RESUMO
Nitrosomonas europaea is a chemolithotroph that obtains energy through the oxidation of ammonia to hydroxylamine while assimilates atmospheric CO2 to cover the cell carbon demands for growth. This microorganism plays a relevant role in the nitrogen biogeochemical cycle on Earth but its carbon metabolism remains poorly characterized. Based on sequence homology, we identified two genes (cbbG and gabD) coding for redox enzymes in N. europaea. Cloning and expression of the genes in Escherichia coli, allowed the production of recombinant enzymes purified to determine their biochemical properties. The protein CbbG is a glyceraldehyde-3-phosphate (Ga3P) dehydrogenase (Ga3PDHase) catalyzing the reversible oxidation of Ga3P to 1,3-bis-phospho-glycerate (1,3bisPGA), using specifically NAD+/NADH as cofactor. CbbG showed â¼6-fold higher Km value for 1,3bisPGA but â¼5-fold higher kcat for the oxidation of Ga3P. The protein GabD irreversibly oxidizes Ga3P to 3Pglycerate using NAD+ or NADP+, thus resembling a non-phosphorylating Ga3PDHase. However, the enzyme showed â¼6-fold higher Km value and three orders of magnitude higher catalytic efficiency with succinate semialdehyde (SSA) and NADP+. Indeed, the GabD protein identity corresponds to an SSA dehydrogenase (SSADHase). CbbG seems to be the only Ga3PDHase present in N. europaea; which would be involved in reducing triose-P during autotrophic carbon fixation. Otherwise, in cells grown under conditions deprived of ammonia and oxygen, the enzyme could catalyze the glycolytic step of Ga3P oxidation producing NADH. As an SSADHase, GabD would physiologically act producing succinate and preferentially NADPH over NADH; thus being part of an alternative pathway of the tricarboxylic acid cycle converting α-ketoglutarate to succinate. The properties determined for these enzymes contribute to better identify metabolic steps in CO2 assimilation, glycolysis and the tricarboxylic acid cycle in N. europaea. Results are discussed in the framework of metabolic pathways that launch biosynthetic intermediates relevant in the microorganism to develop and fulfill its role in nature.
Assuntos
Proteínas de Bactérias , Carbono/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , Nitrosomonas europaea , Oxirredutases , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dióxido de Carbono/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Glicólise/fisiologia , Nitrosomonas europaea/enzimologia , Nitrosomonas europaea/genética , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismoRESUMO
Abstract: Background: Mitochondriopathies are multisystem diseases affecting the oxidative phosphorylation (OXPHOS) system. Skin fibroblasts are a good model for the study of these diseases. Fibroblasts with a complex IV mitochondriopathy were used to determine the molecular mechanism and the main affected functions in this disease. Methods: Skin fibroblast were grown to assure disease phenotype. Mitochondria were isolated from these cells and their proteome extracted for protein identification. Identified proteins were validated with the MitoMiner database. Results: Disease phenotype was corroborated on skin fibroblasts, which presented a complex IV defect. The mitochondrial proteome of these cells showed that the most affected proteins belonged to the OXPHOS system, mainly to the complexes that form supercomplexes or respirosomes (I, III, IV, and V). Defects in complex IV seemed to be due to assembly issues, which might prevent supercomplexes formation and efficient substrate channeling. It was also found that this mitochondriopathy affects other processes that are related to DNA genetic information flow (replication, transcription, and translation) as well as beta oxidation and tricarboxylic acid cycle. Conclusions: These data, as a whole, could be used for the better stratification of these diseases, as well as to optimize management and treatment options.
Resumen: Introducción: Las mitocondriopatías son enfermedades multisistémicas que afectan el funcionamiento de la fosforilación oxidativa (OXPHOS). Un buen modelo de estudio para estas enfermedades es el cultivo primario de fibroblastos. En este trabajo se utilizaron fibroblastos con mitocondriopatía del complejo IV para determinar cuáles son las principales funciones afectadas en esta enfermedad. Métodos: Se realizaron cultivos primarios de fibroblastos para corroborar el fenotipo de la enfermedad. Las mitocondrias se aislaron de estas células y se extrajo su proteoma para su identificación. Las proteínas identificadas se validaron con la base de datos de MitoMiner. Resultados: Los fibroblastos conservaron el fenotipo de la enfermedad que incluye un defecto del complejo IV. El proteoma mitocondrial de estas células mostró que las proteínas más afectadas pertenecen al sistema de OXPHOS, principalmente los complejos que forman supercomplejos o respirosomas (I, III, IV y V). El defecto en el complejo IV al parecer se debió a problemas de ensamblaje que pueden evitar la formación de los supercomplejos y la eficiente canalización de sustratos. También se observó que esta mitocondriopatía afecta otros procesos relacionados con el flujo de información genética del DNA (replicación, transcripción y traducción), así como con la beta oxidación y el ciclo de los ácidos tricarboxílicos (TCA). Conclusiones: En conjunto, estos datos podrían utilizarse para una mejor clasificación de estas enfermedades, así como para la optimización de las opciones de manejo y tratamiento.
