-
[show abstract]
[hide abstract]
ABSTRACT: Cytochrome c oxidase (CcO) is the terminal oxidase of the mitochondrial electron transport chain. This bigenomic enzyme in mammals contains 13 subunits of which the 3 catalytic subunits are encoded by the mitochondrial genes. The remaining 10 subunits with suspected roles in the regulation, and/or assembly, are coded by the nuclear genome. The enzyme contains two heme groups (heme a and a3) and two Cu(2+) centers (Cu(2+) A and Cu(2+) B) as catalytic centers and handles more than 90% of molecular O(2) respired by the mammalian cells and tissues. CcO is a highly regulated enzyme which is believed to be the pacesetter for mitochondrial oxidative metabolism and ATP synthesis. The structure and function of the enzyme are affected in a wide variety of diseases including cancer, neurodegenerative diseases, myocardial ischemia/reperfusion, bone and skeletal diseases, and diabetes. Despite handling a high O(2) load the role of CcO in the production of reactive oxygen species still remains a subject of debate. However, a volume of evidence suggests that CcO dysfunction is invariably associated with increased mitochondrial reactive oxygen species production and cellular toxicity. In this paper we review the literature on mechanisms of multimodal regulation of CcO activity by a wide spectrum of physiological and pathological factors. We also review an array of literature on the direct or indirect roles of CcO in reactive oxygen species production.
Free radical biology & medicine 07/2012; 53(6):1252-63. · 5.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Alcohol treatment induces oxidative stress by a combination of increased production of partially reduced oxygen species and decreased cellular antioxidant pool, including GSH. Recently, we showed that mitochondrion-targeted CYP2E1 augments alcohol-mediated toxicity, causing an increase in reactive oxygen species production and oxidative stress. Here, we show that cytochrome c oxidase (CcO), the terminal oxidase of the mitochondrial respiratory chain, is a critical target of CYP2E1-mediated alcohol toxicity. COS-7 and Hep G2 cell lines expressing predominantly mitochondrion-targeted (Mt(++)) CYP2E1 and livers from alcohol-treated rats showed loss of CcO activity and increased protein carbonylation, which was accompanied by a decline in the steady state levels of subunits I, IVI1, and Vb of the CcO complex. This was also accompanied by reduced mitochondrial DNA content and reduced mitochondrial mRNA. These changes were more prominent in Mt(++) cells in comparison with wild type (WT) CYP2E1-expressing or ER(+) (mostly microsome-targeted) cells. In addition, mitochondrion-specific antioxidants, ubiquinol conjugated to triphenyl phosphonium, triphenylphosphonium conjugated carboxyl proxyl, and the CYP2E1 inhibitor diallyl sulfide prevented the loss of CcO activity and the CcO subunits, most likely through reduced oxidative damage to the enzyme complex. Our results suggest that damage to CcO and dissociation of respirosome complexes are critical factors in alcohol-induced toxicity, which is augmented by mitochondrion-targeted CYP2E1. We propose that CcO is one of the direct and immediate targets of alcohol-induced toxicity causing respiratory dysfunction.
Journal of Biological Chemistry 03/2012; 287(19):15284-97. · 4.77 Impact Factor
-
Ling Ren,
Sung-Hyeok Hong,
Qing-Rong Chen,
Joseph Briggs,
Jessica Cassavaugh, Satish Srinivasan,
Michael M Lizardo,
Arnulfo Mendoza,
Ashley Y Xia,
Narayan Avadhani,
Javed Khan,
Chand Khanna
[show abstract]
[hide abstract]
ABSTRACT: Ezrin links the plasma membrane to the actin cytoskeleton where it plays a pivotal role in the metastatic progression of several human cancers; however, the precise mechanistic basis for its role remains unknown. Here, we define transitions between active (phosphorylated open) and inactive (dephosphorylated closed) forms of Ezrin that occur during metastatic progression in osteosarcoma. In our evaluation of these conformations we expressed C-terminal mutant forms of Ezrin that are open (phosphomimetic T567D) or closed (phosphodeficient T567A) and compared their biologic characteristics to full-length wild-type Ezrin in osteosarcoma cells. Unexpectedly, cells expressing open, active Ezrin could form neither primary orthotopic tumors nor lung metastases. In contrast, cells expressing closed, inactive Ezrin were also deficient in metastasis but were unaffected in their capacity for primary tumor growth. By imaging single metastatic cells in the lung, we found that cells expressing either open or closed Ezrin displayed increased levels of apoptosis early after their arrival in the lung. Gene expression analysis suggested dysregulation of genes that are functionally linked to carbohydrate and amino acid metabolism. In particular, cells expressing closed, inactive Ezrin exhibited reduced lactate production and basal or ATP-dependent oxygen consumption. Collectively, our results suggest that dynamic regulation of Ezrin phosphorylation at amino acid T567 that controls structural transitions of this protein plays a pivotal role in tumor progression and metastasis, possibly in part by altering cellular metabolism.
Cancer Research 12/2011; 72(4):1001-12. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Previously we showed that hypoxia-induced mitochondrial respiratory stress in RAW 264.7 macrophages and other cells caused activation of retrograde signaling (also known as mitochondrial respiratory stress signaling) and the appearance of tartrate-resistant acid phosphatase (TRAP)-positive cells. In the present study, we used N-acetyl cysteine and ascorbate (general antioxidants) and MitoQ, a mitochondria-specific antioxidant, to investigate the role of intracellular reactive oxygen species (ROS) in osteoclast differentiation. Our results show that hypoxia-mediated mitochondrial dysfunction, as tested by disruption of mitochondrial transmembrane potential, was suppressed by MitoQ as well as by the other antioxidants. These agents also suppressed the activation of mitochondrial retrograde signaling. Interestingly, in terms of molar concentrations, MitoQ was more than 1000-fold more effective than general antioxidants in suppressing the receptor activator of nuclear factor-B ligand-induced differentiation of RAW 264.7 cells into multinucleated and TRAP-positive osteoclasts. We propose that mitochondrial function and intramitochondrial ROS play important roles in osteoclastogenesis.
Annals of the New York Academy of Sciences 03/2010; 1192:245-52. · 3.15 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: CcO (cytochrome c oxidase) is a multisubunit bigenomic protein complex which catalyses the last step of the mitochondrial electron transport chain. The nuclear-encoded subunits are thought to have roles either in regulation or in the structural stability of the enzyme. Subunit Vb is a peripheral nuclear-encoded subunit of mammalian CcO that is dramatically reduced under hypoxia. Although it has been shown to contain different ligand-binding sites and undergo modifications, its precise function is not known. In the present study we generated a cell line from RAW 264.7 murine macrophages that has a more than 80% reduced level of Vb. Functional analysis of these cells showed a loss of CcO activity, membrane potential and less ability to generate ATP. Resolution of complexes on blue native gel and two-dimensional electrophoretic analysis showed an accumulation of subcomplexes of CcO and also reduced association with supercomplexes of the electron transfer chain. Furthermore, the mitochondria from CcO Vb knock-down cells generated increased ROS (reactive oxygen species), and the cells were unable to grow on galactose-containing medium. Pulse-chase experiments suggest the role of the CcO Vb subunit in the assembly of the complex. We show for the first time the role of a peripheral, non-transmembrane subunit in the formation as well as function of the terminal CcO complex.
Biochemical Journal 05/2009; 420(3):439-49. · 4.90 Impact Factor
-
Karunakaran Chandran,
Deepika Aggarwal,
Raymond Q Migrino,
Joy Joseph,
Donna McAllister,
Eugene A Konorev,
William E Antholine,
Jacek Zielonka, Satish Srinivasan,
Narayan G Avadhani,
B Kalyanaraman
[show abstract]
[hide abstract]
ABSTRACT: Doxorubicin (DOX) is used for treating various cancers. Its clinical use is, however, limited by its dose-limiting cardiomyopathy. The exact mechanism of DOX-induced cardiomyopathy still remains unknown. The goals were to investigate the molecular mechanism of DOX-induced cardiomyopathy and cardioprotection by mitoquinone (Mito-Q), a triphenylphosphonium-conjugated analog of coenzyme Q, using a rat model. Rats were treated with DOX, Mito-Q, and DOX plus Mito-Q for 12 weeks. The left ventricular function as measured by two-dimensional echocardiography decreased in DOX-treated rats but was preserved during Mito-Q plus DOX treatment. Using low-temperature ex vivo electron paramagnetic resonance (EPR), a time-dependent decrease in heme signal was detected in heart tissues isolated from rats administered with a cumulative dose of DOX. DOX attenuated the EPR signals characteristic of the exchange interaction between cytochrome c oxidase (CcO)-Fe(III) heme a3 and CuB. DOX and Mito-Q together restored these EPR signals and the CcO activity in heart tissues. DOX strongly downregulated the stable expression of the CcO subunits II and Va and had a slight inhibitory effect on CcO subunit I gene expression. Mito-Q restored CcO subunit II and Va expressions in DOX-treated rats. These results suggest a novel cardioprotection mechanism by Mito-Q during DOX-induced cardiomyopathy involving CcO.
