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1.
Int J Mol Sci ; 23(9)2022 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-35563503

RESUMO

The oxidation of proline to pyrroline-5-carboxylate (P5C) leads to the transfer of electrons to ubiquinone in mitochondria that express proline dehydrogenase (ProDH). This electron transfer supports Complexes CIII and CIV, thus generating the protonmotive force. Further catabolism of P5C forms glutamate, which fuels the citric acid cycle that yields the reducing equivalents that sustain oxidative phosphorylation. However, P5C and glutamate catabolism depend on CI activity due to NAD+ requirements. NextGen-O2k (Oroboros Instruments) was used to measure proline oxidation in isolated mitochondria of various mouse tissues. Simultaneous measurements of oxygen consumption, membrane potential, NADH, and the ubiquinone redox state were correlated to ProDH activity and F1FO-ATPase directionality. Proline catabolism generated a sufficiently high membrane potential that was able to maintain the F1FO-ATPase operation in the forward mode. This was observed in CI-inhibited mouse liver and kidney mitochondria that exhibited high levels of proline oxidation and ProDH activity. This action was not observed under anoxia or when either CIII or CIV were inhibited. The duroquinone fueling of CIII and CIV partially reproduced the effects of proline. Excess glutamate, however, could not reproduce the proline effect, suggesting that processes upstream of the glutamate conversion from proline were involved. The ProDH inhibitors tetrahydro-2-furoic acid and, to a lesser extent, S-5-oxo-2-tetrahydrofurancarboxylic acid abolished all proline effects. The data show that ProDH-directed proline catabolism could generate sufficient CIII and CIV proton pumping, thus supporting ATP production by the F1FO-ATPase even under CI inhibition.


Assuntos
Prolina Oxidase , Ubiquinona , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Complexo I de Transporte de Elétrons/metabolismo , Ácido Glutâmico/metabolismo , Camundongos , Mitocôndrias/metabolismo , Prolina/metabolismo , Prolina Oxidase/metabolismo , Ubiquinona/metabolismo
2.
Neurochem Res ; 44(10): 2301-2306, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30810978

RESUMO

The reaction catalyzed by succinate-CoA ligase in the mitochondrial matrix yields a high-energy phosphate when operating towards hydrolysis of the thioester bond of succinyl-CoA, known as mitochondrial substrate-level phosphorylation (mSLP). The catabolism of several metabolites converge to succinyl-CoA but through different biochemical pathways. Among them, threonine, serine and methionine catabolize to succinyl-CoA through the common intermediate, 2-ketobutyrate. During the course of this pathway 2-ketobutyrate will become succinyl-CoA through propionyl-CoA catabolism, obligatorily passing through an ATP-consuming step substantiated by propionyl-CoA carboxylase. Here, by recording the directionality of the adenine nucleotide translocase while measuring membrane potential we tested the hypothesis that catabolism of 2-ketobutyrate negates mSLP due to the ATP-consuming propionyl-CoA carboxylase step in rotenone-treated, isolated mouse liver and brain mitochondria. 2-Ketobutyrate produced a less negative membrane potential compared to NADH or FADH2-linked substrates, which was sensitive to inhibition by rotenone, atpenin and arsenate, implying the involvement of complex I, complex II and a dehydrogenase-most likely branched chain keto-acid dehydrogenase, respectively. Co-addition of 2-ketobutyrate with NADH- or FADH2-linked substrates yielded no greater membrane potential than in the presence of substrates alone. However, in the presence of NADH-linked substrates, 2-ketobutyrate prevented mSLP in a dose-dependent manner. Our results imply that despite that 2-ketobutyrate leads to succinyl-CoA formation, obligatory metabolism through propionyl-CoA carboxylase associated with ATP expenditure abolishes mSLP. The provision of metabolites converging to 2-ketobutyrate may be a useful way for manipulating mSLP without using pharmacological or genetic tools.


Assuntos
Acil Coenzima A/metabolismo , Butiratos/farmacologia , Mitocôndrias/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Acil Coenzima A/efeitos dos fármacos , Animais , Camundongos , Mitocôndrias/metabolismo , Fosforilação/fisiologia , Rotenona/farmacologia , Especificidade por Substrato/efeitos dos fármacos
3.
Biochim Biophys Acta Bioenerg ; 1859(9): 909-924, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29746824

RESUMO

Provision of NAD+ for oxidative decarboxylation of alpha-ketoglutarate to succinyl-CoA by the ketoglutarate dehydrogenase complex (KGDHC) is critical for maintained operation of succinyl-CoA ligase yielding high-energy phosphates, a process known as mitochondrial substrate-level phosphorylation (mSLP). We have shown previously that when NADH oxidation by complex I is inhibited by rotenone or anoxia, mitochondrial diaphorases yield NAD+, provided that suitable quinones are present (Kiss G et al., FASEB J 2014, 28:1682). This allows for KGDHC reaction to proceed and as an extension of this, mSLP. NAD(P)H quinone oxidoreductase 1 (NQO1) is an enzyme exhibiting diaphorase activity. Here, by using Nqo1-/- and WT littermate mice we show that in rotenone-treated, isolated liver mitochondria 2-methoxy-1,4-naphtoquinone (MNQ) is preferentially reduced by matrix Nqo1 yielding NAD+ to KGDHC, supporting mSLP. This process was sensitive to inhibition by specific diaphorase inhibitors. Reduction of idebenone and its analogues MRQ-20 and MRQ-56, menadione, mitoquinone and duroquinone were unaffected by genetic disruption of the Nqo1 gene. The results allow for the conclusions that i) MNQ is a Nqo1-preferred substrate, and ii) in the presence of suitable quinones, mitochondrially-localized diaphorases other than Nqo1 support NADH oxidation when complex I is inhibited. Our work confirms that complex I bypass can occur by quinones reduced by intramitochondrial diaphorases oxidizing NADH, ultimately supporting mSLP. Finally, it may help to elucidate structure-activity relationships of redox-active quinones with diaphorase enzymes.


Assuntos
Acil Coenzima A/metabolismo , Mitocôndrias Hepáticas/enzimologia , NAD(P)H Desidrogenase (Quinona)/fisiologia , NAD/metabolismo , Naftoquinonas/química , Animais , Respiração Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Fosforilação , Especificidade por Substrato
4.
Biochim Biophys Acta ; 1861(11): 1727-1735, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27542539

RESUMO

Encysted embryos (cysts) of the crustacean Artemia franciscana exhibit enormous tolerance to adverse conditions encompassing high doses of radiation, years of anoxia, desiccation and extreme salinity. So far, several mechanisms have been proposed to contribute to this extremophilia, however, none were sought in the lipid profile of the cysts. Here in, we used high resolution shotgun lipidomics suited for detailed quantitation and analysis of lipids in uncharacterized biological membranes and samples and assembled the total, mitochondrial and mitoplastic lipidome of Artemia franciscana cysts. Overall, we identified and quantitated 1098 lipid species dispersed among 22 different classes and subclasses. Regarding the mitochondrial lipidome, most lipid classes exhibited little differences from those reported in other animals, however, Artemia mitochondria harboured much less phosphatidylethanolamine, plasmenylethanolamines and ceramides than mitochondria of other species, some of which by two orders of magnitude. Alternatively, Artemia mitochondria exhibited much higher levels of phosphatidylglycerols and phosphatidylserines. The identification and quantitation of the total and mitochondrial lipidome of the cysts may help in the elucidation of actionable extremophilia-affording proteins, such as the 'late embryogenesis abundant' proteins, which are known to interact with lipid membranes.


Assuntos
Artemia/embriologia , Artemia/metabolismo , Embrião não Mamífero/metabolismo , Metabolismo dos Lipídeos , Metaboloma , Mitocôndrias/metabolismo , Animais , Western Blotting , Cardiolipinas/metabolismo , Análise por Conglomerados
5.
FASEB J ; 30(1): 286-300, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26358042

RESUMO

Itaconate is a nonamino organic acid exhibiting antimicrobial effects. It has been recently identified in cells of macrophage lineage as a product of an enzyme encoded by immunoresponsive gene 1 (Irg1), acting on the citric acid cycle intermediate cis-aconitate. In mitochondria, itaconate can be converted by succinate-coenzyme A (CoA) ligase to itaconyl-CoA at the expense of ATP (or GTP), and is also a weak competitive inhibitor of complex II. Here, we investigated specific bioenergetic effects of increased itaconate production mediated by LPS-induced stimulation of Irg1 in murine bone marrow-derived macrophages (BMDM) and RAW-264.7 cells. In rotenone-treated macrophage cells, stimulation by LPS led to impairment in substrate-level phosphorylation (SLP) of in situ mitochondria, deduced by a reversal in the directionality of the adenine nucleotide translocase operation. In RAW-264.7 cells, the LPS-induced impairment in SLP was reversed by short-interfering RNA(siRNA)-but not scrambled siRNA-treatment directed against Irg1. LPS dose-dependently inhibited oxygen consumption rates (61-91%) and elevated glycolysis rates (>21%) in BMDM but not RAW-264.7 cells, studied under various metabolic conditions. In isolated mouse liver mitochondria treated with rotenone, itaconate dose-dependently (0.5-2 mM) reversed the operation of adenine nucleotide translocase, implying impairment in SLP, an effect that was partially mimicked by malonate. However, malonate yielded greater ADP-induced depolarizations (3-19%) than itaconate. We postulate that itaconate abolishes SLP due to 1) a "CoA trap" in the form of itaconyl-CoA that negatively affects the upstream supply of succinyl-CoA from the α-ketoglutarate dehydrogenase complex; 2) depletion of ATP (or GTP), which are required for the thioesterification by succinate-CoA ligase; and 3) inhibition of complex II leading to a buildup of succinate which shifts succinate-CoA ligase equilibrium toward ATP (or GTP) utilization. Our results support the notion that Irg1-expressing cells of macrophage lineage lose the capacity of mitochondrial SLP for producing itaconate during mounting of an immune defense.


