T cell Activation Is Driven by an ADP-Dependent Glucokinase Linking Enhanced Glycolysis with Mitochondrial Reactive Oxygen Species Generation (original) (raw)
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Mediator-assisted simultaneous probing of cytosolic and mitochondrial redox activity in living cells
Analytical Biochemistry, 2009
This work describes an electron transfer mediator-assisted amperometric flow injection method for assessing redox enzyme activity in different subcellular compartments of the phosphoglucose isomerase deletion mutant strain of Saccharomyces cerevisiae, EBY44. The method is demonstrated using the ferricyanide-menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pentose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric in vitro assays show no difference between the cofactors. Respiratory competent cells show cytosolic inhibition only when NADPH is produced, whereas production of NADH reveals uncoupling at low dicoumarol concentrations and inhibition of complexes III and IV at higher concentrations. Spectrophotometric assays only indicate the presence of cytosolic inhibition regardless of the reduced cofactor used. This article shows the applicability of the amperometric method and emphasizes the significance of determining biological effects of chemicals in living cells.
Journal of Biological Chemistry
The enzymatic activity underlying the respiratory burst in human neutrophils was examined in a subcellular fraction with high specific activity and shown to be a membrane-associated complex of a flavoprotein, ubiquinone-10, and cytochrome baas in an approximate 1.3:1:2 molar ratio. Study of the redox poise of these electron carriers indicated that electron flow in the intact complex from unstimulated cells proceeded: NADPH + E-FAD -P ubiquinone-10. Similar studies on the complex prepared from stimulated neutrophils indicated that electron flow proceeded NADPH + E-FAD + ubiquinone-10 + cytochrome bass + oxygen. The active enzyme complex was inhibited by p-chloromercuribenzoate. Inhibition persisted after removal of excess inhibitor, was reversed by dithiothreitol, and could be blocked by prior addition of substrate (NADPH). Inhibition of the active oxidase complex by p-chloromercuribenzoate also inhibited electron flow from NADPH to all purported electron carriers in the chain (Le. E-FAD, ubiquinone-10, and cytochrome bass). We conclude that activation of the oxidase enzyme complex in the intact neutrophil resulted in linkage of electron carrier function between endogenous ubiquinone-10 and cytochrome bass and was without demonstrable effect on proximal electron flow. The pchloromercuribenzoate sensitive site(s) proximal to the initial electron acceptor (E-FAD) did not appear to be altered by the cellular activation process.
F1000 - Post-publication peer review of the biomedical literature, 2000
It is widely appreciated that T cells increase glycolytic flux during activation, however the role of mitochondrial flux is unclear. Here we have shown that mitochondrial metabolism, in the absence of glucose metabolism, was sufficient to support interleukin-2 (IL-2) induction. Furthermore, we used mice with reduced mitochondrial reactive oxygen species (mROS) production in T cells (T-Uqcrfs −/− mice) to show that mitochondria are required for T cell activation to produce mROS for activation of nuclear factor of activated T cells (NFAT) and subsequent IL-2 induction. These mice could not induce antigen-specific expansion of T cells in vivo, however Uqcrfs1 −/− T cells retained the ability to proliferate in vivo under lymphopenic conditions. This suggests that Uqcrfs1 −/− T cells were not lacking bioenergetically, but rather lacked specific ROS-dependent signaling events needed for antigen-specific expansion. Thus, mitochondrial metabolism is a critical component of T cell activation through production of complex III ROS.
FEBS Letters, 1985
The reductton of ubrqumone-5 (Ql) by the phagocytosn-specific NADPH oxidase of gumea pig macrophages was not mhrbrted by superoxide dismutase (SOD) at concentrations usually used for 0; assay but was mhrbtted at about lOO-times higher concentratrons Titration of the reaction with SOD and a companson with that of xanthme oxidase showed that the mhrbition was not due to the semrqumone oxrdatron accelerated by a removal of 0; but due to the accelerated drsmutation of 0; which otherwise reduces the qumone Molecular oxygens are therefore preferential electron acceptors m the NADPH oxidase even m the presence of Q 1
Arjuna (Terminalia arjuna) is a medicinal plant used in many polyherbal hepatoprotective formulations. Although widely claimed to be antioxidant, data supporting such actions of Arjuna are limited. In the present study, we have investigated the efficacy of the aqueous extract of T. arjuna (AETA) using a standard pro-oxidant [tertiary butyl hydroperoxide (TBHP)] in HepG2 cells. Cells were incubated with AETA (5-100 mg/ml) for a range of time points (4-24 h) with or without TBHP (500 mM), and biochemical markers of oxidative stress (OS) were determined. Cells incubated with TBHP showed the significant induction of OS response in cytosol manifested as lipid hydroperoxide (76%-198%) and the generation of reactive oxygen species (60%-127%). Diminished levels of reduced glutathione (35%-60%) and total antioxidant capacity (20%-61%) suggested an altered redox state. Significant perturbations in the activities of antioxidant enzymes such as catalase (30%-56%), superoxide dismutase (25%-68%), glutathione S-transferase (29%-67%), glutathione peroxidase (24%-68%) and glutathione reductase (38%-49%) were discernible suggesting the ongoing OS in the cells. However, cells treated with AETA (100 mg/ml) along with TBHP offered significant protection by reducing levels of lipid hydroperoxide (33%-62%) and ROS (69%) and by increasing antioxidant capacity (54%-81%) and levels of reduced glutathione (49%-82%). Further, it also enhanced the activities of endogenous antioxidant enzymes (superoxide dismutase, 60%; catalase, 35%-82%; glutathione peroxidase, 42-65 %; glutathione reductase, 48%-62%; and glutathione S-transferase, 22%-100%). Taken together, these data suggest that Arjuna can protect against the oxidative damage induced by TBHP and may be effectively used as a hepatoprotective adjuvant to abrogate OS in vivo.
Mitochondrial function is differentially affected upon oxidative stress
Free Radical Biology and Medicine, 1999
The mechanisms that lead to mitochondrial damage under oxidative stress conditions were examined in synaptosomes treated with ascorbate/iron. A loss of membrane integrity, evaluated by electron microscopy and by LDH leakage, was observed in peroxidized synaptosomes and it was prevented by pre-incubation with vitamin E (150 M) and idebenone (50 M). ATP levels decreased, in synaptosomes exposed to ascorbate/iron, as compared to controls. NADH-ubiquinone oxidoreductase (Cx I) and cytochrome c oxidase (Cx IV) activities were unchanged after ascorbate/ iron treatment, whereas succinate-ubiquinone oxidoreductase (Cx II), ubiquinol cytochrome c reductase (Cx III) and ATP-synthase (Cx V) activities were reduced by 55%, 40%, and 55%, respectively. The decrease of complex II and ATP-synthase activities was prevented by reduced glutathione (GSH), whereas the other antioxidants tested (vitamin E and idebenone) were ineffective. However, vitamin E, idebenone and GSH prevented the reduction of complex III activity observed in synaptosomes treated with ascorbate/iron. GSH protective effect suggests that the oxidation of protein SH-groups is involved in the inhibition of complexes II, III and V activity, whereas vitamin E and idebenone protection suggests that membrane lipid peroxidation is also involved in the reduction of complex III activity. These results may indicate that the inhibition of the mitochondrial respiratory chain enzymatic complexes, that are differentially affected by oxidative stress, can be recovered by specific antioxidants.
Methods and Protocols
Mitochondrial dysfunction and cytosolic oxidative stress are pathological biomarkers interlinked in several chronic diseases and cellular toxicity promoted by high-energy radiation or xenobiotics. Thus, assessing the activities of the mitochondrial redox chain complexes and the cytosolic antioxidant enzymes in the same cell culture system is a valuable approach to addressing the challenge of chronic diseases or unveiling the molecular mechanisms underlying the toxicity of physical and chemical stress agents. The present article gathers the experimental procedures to obtain, from isolated cells, a mitochondria-free cytosolic fraction and a mitochondria-rich fraction. Furthermore, we describe the methodologies to evaluate the activity of the main antioxidant enzymes in the mitochondria-free cytosolic fraction (superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase), and the activity of the individual mitochondrial complexes I, II and IV, as well as the conjug...
Biochemical and Biophysical Research Communications, 2003
Respiratory chain complex I (NADH:ubiquinone oxidoreductase) deficiency is one of the most frequent causes of mitochondrial disease in humans. The activity of this complex can be confidently measured in most tissue samples, but not in cultured skin fibroblasts or circulating lymphocytes. Highly contaminating non-mitochondrial NADH-quinone oxidoreductase activity in fibroblasts and the limited access of substrates to complex I in lymphocytes hinder its measurement in permeabilized cells. Complex I assay in these cells requires the isolation of mitochondria, which in turn necessitates large quantities of cells and is not feasible when studying circulating lymphocytes. Here we report a simple method to measure complex I activity in a minute amount of either cell type. The procedure strongly reduces contaminating NADH:quinone oxidoreductase activity and permits measuring high rates of rotenone-sensitive complex I activity thanks to effective cell permeabilization.