Biophysical Journal 03/2009; 96(4):1388-98. · 3.65 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Bimodal targeting of the endoplasmic reticular protein, cytochrome P4501A1 (CYP1A1), to mitochondria involves activation of a cryptic mitochondrial targeting signal through endoprotease processing of the protein. Here, we characterized the endoprotease that regulates mitochondrial targeting of CYP1A1. The endoprotease, which was induced by beta-naphthoflavone, was a dimer of 90 kDa and 40 kDa subunits, each containing Ser protease domains. The purified protease processed CYP1A1 in a sequence-specific manner, leading to its mitochondrial import. The glucocorticoid receptor, retinoid X receptor, and p53 underwent similar processing-coupled mitochondrial transport. The inducible 90 kDa subunit was a limiting factor in many cells and some tissues and, thus, regulates the mitochondrial levels of these proteins. A number of other mitochondria-associated proteins with noncanonical targeting signals may also be substrates of this endoprotease. Our results describe a new mechanism of mitochondrial protein import that requires an inducible cytoplasmic endoprotease for activation of cryptic mitochondrial targeting signals.
Molecular cell 11/2008; 32(1):32-42. · 14.61 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Here we report that ferricytochrome c (cyt c(3+)) induces oxidation of hydroethidine (HE) and mitochondria-targeted hydroethidine (Mito-HE or MitoSOX Red) forming highly characteristic homo- and heterodimeric products. Using an HPLC-electrochemical (EC) method, several products were detected from cyt c(3+)-catalyzed oxidation of HE and Mito-HE and characterized by mass spectrometry and NMR techniques as follows: homodimers (HE-HE, E(+)-E(+), Mito-HE-Mito-HE, and Mito-E(+)-Mito-E(+)) and heterodimers (HE-E(+) and Mito-HE-Mito-E(+)), as well as the monomeric ethidium (E(+)) and mito-ethidium (Mito-E(+)). Similar products were detected when HE and Mito-HE were incubated with mitochondria. In contrast, mitochondria depleted of cyt c(3+) were much less effective in oxidizing HE or Mito-HE to corresponding dimeric products. Unlike E(+) or Mito-E(+), the dimeric analogs (E(+)-E(+) and Mito-E(+)-Mito-E(+)) were not fluorescent. Superoxide (O(2)(*-)) or Fremy's salt reacts with Mito-HE to form a product, 2-hydroxy-mito-ethidium (2-OH-Mito-E(+)) that was detected by HPLC. We conclude that HPLC-EC but not the confocal and fluorescence microscopy is a viable technique for measuring superoxide and cyt c(3+)-dependent oxidation products of HE and Mito-HE in cells. Superoxide detection using HE and Mito-HE could be severely compromised due to their propensity to undergo oxidation.
Free Radical Biology and Medicine 04/2008; 44(5):835-46. · 5.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The environmental toxin 2,3,7,8-tetrachlorodibenzodioxin (TCDD) is a known human carcinogen; however, its precise mechanism of action remains unclear. Here we show that TCDD induces mitochondrial dysfunction, stress signaling, and tumor invasion by a mechanism similar to that described for mtDNA-depleted cells. Treatment of C2C12 cells with TCDD disrupted mitochondrial transmembrane potential in a time-dependent fashion and inhibited mitochondrial transcription and translation. TCDD also increased cytosolic [Ca(2+)](c) and RyR1-specific Ca(2+) release. These changes were associated with increased calcineurin (CnA) levels and activation of CnA-sensitive NF-kappaB/Rel (IkappaBbeta-dependent) factors. Cells treated with TCDD displayed resistance to apoptosis, increased expression of the tumor marker cathepsin L, and a high degree of invasiveness as tested by the Matrigel membrane invasion assay. These effects were reversed by the CnA inhibitor FK506, and CnA mRNA silencing suggesting that TCDD triggers a signaling pathway similar to mtDNA depletion. Taken together, these results reveal that TCDD may promote tumor progression in vivo by directly targeting mitochondrial transcription and induction of mitochondrial stress signaling.
Proceedings of the National Academy of Sciences 02/2008; 105(1):186-91. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Previously we showed that mitochondrial dysfunction induced by mitochondrial DNA depletion or treatment with electron transport chain inhibitors triggers a stress signaling involving activation of calcineurin and Ca2+-responsive factors. In this study we show that exposure of RAW 264.7 cells to hypoxia, causing increased reactive oxygen species (ROS) production and disruption of mitochondrial transmembrane potential, also induced a similar stress signaling. Hypoxia caused increased [Ca2+]c, activation of cytosolic calcineurin and induced expression of Ryanodine Receptor 2 (RyR2) gene. Prolonged hypoxia (5% O2 for 5-6 days) also induced the expression of calcitonin receptor at high levels, and those of cathepsin K, and tartarate-resistant alkaline phosphatase (TRAP) at low-moderate levels in macrophage cells. Addition of RANKL had an additive effect suggesting different mechanisms of activation. Consistent with this possibility, prolonged hypoxia induced the formation of TRAP-positive osteoclast-like cells suggesting the occurrence of an autocrine mechanism for osteoclastogenesis.
