Arrangement of subunit IV in beef heart cytochrome c oxidase probed by chemical labeling and protease digestion experiments

The arrangement of subunit IV in beef heart cytochrome c oxidase has been explored by chemical labeling and protease digestion studies. This subunit has been purified from four samples of cytochrome c oxidase that had been reacted with N-(4-azido-2-nitrophenyl)-2-aminoethyl[35S]-sulfonate (NAP-taurine), diazobenzene[35S]sulfonate, 1-myristoyl-2-[12-[(4-azido-2-nitrophenyl)amino]lauroyl]-sn-glycero-3- [14C]phosphocholine (I), and 1-palmitoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-[3H]phosphocholine (II), respectively. The labeled polypeptide was then fragmented by cyanogen bromide, at arginyl side chains with trypsin (after maleylation), and the distribution of the labeling within the sequence was analyzed. The N-terminal part of subunit IV (residues 1-71) was shown to be heavily labeled by water-soluble, lipid-insoluble reagents but not by the phospholipid derivatives. These latter reagents labeled only in the region of residues 62-122, containing the long hydrophobic and putative membrane-spanning stretch. Trypsin cleavage of native cytochrome c oxidase complex at pH 8.2 was shown to clip the first seven amino acids from subunit IV. This cleavage was found to occur in submitochondrial particles but not in mitochondria or mitoplasts. These results are interpreted to show that subunit IV is oriented with its N terminus on the matrix side of the mitochondrial inner membrane and spans the membrane with the extended sequence of hydrophobic lipid residues 79-98 buried in the bilayer.     

Weight loss and race modulate nitric oxide metabolism in overweight women

Weight reduction is associated with a decrease in the risk of developing cardiovascular disease. We hypothesized that, given the central role of reactive oxygen and nitrogen species in vascular biology, changes in nitric oxide (NO) metabolism contribute to benefits of weight loss. In a controlled weight loss trial involving overweight (body mass index (BMI) = 27-30 kg/m(2)), otherwise healthy premenopausal Caucasian and African-American women, serum levels of nitrite and nitrate, as an index of NO production, and protein 3-nitrotyrosine and myeloperoxidase (MPO), as markers of inflammation, were determined. Testing was performed before and after reduction to normal body weight (BMI < 25) under standardized conditions, with controlled diet, and following 1 month of weight maintenance. After weight loss there was an increase in nitrite and nitrate, and levels were higher among African-American women relative to Caucasian counterparts. Whereas weight loss was associated with a decrease in 3-nitrotyrosine in Caucasian women, no change was observed among African-Americans. Furthermore, MPO levels increased in response to weight loss for African-Americans, but did not change in Caucasian women. These data indicate that vascular production of reactive nitrogen species can be modulated by race and weight loss and highlight important racial differences in these responses and are discussed in the context of risk for developing vascular disease.     

Oxidative modification of hepatic mitochondria protein thiols: effect of chronic alcohol consumption

Redox modification of mitochondrial proteins is thought to play a key role in regulating cellular function, although direct evidence to support this hypothesis is limited. Using an in vivo model of mitochondrial redox stress, ethanol hepatotoxicity, the modification of mitochondrial protein thiols was examined using a proteomics approach. Specific labeling of reduced thiols in the mitochondrion from the livers of control and ethanol-fed rats was achieved by using the thiol reactive compound (4-iodobutyl)triphenylphosphonium (IBTP). This molecule selectively accumulates in the organelle and can be used to identify thiol-containing proteins. Mitochondrial proteins that have been modified are identified by decreased labeling with IBTP using two-dimensional SDS-PAGE followed by immunoblotting with an antibody directed against the triphenylphosphonium moiety of the IBTP molecule. Analyses of these data showed a significant decrease in IBTP labeling of thiols present in specific mitochondria matrix proteins from ethanol-fed rats compared with their corresponding controls. These proteins were identified as the low-K(m) aldehyde dehydrogenase and glucose-regulated protein 78. The decrease in IBTP labeling in aldehyde dehydrogenase was accompanied by a decrease in specific activity of the enzyme. These data demonstrate that mitochondrial protein thiol modification is associated with chronic alcohol intake and might contribute to the pathophysiology associated with hepatic injury. Taken together, we have developed a protocol to chemically tag and select thiol-modified proteins that will greatly enhance efforts to establish posttranslational redox modification of mitochondrial protein in in vivo models of oxidative or nitrosative stress.     

