[Show abstract][Hide abstract] ABSTRACT: Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species, which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies, but its role remains controversial. Using high-throughput screening, we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.
Full-text · Article · Sep 2015 · Nature Chemical Biology
[Show abstract][Hide abstract] ABSTRACT: Superoxide and H 2 O 2 are important in redox signaling, disease and aging. However, there are ten sites associated with the mitochondrial respiratory chain that are known to produce these species at measurable rates, making it hard to assess the contribution of particular sites in cell physiology or pathology. We have defined the maximum capacities and many kinetic aspects of each site in skeletal muscle mitochondria. In order of capacity they are sites IIIQo in complex III; IQ and IIF in complexes I and II; OF, PF and BF in the 2-oxoglutarate, pyruvate and branched chain 2-oxoacid dehydrogenase complexes; GQ in mitochondrial glycerol phosphate dehydrogenase (mGPDH); IF in complex I; EF in ETF/ETF:Q oxidoreductase and DQ in dihydroorotate dehydrogenase. By screening libraries of small molecules against these sites, we identified a novel suppressor of superoxide/H O production at site IQ that does not affect oxidative phosphorylation (CN-POBS), and a novel inhibitor of mGPDH (iGP-1). The rate of superoxide/H2O2 production at any site depends on its redox state, which can be measured for sites IIIQo and IF. Therefore, we can assess the rates from these sites in complex substrate mixes in different conditions. Using specific inhibitors with correction for changes at sites IIIQo and IF, and by measuring the suppression of signal by CN-POBS, the rates from all significant sites can be estimated. In this way we analyzed the rates of superoxide/H 2 O 2 production from different sites in mitochondria oxidizing conventional substrates. The overall rates differed tenfold and, importantly, the contribution of each site varied with substrate; e.g. with succinate, most of the signal was from site IQ, with a little from IF and IIIQo, whereas with glutamate + malate, it was shared between sites IF, IIIQo and OF. In a complex mix of substrates mimicking resting muscle cytosol, the major contributors were sites IQ and IIF, with smaller contributions from IF, IIIQo and maybe EF and GQ. In a medium mimicking contracting muscle at about 30% VO 2 max the overall rate was fivefold lower and site IF was dominant, with smaller contributions from IQ, IIF, IIIQo and maybe EF, PF and GQ. These results highlight the sites that may operate in intact muscle at rest and during exercise. This new suite of approaches revolutionizes our ability to assess and control production of superoxide and H 2 O 2 from specific mitochondrial sites that are important in physiology and pathology.
[Show abstract][Hide abstract] ABSTRACT: Dehydrogenases that use ubiquinone as electron acceptor, including complex I of the respiratory chain, complex II, and glycerol 3-phosphate dehydrogenase, are known to be direct generators of superoxide and/or H2O2. Dihydroorotate dehydrogenase oxidizes dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H2O2 directly or only does so indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that dihydroorotate oxidation leads to superoxide/H2O2 production at a fairly high rate of about 300pmol H2O2·min(-1)·mg protein(-1) when oxidation of ubiquinol is prevented and complex II is uninhibited. This H2O2 production is abolished by brequinar or leflunomide, known inhibitors of dihydroorotate dehydrogenase. 80% of this rate is indirect, originating from site IIF of complex II, since it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H2O2 production (rotenone to inhibit sites in complex I (site IQ and, indirectly, site IF); myxothiazol to inhibit site IIIQo in complex III, and malonate plus atpenin A5 to inhibit site IIF in complex II), dihydroorotate dehydrogenase generates superoxide/H2O2 at a small but significant rate (23pmol H2O2·min(-1)·mg protein(-1)), from the ubiquinone-binding site. We conclude that dihydroorotate dehydrogenase can generate superoxide and/or H2O2 directly at low rates, and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.
Full-text · Article · Apr 2014 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a ubiquinone-linked enzyme in the mitochondrial inner membrane best characterized as part of the glycerol phosphate shuttle that transfers reducing equivalents from cytosolic NADH into the mitochondrial electron transport chain. Despite the widespread expression of mGPDH and the availability of mGPDH-null mice, the physiological role of this enzyme remains poorly defined in many tissues, likely because of compensatory pathways for cytosolic regeneration of NAD(+) and mechanisms for glycerol phosphate metabolism. Here we describe a novel class of cell-permeant small-molecule inhibitors of mGPDH (iGP) discovered through small-molecule screening. Structure-activity analysis identified a core benzimidazole-phenyl-succinamide structure as being essential to inhibition of mGPDH while modifications to the benzimidazole ring system modulated both potency and off-target effects. Live-cell imaging provided evidence that iGPs penetrate cellular membranes. Two compounds (iGP-1 and iGP-5) were characterized further to determine potency and selectivity and found to be mixed inhibitors with IC50 and K i values between ∼1-15 µM. These novel mGPDH inhibitors are unique tools to investigate the role of glycerol 3-phosphate metabolism in both isolated and intact systems.