Annals of the New York Academy of Sciences 12/2007; 1117:51-61. · 3.15 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We report here the detection and characterization of spin adducts formed from the trapping of reactive oxygen species (superoxide and hydroxyl radicals) and glutathiyl and carbon-centered radicals by a newly synthesized nitrone, Mito-DEPMPO. This is a cationic nitrone spin trap with a triphenyl phosphonium cation conjugated to the DEPMPO analogue. The Mito-DEPMPO-OOH adduct, formed from the trapping of superoxide by Mito-DEPMPO, was enzymatically generated using xanthine/xanthine oxidase and neuronal nitric oxide synthase, and chemically generated by KO2 in 18-crown-6. The Mito-DEPMPO-OOH adduct exhibits an eight-line EPR spectrum with partial asymmetry arising from the alternate line-width effect. The half-life of the Mito-DEPMPO-OOH adduct is 2-2.5-times greater than that of the DEPMPO-OOH. The Mito-DEPMPO-SG adduct, formed from the trapping of glutathiyl radicals by Mito-DEPMPO, is 3-times more persistent than the analogue DEPMPO-SG adduct. In this study, we describe the EPR characterization of spin adducts formed from Mito-DEPMPO. The EPR parameters of Mito-DEPMPO adducts are distinctly different and highly characteristic. The detection of superoxide from an intact mitochondrion was feasible with Mito-DEPMPO but not with DEPMPO. We conclude that Mito-DEPMPO nitrone and its analogues are more effective than most nitrone spin traps for trapping superoxide, hydroxyl, and thiyl radicals formed in biological systems, including mitochondria.
Chemical Research in Toxicology 08/2007; 20(7):1053-60. · 3.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have previously shown that disruption of mitochondrial membrane potential by depletion of mitochondrial DNA (mtDNA) or treatment with a mitochondrial ionophore, carbonyl cyanide m-chlorophenylhydrazone, initiates a stress signaling, which causes resistance to apoptosis, and induces invasive behavior in C2C12 myocytes and A549 cells. In the present study we show that calcineurin (Cn), activated as part of this stress signaling, plays an important role in increased glucose uptake and glycolysis. Here we report that, although both insulin and insulin-like growth factor-1 receptor levels (IR and IGF1R, respectively) are increased in response to mitochondrial stress, autophosphorylation of IGF1R was selectively increased suggesting a shift in receptor pathways. Using an approach with FK506, an inhibitor of Cn, and mRNA silencing by small interference RNA we show that mitochondrial stress-activated Cn is critical for increased GLUT 4 and IGF1R expression and activation. The importance of the IGF1R pathway in cell survival under mitochondrial stress is demonstrated by increased apoptosis either by IGF1R mRNA silencing or by treatment with IGF1R inhibitors (AG1024 and picropodophyllin). This study describes a novel mechanism of mitochondrial stress-induced metabolic shift involving Cn with implications in resistance to apoptosis and tumor proliferation.
Journal of Biological Chemistry 06/2007; 282(19):14536-46. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have investigated the effects of hypoxia and myocardial ischemia/reperfusion on the structure and function of cytochrome c oxidase (CcO). Hypoxia (0.1% O(2) for 10 h) and cAMP-mediated inhibition of CcO activity were accompanied by hyperphosphorylation of subunits I, IVi1, and Vb and markedly increased reactive O(2) species production by the enzyme complex in an in vitro system that uses reduced cytochrome c as an electron donor. Both subunit phosphorylation and enzyme activity were effectively reversed by 50 nm H89 or 50 nm myristoylated peptide inhibitor (MPI), specific inhibitors of protein kinase A, but not by inhibitors of protein kinase C. In rabbit hearts subjected to global and focal ischemia, CcO activity was inhibited in a time-dependent manner and was accompanied by hyperphosphorylation as in hypoxia. Additionally, CcO activity and subunit phosphorylation in the ischemic heart were nearly completely reversed by H89 or MPI added to the perfusion medium. Hyperphosphorylation of subunits I, IVi1, and Vb was accompanied by reduced subunit contents of the immunoprecipitated CcO complex. Most interestingly, both H89 and MPI added to the perfusion medium dramatically reduced the ischemia/reperfusion injury to the myocardial tissue. Our results pointed to an exciting possibility of using CcO activity modulators for controlling myocardial injury associated with ischemia and oxidative stress conditions.
Journal of Biological Chemistry 02/2006; 281(4):2061-70. · 4.77 Impact Factor
