doi: 10.1074/jbc.M801954200. Epub 2008 Jun 5.
Tumor necrosis factor alpha inhibits oxidative phosphorylation through tyrosine phosphorylation at subunit I of cytochrome c oxidase
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- PMID:18534980
- PMCID: PMC3258931
- DOI: 10.1074/jbc.M801954200
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Tumor necrosis factor alpha inhibits oxidative phosphorylation through tyrosine phosphorylation at subunit I of cytochrome c oxidase
Lobelia Samavati et al. J Biol Chem..
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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 alpha (TNFalpha) has been shown to play a central role in inflammation. We hypothesized that TNFalpha-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 TNFalpha treatment led to an approximately 60% reduction in CcO activity. In contrast, there was no direct effect of TNFalpha on CcO activity using isolated mitochondria and purified CcO, indicating that a TNFalpha-triggered intracellular signaling cascade mediates CcO inhibition. CcO isolated after TNFalpha treatment showed tyrosine phosphorylation on CcO catalytic subunit I and was approximately 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 TNFalpha. Concomitantly, cellular ATP was more than 35 and 64% reduced in murine hepatocytes and H2.35 cells. We postulate that an important contributor in TNFalpha-mediated pathologies, such as sepsis, is energy paucity, which parallels the poor tissue oxygen extraction and utilization found in such patients.
Figures
TNFαleads to CcO inhibition in cow liver tissue.A, cow liver tissue was partially homogenized leaving most cells intact and incubated in the presence (squares) or absence (triangles) of 20 ng/ml TNFα for 5 min at 20 °C. CcO activity was measured in the presence of 5 mm ATP (closed symbols, inhibited) or ADP (open symbols, stimulated) by the addition of increasing amounts of cytochromec. Specific activity is defined as consumed O2 (μmol)/(min·total protein (mg)). Shown are representative results of three independent experiments.TN, turnover number.B, Western analysis using an antibody against CcO subunit IV (isoform 1) indicates no changes of CcO amount after short term TNFα treatment. The mitochondria were isolated with or without TNFα treatment (see above). Thirty μg of total mitochondrial protein was loaded, and CcO subunit IV was detected with a monoclonal antibody.
Effect of TNFαon CcO activity is not species-dependent. Fresh mouse liver tissue was partially homogenized and incubated in the presence or absence of 20 ng/ml TNFα for 5 min at 20 °C. CcO activity was measured in the presence of allosteric effectors ATP (closed symbols) or ADP (open symbols) as described in Fig. 1.TN, turnover number.
TNFαdoes not inhibit isolated CcO. CcO isolated under standard conditions (see “Experimental Procedures”) was incubated in the presence or absence of TNFα. Turnover number (TN) is defined as consumed O2 (μmol)/(s·CcO (μmol)).
TNFαdoes not lead to CcO inhibition in isolated mitochondria. Mouse liver mitochondria were freshly isolated. TNFα-treated (squares) and untreated (triangles) mitochondria showed no difference for ADP-stimulated (open symbols) and ATP-inhibited (closed symbols) respiration (see Fig. 1 for details).TN, turnover number.
Isolation of CcO in the presence of TNFαleads to tyrosine phosphorylation of CcO subunit I at tyrosine 304. Cow liver CcO was isolated side by side after incubation of tissue with (+) or without (-) TNFα.A, CcO subunits were separated by SDS-PAGE, and protein bands were stained with Coomassie Blue. Subunits are as indicated.B, Western analysis with a phosphotyrosine-specific antibody revealed a strong signal only for the TNFα-treated sample at the size of subunit I (lane 4).Lane 1, phosphotyrosine-conjugated BSA (positive control; produces a broad, smeared signal);lane 2, ovalbumin (negative control);lane 3, CcO isolated in the absence of TNFα.C, a customized polyclonal antibody raised against the phosphorylated tyrosine 304 epitope of CcO subunit I produced a strong signal for the TNFα treated sample (lane 4) but not for control CcO (lane 3).Lanes 1-4 as inB. Note that in contrast toB, phosphotyrosine-conjugated BSA serves as a negative control inC, indicating that the epitope surrounding phosphotyrosine 304 is required for binding.Box, the synthetic phosphopeptide GMDVDTRApYFTSAC (+), which was used to generate the phosphoepitope-specific antibody and the unphosphorylated peptide GMDVDTRAYFTSAC (-; 100 ng each) were spotted on a membrane and subjected to Western analysis using the phosphotyrosine 304-specific antibody. A strong and specific signal was observed for the phosphorylated peptide (+). Shown are representative results of five independent experiments.
CcO isolated in the presence of TNFαis inhibited and shows pronounced sigmoidal kinetics. Cow liver tissue was incubated in the presence or absence of TNFα. Mitochondria and subsequently CcO were isolated under conditions that preserve the physiological phosphorylation status (see “Experimental Procedures”). The activity of solubilized CcO pretreated to restore regulatory properties as described (23) was measured in the presence of 5 mm ATP (closed symbols) or ADP (open symbols). Turnover number (TN) is defined as consumed O2 (μmol)/(s·CcO (μmol)).
Phosphorylation of tyrosine 304 correlates with changes in CcO activity. To assess a time-dependent change in CcO phosphorylation (A) and respiration (B), bovine liver tissue was treated with a lower concentration of 10 ng/ml TNFα for the indicated time periods, followed by immediate mitochondria isolation and measurement of respiration. Specific activity is defined as consumed O2 (μmol)/(min·total mitochondrial protein (mg)) (B). Western blot analysis of subsequently isolated CcO (6 μg/lane) using the phosphotyrosine 304-specific antibody showed a time-dependent increase of CcO subunit I phosphorylation, which was maximal after 12 min (A).C, correlation between tyrosine 304 phosphorylation (A) and CcO specific activity at maximal turnover (B, at 30 μm cytochromec) are presented time-dependently. Films from Western analysis were scanned and intensities of individual bands analyzed with ImageQuant software (version 5, Molecular Dynamics) correcting for background signal. The signal obtained after 12 min was arbitrarily set to 100%.TN, turnover number.
TNFαtreatment leads to a decrease in the mitochondrial membrane potential. Murine H2.35 cells were incubated for 5 min with TNFα or potassium ionophore valinomycin in PBS. Relative membrane potentials were determined using the fluorescent probe JC-1. Measurements were performed with a FACScan (Becton-Dickinson) flow cytometer equipped with a 488-nm argon ion laser, and data obtained were analyzed with the Cell Quest software. Fluorescence was measured using a 585 ± 42-nm band-pass filter, and 10,000 cells were analyzed in each run. In comparison with the untreated cells (Control), TNFα treatment leads to membrane depolarization (B) similar to that observed with valinomycin (A).
Effect of TNFαon cellular ATP levels. Mouse liver tissue and H2.35 cells were incubated with 20 ng/ml TNFα for 5 min at 20 °C. ATP concentrations were measured using the bioluminescence method. ATP concentrations of the control samples were set to 100%. ATP levels were reduced by 35 and 64% in liver tissue and H2.35 cells, respectively.
Model of TNFα-mediated CcO inhibition. TNFα binds to its cognate cell surface receptors, which initiates receptor ligation triggering the intracellular TNFα signaling cascade. As part of this process a mitochondrial tyrosine kinase (TK) is activated, which phosphorylates CcO subunit I on tyrosine 304. CcO inhibition leads to a decrease of the mitochondrial membrane potential ΔΨm. This in turn provides fewer substrate protons for ATP synthase, resulting in lowered ATP levels, which over extended periods of time leads to cell death caused by energy depletion.
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