Assuntos
Hidroliases/metabolismo , Macrófagos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Succinatos/farmacologia , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Feminino , Glicólise , Hidroliases/genética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Malonatos/farmacologia , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/metabolismo , Fosforilação Oxidativa , Rotenona/farmacologia , Succinato-CoA Ligases/metabolismo
6.
Biochem J ; 473(20): 3463-3485, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27496549

RESUMO

Succinate-CoA ligase (SUCL) is a heterodimer enzyme composed of Suclg1 α-subunit and a substrate-specific Sucla2 or Suclg2 ß-subunit yielding ATP or GTP, respectively. In humans, the deficiency of this enzyme leads to encephalomyopathy with or without methylmalonyl aciduria, in addition to resulting in mitochondrial DNA depletion. We generated mice lacking either one Sucla2 or Suclg2 allele. Sucla2 heterozygote mice exhibited tissue- and age-dependent decreases in Sucla2 expression associated with decreases in ATP-forming activity, but rebound increases in cardiac Suclg2 expression and GTP-forming activity. Bioenergetic parameters including substrate-level phosphorylation (SLP) were not different between wild-type and Sucla2 heterozygote mice unless a submaximal pharmacological inhibition of SUCL was concomitantly present. mtDNA contents were moderately decreased, but blood carnitine esters were significantly elevated. Suclg2 heterozygote mice exhibited decreases in Suclg2 expression but no rebound increases in Sucla2 expression or changes in bioenergetic parameters. Surprisingly, deletion of one Suclg2 allele in Sucla2 heterozygote mice still led to a rebound but protracted increase in Suclg2 expression, yielding double heterozygote mice with no alterations in GTP-forming activity or SLP, but more pronounced changes in mtDNA content and blood carnitine esters, and an increase in succinate dehydrogenase activity. We conclude that a partial reduction in Sucla2 elicits rebound increases in Suclg2 expression, which is sufficiently dominant to overcome even a concomitant deletion of one Suclg2 allele, pleiotropically affecting metabolic pathways associated with SUCL. These results as well as the availability of the transgenic mouse colonies will be of value in understanding SUCL deficiency.


Assuntos
Succinato-CoA Ligases/metabolismo , Alelos , Animais , Western Blotting , Carnitina/análogos & derivados , Carnitina/metabolismo , Células Cultivadas , DNA Mitocondrial/genética , Heterozigoto , Humanos , Técnicas In Vitro , Potencial da Membrana Mitocondrial/genética , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Knockout , Camundongos Mutantes , Mitocôndrias/genética , Fosforilação/genética , Fosforilação/fisiologia , RNA Mensageiro/genética , Succinato-CoA Ligases/genética
7.
Sci Rep ; 14(1): 1729, 2024 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-38242919

RESUMO

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in the mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in isolated mitochondria in real-time, we demonstrate that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. 13C metabolic tracing or untargeted analysis of metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system showed that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase Complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Complex II operated amphidirectionally during the anoxic event, providing quinones to Complex I and reducing fumarate to succinate. Our results highlight the importance of quinone provision to Complex I oxidizing NADH maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.


Assuntos
Mitocôndrias , NAD , Humanos , NAD/metabolismo , Mitocôndrias/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Quinonas/metabolismo , Fosforilação Oxidativa , Succinatos/metabolismo , Hipóxia/metabolismo , Oxirredução
8.
Antioxidants (Basel) ; 11(8)2022 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-36009207