[Show abstract][Hide abstract] ABSTRACT: Mitochondria constitute an important topic of biomedical enquiry (one paper in every 154 indexed in PubMed since 1998 is retrieved by the keyword 'mitochon-dria') because of widespread recognition of their importance in cell physiology and pathology. Mitochondrial dysfunction is widely implicated in ageing and in the diseases of ageing, through dysfunction in adenosine triphosphate (ATP) syn-thesis, Ca 2+ homeostasis, central metabolic pathways or radical production. Nonetheless, the mechanisms and regulation of superoxide and hydrogen perox-ide formation by mitochondria remain poorly described. Measurement of the capacities of different sites of superoxide and hydrogen peroxide production in isolated skeletal muscle mitochondria show that the maximum capacities of sites in complexes I, II and III and in several associated redox enzymes greatly exceed the native rates observed in the absence of respiratory chain inhibitors. In vitro, the native rates and the relative importance of different sites both depend on the substrate being oxidized, with sites I Q , II F , GPDH, I F and III Qo each being impor-tant with particular substrates. The techniques involved in measuring rates from each site should become applicable to cell cultures and in vivo in the future.
Full-text · Article · Oct 2013 · British Journal of Dermatology
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial production of reactive oxygen species is often considered an unavoidable consequence of aerobic metabolism and currently cannot be manipulated without perturbing oxidative phosphorylation. Antioxidants are widely used to suppress effects of reactive oxygen species after formation, but they can never fully prevent immediate effects at the sites of production. To identify site-selective inhibitors of mitochondrial superoxide/H2O2 production that do not interfere with mitochondrial energy metabolism, we developed a robust small-molecule screen and secondary profiling strategy. We describe the discovery and characterization of a compound (N-cyclohexyl-4-(4-nitrophenoxy)benzenesulfonamide; CN-POBS) that selectively inhibits superoxide/H2O2 production from the ubiquinone-binding site of complex I (site IQ) with no effects on superoxide/H2O2 production from other sites or on oxidative phosphorylation. Structure/activity studies identified a core structure that is important for potency and selectivity for site IQ. By employing CN-POBS in mitochondria respiring on NADH-generating substrates, we show that site IQ does not produce significant amounts of superoxide/H2O2 during forward electron transport on glutamate plus malate. Our screening platform promises to facilitate further discovery of direct modulators of mitochondrially-derived oxidative damage and advance our ability to understand and manipulate mitochondrial reactive oxygen species production in both normal and pathological conditions.
Full-text · Article · Aug 2013 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial radical production is important in redox signaling, aging and disease, but the relative contributions of different production sites are poorly understood. We analyzed the rates of superoxide/H2O2 production from different defined sites in rat skeletal muscle mitochondria oxidizing a variety of conventional substrates in the absence of added inhibitors: succinate; glycerol 3-phosphate; palmitoylcarnitine plus carnitine; or glutamate plus malate. In all cases, the sum of the estimated rates accounted fully for the measured overall rates. There were two striking results. First, the overall rates differed by an order of magnitude between substrates. Second, the relative contribution of each site was very different with different substrates. During succinate oxidation, most of the superoxide production was from the site of quinone reduction in complex I (site IQ), with small contributions from the flavin site in complex I (site IF) and the quinol oxidation site in complex III (site IIIQo). However, with glutamate plus malate as substrate, site IQ made little or no contribution, and production was shared between site IF, site IIIQo and 2-oxoglutarate dehydrogenase. With palmitoylcarnitine as substrate, the flavin site in complex II (site IIF) was a major contributor (together with sites IF and IIIQo), and with glycerol 3-phosphate as substrate, five different sites all contributed, including glycerol 3-phosphate dehydrogenase. Thus, the relative and absolute contributions of specific sites to the production of reactive oxygen species in isolated mitochondria depend very strongly on the substrates being oxidized, and the same is likely true in cells and in vivo.
[Show abstract][Hide abstract] ABSTRACT: RING finger protein 11 (RNF11), a negative regulator of NF-κB signaling pathway, colocalizes with α-synuclein and is sequestered in Lewy bodies in Parkinson's disease (PD). Since persistent NF-κB activation is reported in PD, in this report we investigated if RNF11 expression level is correlated to activated NF-κB in PD. We examined RNF11 expression levels in correlation to phospho-p65, a marker for activated NF-κB, in control and PD brain tissue from cerebral cortex. In addition we performed double immunofluorescence labeling experiments to confirm this correlation. Our investigations demonstrated that the neuronal RNF11 expression was down-regulated in PD and was usually associated with increased expression of phospho-p65. Double labeling confirmed that loss of neuronal RNF11 was linked to increased phospho-p65 expression, suggesting that persistent presence of NF-κB activation could be due to decreased levels of its negative regulator. Our data exemplifies the relevance of RNF11 and persistent NF-κB activation in PD.