Mitochondria,nitric oxide,and cardiovascular dysfunction

Cardiovascular diseases encompass a wide spectrum of abnormalities with diverse etiologies. The molecular mechanisms underlying these disorders include a variety of responses such as changes in nitric oxide- (NO) dependent cell signaling and increased apoptosis. An interesting aspect that has received little or no attention is the role mitochondria may play in the vascular changes that occur in both atherosclerosis and hypertension. With the changing perspective of the organelle from simply a role in metabolism to a contributor to signal transduction pathways, the role of mitochondria in cells with relatively low energy demands such as the endothelium has become important to understand. In this context, the definition of the NO-cytochrome c oxidase signaling pathway and the influence this has on cytochrome c release is particularly important in understanding apoptotic mechanisms involving the mitochondrion. This review examines the role of compromised mitochondrial function in a variety of vascular pathologies and the modulation of these effects by NO. The interaction of NO with the various mitochondrial respiratory complexes and the role NO plays in modulating mitochondrial-mediated apoptosis in these systems will be discussed.     

Endothelial dysfunction is induced by proinflammatory oxidant hypochlorous acid

The myeloperoxidase (MPO)-derived oxidant hypochlorous acid (HOCl) plays a role in tissue injury under inflammatory conditions. The present study tests the hypothesis that HOCl decreases nitric oxide (NO) bioavailability in the vasculature of Sprague-Dawley rats. Aortic ring segments were pretreated with HOCl (1-50 microM) followed by extensive washing. Endothelium-dependent relaxation was then assessed by cumulative addition of acetylcholine (ACh) or the calcium ionophore A23187. HOCl treatment significantly impaired both ACh- and A23187-mediated relaxation. In contrast, endothelium-independent relaxation induced by sodium nitroprusside was unaffected. The inhibitory effect of HOCl on ACh-induced relaxation was reversed by exposure of ring segments to L-arginine but not D-arginine. In cellular studies, HOCl did not alter endothelial NO synthase (NOS III) protein or activity, but inhibited formation of the NO metabolites nitrate (NO3(-) and nitrite (NO2(-). The reduction in total NO metabolite production in bovine aortic endothelial cells was also reversed by addition of L-arginine. These data suggest that HOCl induces endothelial dysfunction via modification of L-arginine.     

Metabolic Plasticity in Resting and Thrombin Activated Platelets

Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.     

Hydrogen sulfide mediates vasoactivity in an O2-dependent manner

Hydrogen sulfide (H(2)S) has recently been shown to have a signaling role in vascular cells. Similar to nitric oxide (NO), H(2)S is enzymatically produced by amino acid metabolism and can cause posttranslational modification of proteins, particularly at thiol residues. Molecular targets for H(2)S include ATP-sensitive K(+) channels, and H(2)S may interact with NO and heme proteins such as cyclooxygenase. It is well known that the reactions of NO in the vasculature are O(2) dependent, but this has not been addressed in most studies designed to elucidate the role of H(2)S in vascular function. This is important, since H(2)S reactions can be dramatically altered by the high concentrations of O(2) used in cell culture and organ bath experiments. To test the hypothesis that the effects of H(2)S on the vasculature are O(2) dependent, we have measured real-time levels of H(2)S and O(2) in respirometry and vessel tension experiments, as well as the associated vascular responses. A novel polarographic H(2)S sensor developed in our laboratory was used to measure H(2)S levels. Here we report that, in rat aorta, H(2)S concentrations that mediate rapid contraction at high O(2) levels cause rapid relaxation at lower physiological O(2) levels. At high O(2), the vasoconstrictive effect of H(2)S suggests that it may not be H(2)S per se but, rather, a putative vasoactive oxidation product that mediates constriction. These data are interpreted in terms of the potential for H(2)S to modulate vascular tone in vivo.     

Acquisition of Temozolomide Chemoresistance in Gliomas Leads to Remodeling of Mitochondrial Electron Transport Chain

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients.