using a linear gradient from 50 mm to 1 m KH2PO4 (pH 7.4, 0.1% Triton X-100). Eluted fractions were analyzed by spectrophotometer, and CcO-containing fractions were combined. Sodium cholate was added (1% w/v) with stirring and pH was adjusted to pH 7.4. Fractionations were performed with 28% ammonium sulfate for 14 h, 37% for 1 h, and 45% for 5 min; the proteins were collected after each step by centrifugation for 15 min at 27,000 × g. Precipitated proteins were dissolved in 250 mm sucrose, 20 mm Tris-Cl (pH 7.4), 2 mm EDTA and stored at -80 °C after spectrophotometric determination of CcO concentration (29von Jagow G. Klingenberg M. FEBS Lett. 1972; 24: 278-282Crossref PubMed Scopus (54) Google Scholar) and purity (30Kadenbach B. Stroh A. Ungibauer M. Kuhn-Nentwig L. Buge U. Jarausch J. Methods Enzymol. 1986; 126: 32-45Crossref PubMed Scopus (89) Google Scholar).Cytochrome c Oxidase Activity Measurements with Isolated CcO, Mitochondria, and Homogenates from Tissue and Cultured Cells—To obtain regulatory-competent CcO, cholate has to be removed because it tightly binds to purified CcO (31Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1996; 272: 1136-1144Crossref PubMed Scopus (1898) Google Scholar) at nucleotide-binding sites (10Napiwotzki J. Shinzawa-Itoh K. Yoshikawa S. Kadenbach B. Biol. Chem. 1997; 378: 1013-1021Crossref PubMed Scopus (91) Google Scholar), and cardiolipin that was damaged or removed during enzyme isolation has to be replaced (32Lee I. Kadenbach B. Eur. J. Biochem. 2001; 268: 6329-6334Crossref PubMed Scopus (38) Google Scholar). CcO was dialyzed in the presence of 0.1 mm ATP and a 40-fold molar excess of cardiolipin in 50 mm KH2PO4 (pH 7.4), 1% Tween 20, 2 mm EGTA, 10 mm KF. CcO activity was analyzed in a closed 200-μl chamber equipped with a micro Clark-type oxygen electrode (Oxygraph system; Hansatech). Measurements were performed in the presence of 5 mm ADP, an allosteric activator of CcO, or 5 mm ATP, an allosteric inhibitor of CcO, after incubation with an ATP regenerating system as described (23Lee I. Salomon A.R. Ficarro S. Mathes I. Lottspeich F. Grossman L.I. Hüttemann M. J. Biol. Chem. 2005; 280: 6094-6100Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The measurements were carried out with 2 μm CcO at 25 °C in the presence of 20 mm ascorbate and increasing amounts of cow heart cytochrome c from 0-40 μm. Oxygen consumption was recorded on a computer and analyzed with the Oxygraph plus software. Turnover number is defined as oxygen consumed (μmol)/(s·CcO (μmol)). Mitochondria, tissue, and cell homogenates were incubated in the presence or absence of TNFα in incubation buffer (250 mm sucrose, 20 mm K-HEPES, pH 7.4, 10 mm MgSO4, 2 mm KH2PO4, 1 mm PMSF, 10 mm KF, 2 mm EGTA, 2 μm oligomycin). CcO activity measurements were performed in 10 mm K-HEPES (pH 7.4), 40 mm KCl, 1% Tween 20, 2 μm oligomycin, 1 mm PMSF, 10 mm KF, 2 mm EGTA using the same experimental setup as above after washing, sonication, and centrifugation of the samples as described (23Lee I. Salomon A.R. Ficarro S. Mathes I. Lottspeich F. Grossman L.I. Hüttemann M. J. Biol. Chem. 2005; 280: 6094-6100Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Murine hepatocytes were freshly obtained from mice. Protein concentration was determined with the DC protein assay kit (Bio-Rad).Cell Culture—H2.35 murine hepatocytes were grown in modified Dulbecco's medium (Invitrogen) supplemented with 10% fetal bovine serum and 1000 units penicillin/streptomycin at 37 °C and a 5% CO2 atmosphere. For CcO activity measurements, the cells were washed with phosphate-buffered saline (PBS), harvested by scraping in the presence of 10 ml of PBS, collected by centrifugation (50 × g, 5 min), and washed once more with PBS.Antibody Production—Customized rabbit polyclonal antibodies were generated against the phosphotyrosine 304 epitope of CcO subunit I by Abgent (San Diego, CA). Two rabbits were immunized with the cysteine-conjugated synthetic peptide GMDVDTRApYFTSAC according to the vendor's protocol. Antibodies recognizing the unphosphorylated peptide were removed by affinity absorption against column-bound cysteine-conjugated synthetic GMDVDTRAYFTSAC peptide. Antibodies were tested on the synthetic phosphorylated and unphosphorylated peptides, on Tyr304-phosphorylated and unphosphorylated CcO (23Lee I. Salomon A.R. Ficarro S. Mathes I. Lottspeich F. Grossman L.I. Hüttemann M. J. Biol. Chem. 2005; 280: 6094-6100Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), and phosphotyrosine-conjugated BSA by Western analysis, revealing a specific signal for the phosphorylated peptide and Tyr304-phosphorylated CcO subunit I in the expected size.Western Analysis—SDS-PAGE with purified liver CcO or isolated mitochondria was carried out as described previously (33Kadenbach B. Jarausch J. Hartmann R. Merle P. Anal. Biochem. 1983; 129: 517-521Crossref PubMed Scopus (376) Google Scholar). Protein transfer time on a nitrocellulose membrane was 60 min to facilitate efficient transfer of larger CcO subunits. Antiphosphoserine and -threonine antibodies were sets of four (1C8, 4A3, 4A9, and 16B4) and three (1E11, 4D11, and 14B3) individual monoclonal antibodies (Calbiochem), respectively, whereas a single antiphosphotyrosine antibody was used (4G10; Upstate Biotechnology). CcO subunit IV isoform 1 was detected with a monoclonal antibody (10G8; Molecular Probes). In addition, a customized CcO subunit I phosphotyrosine 304 specific antibody was used. Western analysis was performed with a 1:5000 dilution of primary antibodies and horseradish peroxidase-conjugated secondary antibodies (1:10000 dilution; GE Healthcare), and signals were detected using the ECL plus Western blotting detection kit (GE Healthcare).In-gel Tryptic Digest and Protein Identification by Mass Spectrometry—The tryptic digest was performed as described (34Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7765) Google Scholar). Briefly, the bands were excised from Coomassie-stained gels, cut into 1-mm cubes, and incubated in 50 μl of acetonitrile (Merck) for 10 min. Acetonitrile was removed, and gel particles were swollen in 150 μl of 10 mm dithiothreitol (Merck), 10 mm NH4HCO3 (Merck) and incubated for 1 h at 60 °C. After cooling to room temperature, the solution was replaced by 150 μl of 50 mm iodoacetamide, 10 mm NH4HCO3 and incubated for 45 min at room temperature in the dark with occasional vortexing. The solution was removed, and gel pieces were washed with 150 μl of 10 mm NH4HCO3, which was replaced by acetonitrile to dehydrate gel pieces, which were dried under vacuum. Trypsin (bovine, sequencing grade; Roche Applied Science) was diluted in digestion buffer (10 mm NH4HCO3) to 0.1 μg/μl. Gel pieces were covered with digestion buffer and incubated overnight at 37 °C. The supernatant was transferred to a tube, and gel pieces were incubated for 10 min with occasional mixing in 50 μl of elution buffer (50% CH3CN/50% HCOOH; Merck). Elution was repeated two more times, and supernatants were combined and dried under vacuum. For further cleavage of peptides, tryptic fragments were incubated with 10% cyanogen bromide (w/v) in 100 μl of 70% formic acid (Merck) overnight at room temperature in the dark. 300 μl of dH2O were added, and the samples were dried under vacuum. The peptide mixture was dissolved in 0.1% trifluoroacetic acid/dH2O (Applied Biosystems) and separated by reversed phase HPLC on a Phenomenex 150 × 1-mm column with a flow rate of 0.16 ml/min. The gradient used was 0-60% buffer B in 60 min, 80% buffer B in 70 min (buffer A, 0.1% trifluoroacetic acid/dH2O; buffer B, 0.08% trifluoroacetic acid/CH3CN). Matrix-assisted laser desorption ionization time-of-flight analysis of the tryptic/CNBr HPLC peaks was performed on a Bruker mass spectrometer in positive ion mode. For mass fingerprint analysis, each raw spectrum was opened in Bruker Xtof (version 5.1.5) applying advanced baseline correction and noise removal. The mass spectrometer was calibrated with calibration II standard (Bruker Daltonics), nine peptides (m/z 757.3992, 1046.5418, 1296.6848, 1347.7354, 1619.8223, 1758.9326, 2093.0862, 2465.1983, and 3174.4710; quadratic mode). Filter peak list for monoisotopic masses only was enabled; the peak detection threshold was manually adjusted above background. Peak lists were analyzed with the Mascot public interface and searched against NCBInr data base (parameters: trypsin and CNBr were selected, with one potential missed cleavage; Mascot mass fingerprint matrix-assisted laser desorption ionization was selected as the instrument type, oxidized methionine was selected as variable modification, and carbamidomethylated cysteine was selected as fixed modification; MH+ monoisotopic masses and peptide tolerance were 100 ppm; taxonomy field was set as “other mammalia,” because of the bovine origin of the samples). The results were scored using probability-based Mowse score (protein score) is defined as 10 × log(p), where p is the probability that the observed match is a random event. Only significant protein scores were chosen.ATP Assay—Fresh mouse liver tissue (50 mg) was partially disrupted in 800 μl of incubation buffer with a Teflon microtube pestle by applying 10 strokes. The suspension was incubated for 5 min in the presence or absence of 20 ng/ml TNFα. H2.35 cells were collected by scraping after applying similar incubation conditions and immediately stored in aliquots at -80 °C until measurement. ATP was released using the boiling method, by adding 300 μl of boiling buffer (100 mm Tris-Cl, pH 7.75, 4 mm EDTA) and immediate transfer to a boiling water bath for 2 min. The samples were put on ice and sonicated. The samples were diluted 300-fold, and 50 μl were utilized to determine the ATP concentration using the ATP biolumescence assay kit HS II (Roche Applied Science) according to the manufacturer's protocol. The experiments were performed in triplicates, and the data were standardized to the protein concentration using the DC protein assay kit (Bio-Rad).Measurement of Mitochondrial Membrane Potential ΔΨm—Cultured H2.35 cells were grown to 75% confluence. To assess relative changes in mitochondrial membrane potential (ΔΨm), the cells were collected after trypsinization and washed once with PBS. The cells were incubated for 5 min with 20 ng/ml TNFα or 10 μm valinomycin in PBS at 37 °C. After incubation the cells were washed with PBS and incubated for 10 min in PBS containing 1 μm JC-1 (Molecular Probes). JC-1 is able to selectively enter mitochondria, occurs as a monomer at low concentration or at low membrane potential, and emits green fluorescence (35Reers M. Smith T.W. Chen L.B. Biochemistry. 1991; 30: 4480-4486Crossref PubMed Scopus (869) Google Scholar). At higher membrane potential JC-1 forms aggregates that show red fluorescence. Mitochondrial membrane potential ΔΨm was measured with a FACScan (Becton-Dickinson) flow cytometer equipped with a 488-nm argon ion laser, and the data obtained were analyzed with the Cell Quest software. Fluorescence was measured using a 585 ± 42-nm band-pass filter, and 10,000 events were analyzed in each run.RESULTSTNFα Leads to Strong Cytochrome c Oxidase Inhibition in Bovine Hepatocytes—TNFα activates several pathways such as inflammatory, apoptotic, and survival pathways. Several recent studies suggested that TNFα may target mitochondria. However, most of these studies addressed late effects of TNFα, such as increased apoptosis and decreased ETC activity that was observed after 24-48 h (36Zhu J. Liu M. Kennedy R.H. Liu S.J. Cytokine. 2006; 34: 96-105Crossref PubMed Scopus (53) Google Scholar, 37Lopez-Armada M.J. Carames B. Martin M.A. Cillero-Pastor B. Lires-Dean M. Fuentes-Boquete I. Arenas J. Blanco F.J. Osteoarthritis Cartilage. 2006; 14: 1011-1022Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). TNFα may also exert early effects in the cell at the level of the mitochondrial OxPhos system. Because CcO is the terminal and possibly rate-liming enzyme in the ETC, we investigated the early effect of TNFα on CcO activity. As a model system, we first tested the effect of TNFα on chunky cow liver homogenates. Under those conditions most of the cells present are intact, allowing signal transduction in a physiological cellular context. Both optimal incubation time and the concentration of TNFα were determined in a series of preliminary tests (not shown). Five min of incubation at a concentration of 20 ng/ml TNFα was found to produce a strong and consistent response and was applied during all experiments. CcO activity was measured by adding increasing amounts of substra" @default.
• W1986618045 created "2016-06-24" @default.
• W1986618045 creator A5002735757 @default.
• W1986618045 creator A5037635536 @default.
• W1986618045 creator A5041718533 @default.
• W1986618045 creator A5048054959 @default.
• W1986618045 creator A5069766252 @default.
• W1986618045 date "2008-07-01" @default.
• W1986618045 modified "2023-10-15" @default.
• W1986618045 title "Tumor Necrosis Factor α Inhibits Oxidative Phosphorylation through Tyrosine Phosphorylation at Subunit I of Cytochrome c Oxidase" @default.
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