Full-text · Article · May 2013 · Neuroscience Letters
[Show abstract][Hide abstract] ABSTRACT: H2O2 production by skeletal muscle mitochondria oxidizing palmitoylcarnitine was examined in two conditions: the absence of respiratory chain inhibitors, and the presence of myxothiazol to inhibit complex III. Without inhibitors, respiration and H2O2 production were low unless carnitine or malate was added to limit acetyl CoA accumulation. With palmitoylcarnitine alone, H2O2 production was dominated by complex II (44% from site IIF in the forward reaction); the remainder was mostly from complex I (34%, superoxide from site IF). With added carnitine, H2O2 production was about equally shared between complexes I, II, and III. With added malate, it was 75% from complex III (superoxide from site IIIQo) and 25% from site IF. Thus complex II (site IIF in the forward reaction) is a major source of H2O2 production during oxidation of palmitoylcarnitine±carnitine. In the second condition (myxothiazol present to keep ubiquinone reduced), the rates of H2O2 production were highest in the presence of palmitoylcarnitine±carnitine and were dominated by complex II (site IIF in the reverse reaction). About half the rest was from site IF, but a significant portion, ~40pmol H2O2·min(-1)·mg protein(-1), was not from complexes I, II or III, and was attributed to the proteins of β-oxidation (electron transferring flavoprotein, ETF, and ETF-ubiquinone oxidoreductase, ETFQOR). The maximum rate from the ETF system was ~200pmol H2O2·min(-1)·mg protein(-1) under conditions of compromised antioxidant defense and reduced ubiquinone pool. Thus complex II and the ETF system both contribute to H2O2 production during fatty acid oxidation under appropriate conditions.
Full-text · Article · Apr 2013 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: The oxidation of sn-glycerol 3-phosphate by mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a major pathway for transfer of cytosolic reducing equivalents to the mitochondrial electron transport chain. It is known to generate H2O2 at a range of rates and from multiple sites within the chain. The rates and sites depend upon tissue source, concentrations of glycerol 3-phosphate and calcium, and the presence of different electron transport chain inhibitors. We report a detailed examination of H2O2 production during glycerol 3-phosphate oxidation by skeletal muscle, brown fat, brain, and heart mitochondria with an emphasis on conditions under which mGPDH itself is the source of superoxide and H2O2. Importantly, we demonstrate that a substantial portion of H2O2 production commonly attributed to mGPDH originates instead from electron flow through the ubiquinone pool into complex II. When complex II is inhibited and mGPDH is the sole superoxide producer, the rate of superoxide production depends on the concentrations of glycerol 3-phosphate and calcium and correlates positively with the predicted reduction state of the ubiquinone pool. mGPDH-specific superoxide production plateaus at a rate comparable to the other major sites of superoxide production in mitochondria, the superoxide-producing center shows no sign of being over-reducible, and the maximum superoxide production rate correlates with mGPDH activity in four different tissues. mGPDH produces superoxide approximately equally towards each side of the mitochondrial inner membrane suggesting that the Q-binding pocket of mGPDH is the major site of superoxide generation. These results clarify the maximum rate and mechanism of superoxide production by mGPDH.
Full-text · Article · Nov 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Individual sites of superoxide production in the mitochondrial respiratory chain have previously been defined and partially characterized using specific inhibitors, but the native contribution of each site to total superoxide production in the absence of inhibitors is unknown. We estimated rates of superoxide production (measured as H(2)O(2)) at various sites in rat muscle mitochondria using specific endogenous reporters. The rate of superoxide production by the complex I flavin (site I(F)) was calibrated to the reduction state of endogenous NAD(P)H. Similarly, the rate of superoxide production by the complex III site of quinol oxidation (site III(Qo)) was calibrated to the reduction state of endogenous cytochrome b(566). We then measured the endogenous reporters in mitochondria oxidizing NADH-generating substrates, without added respiratory inhibitors, with and without ATP synthesis. We used the calibrated reporters to calculate the rates of superoxide production from sites I(F) and III(Qo). The calculated rates of superoxide production accounted for much of the measured overall rates. During ATP synthesis, site I(F) was the dominant superoxide producer. Under nonphosphorylating conditions, overall rates were higher, and sites I(F) and III(Qo) and unidentified sites (perhaps the complex I site of quinone reduction, site I(Q)) all made substantial contributions to measured H(2)O(2) production.