RESUMO

α-ketoglutarate dehydrogenase complex (KGDHc), or 2-oxoglutarate dehydrogenase complex (OGDHc) is a rate-limiting enzyme in the tricarboxylic acid cycle, that has been identified in neurodegenerative diseases such as in Alzheimer's disease. The aim of the present study was to establish the role of the KGDHc and its subunits in the bioenergetics and reactive oxygen species (ROS) homeostasis of brain mitochondria. To study the bioenergetic profile of KGDHc, genetically modified mouse strains were used having a heterozygous knock out (KO) either in the dihydrolipoyl succinyltransferase (DLST+/-) or in the dihydrolipoyl dehydrogenase (DLD+/-) subunit. Mitochondrial oxygen consumption, hydrogen peroxide (H2O2) production, and expression of antioxidant enzymes were measured in isolated mouse brain mitochondria. Here, we demonstrate that the ADP-stimulated respiration of mitochondria was partially arrested in the transgenic animals when utilizing α-ketoglutarate (α-KG or 2-OG) as a fuel substrate. Succinate and α-glycerophosphate (α-GP), however, did not show this effect. The H2O2 production in mitochondria energized with α-KG was decreased after inhibiting the adenine nucleotide translocase and Complex I (CI) in the transgenic strains compared to the controls. Similarly, the reverse electron transfer (RET)-evoked H2O2 formation supported by succinate or α-GP were inhibited in mitochondria isolated from the transgenic animals. The decrease of RET-evoked ROS production by DLST+/- or DLD+/- KO-s puts the emphasis of the KGDHc in the pathomechanism of ischemia-reperfusion evoked oxidative stress. Supporting this notion, expression of the antioxidant enzyme glutathione peroxidase was also decreased in the KGDHc transgenic animals suggesting the attenuation of ROS-producing characteristics of KGDHc. These findings confirm the contribution of the KGDHc to the mitochondrial ROS production and in the pathomechanism of ischemia-reperfusion injury.

9.
Sci Rep ; 8(1): 5915, 2018 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-29651047

RESUMO

Heterologous expression is a landmark technique for studying a protein itself or its effect on the expression host, in which membrane-embedded proteins are a common choice. Yet, the impact of inserting a foreign protein to the lipid environment of host membranes, has never been addressed. Here we demonstrated that heterologous expression of the Artemia franciscana adenine nucleotide translocase (ANT) in yeasts altered lipidomic composition of their inner mitochondrial membranes. Along with this, activities of complex II, IV and ATP synthase, all membrane-embedded components, were significantly decreased while their expression levels remained unaffected. Although the results represent an individual case of expressing a crustacean protein in yeast inner mitochondrial membranes, it cannot be excluded that host lipidome alterations is a more widespread epiphenomenon, potentially biasing heterologous expression experiments. Finally, our results raise the possibility that not only lipids modulate protein function, but also membrane-embedded proteins modulate lipid composition, thus revealing a reciprocal mode of regulation for these two biomolecular entities.


Assuntos
Lipídeos/genética , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/genética , Animais , Artemia/enzimologia , Regulação Enzimológica da Expressão Gênica/genética , Transporte de Íons/genética , Lipídeos/química , Proteínas de Membrana/genética , Mitocôndrias/genética , Saccharomyces cerevisiae/genética
10.
Neurochem Int ; 109: 41-53, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28300620

RESUMO

GABA is catabolized in the mitochondrial matrix through the GABA shunt, encompassing transamination to succinic semialdehyde followed by oxidation to succinate by the concerted actions of GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH), respectively. Gamma-hydroxybutyrate (GHB) is a neurotransmitter and a psychoactive drug that could enter the citric acid cycle through transhydrogenation with α-ketoglutarate to succinic semialdehyde and d-hydroxyglutarate, a reaction catalyzed by hydroxyacid-oxoacid transhydrogenase (HOT). Here, we tested the hypothesis that the elevation in matrix succinate concentration caused by exogenous addition of GABA, succinic semialdehyde or GHB shifts the equilibrium of the reversible reaction catalyzed by succinate-CoA ligase towards ATP (or GTP) hydrolysis, effectively negating substrate-level phosphorylation (SLP). Mitochondrial SLP was addressed by interrogating the directionality of the adenine nucleotide translocase during anoxia in isolated mouse brain and liver mitochondria. GABA eliminated SLP, and this was rescued by the GABA-T inhibitors vigabatrin and aminooxyacetic acid. Succinic semialdehyde was an extremely efficient substrate energizing mitochondria during normoxia but mimicked GABA in abolishing SLP in anoxia, in a manner refractory to vigabatrin and aminooxyacetic acid. GHB could moderately energize liver but not brain mitochondria consistent with the scarcity of HOT expression in the latter. In line with these results, GHB abolished SLP in liver but not brain mitochondria during anoxia and this was unaffected by either vigabatrin or aminooxyacetic acid. It is concluded that when mitochondria catabolize GABA or succinic semialdehyde or GHB through the GABA shunt, their ability to perform SLP is impaired.


Assuntos
Mitocôndrias/metabolismo , Oxibato de Sódio/metabolismo , Ácido gama-Aminobutírico/análogos & derivados , Ácido gama-Aminobutírico/metabolismo , Adjuvantes Anestésicos/metabolismo , Animais , Feminino , Masculino , Metabolismo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/fisiologia , Especificidade por Substrato/fisiologia
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