Full-text · Article · Aug 2012 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: Respiratory complex II oxidizes succinate to fumarate as part of the Krebs cycle and reduces ubiquinone in the electron transport chain. Previous experimental evidence suggested that complex II is not a significant contributor to the production of reactive oxygen species (ROS) in isolated mitochondria or intact cells unless mutated. However, we find that when complex I and complex III are inhibited and succinate concentration is low, complex II in rat skeletal muscle mitochondria can generate superoxide or H(2)O(2) at high rates. These rates approach or exceed the maximum rates achieved by complex I or complex III. Complex II generates these ROS in both the forward reaction, with electrons supplied by succinate, and the reverse reaction, with electrons supplied from the reduced ubiquinone pool. ROS production in the reverse reaction is prevented by inhibition of complex II at either the ubiquinone-binding site (by atpenin A5) or the flavin (by malonate), whereas ROS production in the forward reaction is prevented by malonate but not by atpenin A5, showing that the ROS from complex II arises only from the flavin site (site II(F)). We propose a mechanism for ROS production by complex II that relies upon the occupancy of the substrate oxidation site and the reduction state of the enzyme. We suggest that complex II may be an important contributor to physiological and pathological ROS production.
Full-text · Article · Jun 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Activation of innate and adaptive immune responses is tightly regulated, as insufficient activation could result in defective clearance of pathogens, while excessive activation might lead to lethal systemic inflammation or autoimmunity. A20 functions as a negative regulator of innate and adaptive immunity by inhibiting NF-κB activation. A20 mediates its inhibitory function in a complex with other proteins including RNF11 and Itch, both E3 ubiquitin ligases and TAX1BP1, an adaptor protein. Since NF-κB has been strongly implicated in various neuronal functions, we predict that its inhibitor, the A20 complex, is also present in the nervous system. In efforts to better understand the role of A20 complex and NF-κB signaling pathway, we determined regional distribution of A20 mRNA as well as protein expression levels and distribution of RNF11, TAX1BP1 and Itch, in different brain regions. The distribution of TRAF6 was also investigated since TRAF6, also an E3 ligase, has an important role in NF-κB signaling pathway. Our investigations, for the first time, describe and demonstrate that the essential components of the A20 ubiquitin-editing complex are present and mainly expressed in neurons. The A20 complex components are also differentially expressed throughout the human brain. This study provides useful information about region specific expression of the A20 complex components that will be invaluable while determining the role of NF-κB signaling pathway in neuronal development and degeneration.
No preview · Article · May 2012 · Neuroscience Letters
[Show abstract][Hide abstract] ABSTRACT: The RING domain-containing protein RING finger protein 11 (RNF11) is a member of the A20 ubiquitin-editing protein complex and modulates peripheral NF-κB signaling. RNF11 is robustly expressed in neurons and colocalizes with a population of α-synuclein-positive Lewy bodies and neurites in Parkinson disease patients. The NF-κB pathway has an important role in the vertebrate nervous system, where the absence of NF-κB activity during development can result in learning and memory deficits, whereas chronic NF-κB activation is associated with persistent neuroinflammation. We examined the functional role of RNF11 with respect to canonical NF-κB signaling in neurons to gain understanding of the tight association of inflammatory pathways, including NF-κB, with the pathogenesis of neurodegenerative diseases.
Luciferase assays were employed to assess NF-κB activity under targeted short hairpin RNA (shRNA) knockdown of RNF11 in human neuroblastoma cells and murine primary neurons, which suggested that RNF11 acts as a negative regulator of canonical neuronal NF-κB signaling. These results were further supported by analyses of p65 translocation to the nucleus following depletion of RNF11. Coimmunoprecipitation experiments indicated that RNF11 associates with members of the A20 ubiquitin-editing protein complex in neurons. Site-directed mutagenesis of the myristoylation domain, which is necessary for endosomal targeting of RNF11, altered the impact of RNF11 on NF-κB signaling and abrogated RNF11's association with the A20 ubiquitin-editing protein complex. A partial effect on canonical NF-κB signaling and an association with the A20 ubiquitin-editing protein complex was observed with mutagenesis of the PPxY motif, a proline-rich region involved in Nedd4-like protein interactions. Last, shRNA-mediated reduction of RNF11 in neurons and neuronal cell lines elevated levels of monocyte chemoattractant protein 1 and TNF-α mRNA and proteins, suggesting that NF-κB signaling and associated inflammatory responses are aberrantly regulated in the absence of RNF11.
Our findings support the hypothesis that, in the nervous system, RNF11 negatively regulates canonical NF-κB signaling. Reduced or functionally compromised RNF11 could influence NF-κB-associated neuronal functions, including exaggerated inflammatory responses that may have implications for neurodegenerative disease pathogenesis and progression.
Full-text · Article · Apr 2012 · Journal of Neuroinflammation