Assuntos
Humanos , Deficiência de Citocromo-c Oxidase/patologia , Proteômica/métodos , Fibroblastos/patologia , Mitocôndrias/patologia , Fosforilação Oxidativa , DNA/genética , Proteínas/metabolismo , Células Cultivadas , Ciclo do Ácido Cítrico/fisiologiaRESUMO
BACKGROUND: Mitochondriopathies are multisystem diseases affecting the oxidative phosphorylation (OXPHOS) system. Skin fibroblasts are a good model for the study of these diseases. Fibroblasts with a complex IV mitochondriopathy were used to determine the molecular mechanism and the main affected functions in this disease. METHODS: Skin fibroblast were grown to assure disease phenotype. Mitochondria were isolated from these cells and their proteome extracted for protein identification. Identified proteins were validated with the MitoMiner database. RESULTS: Disease phenotype was corroborated on skin fibroblasts, which presented a complex IV defect. The mitochondrial proteome of these cells showed that the most affected proteins belonged to the OXPHOS system, mainly to the complexes that form supercomplexes or respirosomes (I, III, IV, and V). Defects in complex IV seemed to be due to assembly issues, which might prevent supercomplexes formation and efficient substrate channeling. It was also found that this mitochondriopathy affects other processes that are related to DNA genetic information flow (replication, transcription, and translation) as well as beta oxidation and tricarboxylic acid cycle. CONCLUSIONS: These data, as a whole, could be used for the better stratification of these diseases, as well as to optimize management and treatment options.
Assuntos
Deficiência de Citocromo-c Oxidase/patologia , Fibroblastos/patologia , Mitocôndrias/patologia , Proteômica/métodos , Células Cultivadas , Ciclo do Ácido Cítrico/fisiologia , DNA/genética , Humanos , Fosforilação Oxidativa , Proteínas/metabolismoRESUMO
INTRODUCTION: Duchenne muscular dystrophy (DMD) is a degenerative disease of skeletal, respiratory, and cardiac muscles caused by defects in the dystrophin gene. More recently, brain involvement has been verified. Mitochondrial dysfunction and oxidative stress may underlie the pathophysiology of DMD. In this study we evaluate Krebs cycle enzymes activity in the cerebral cortex, diaphragm, and quadriceps muscles of mdx mice. METHODS: Cortex, diaphragm, and quadriceps tissues from male dystrophic mdx and control mice were used. RESULTS: We observed increased malate dehydrogenase activity in the cortex; increased malate dehydrogenase and succinate dehydrogenase activities in the diaphragm; and increased citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase activities in the quadriceps of mdx mice. CONCLUSION: This study showed increased activity of Krebs cycle enzymes in cortex, quadriceps, and diaphragm in mdx mice.
Assuntos
Citrato (si)-Sintase/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Isocitrato Desidrogenase/metabolismo , Malato Desidrogenase/metabolismo , Distrofia Muscular de Duchenne/enzimologia , Animais , Córtex Cerebral/enzimologia , Diafragma/enzimologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/enzimologia , Distrofia Muscular de Duchenne/genéticaRESUMO
The tricarboxylic acid (TCA) cycle, a crucial component of respiratory metabolism, is composed of a set of eight enzymes present in the mitochondrial matrix. However, most of the TCA cycle enzymes are encoded in the nucleus in higher eukaryotes. In addition, evidence has accumulated demonstrating that nuclear genes were acquired from the mitochondrial genome during the course of evolution. For this reason, we here analyzed the evolutionary history of all TCA cycle enzymes in attempt to better understand the origin of these nuclear-encoded proteins. Our results indicate that prior to endosymbiotic events the TCA cycle seemed to operate only as isolated steps in both the host (eubacterial cell) and mitochondria (alphaproteobacteria). The origin of isoforms present in different cell compartments might be associated either with gene-transfer events which did not result in proper targeting of the protein to mitochondrion or with duplication events. Further in silico analyses allow us to suggest new insights into the possible roles of TCA cycle enzymes in different tissues. Finally, we performed coexpression analysis using mitochondrial TCA cycle genes revealing close connections among these genes most likely related to the higher efficiency of oxidative phosphorylation in this specialized organelle. Moreover, these analyses allowed us to identify further candidate genes which might be used for metabolic engineering purposes given the importance of the TCA cycle during development and/or stress situations.
Assuntos
Ciclo do Ácido Cítrico/fisiologia , Evolução Biológica , Ciclo do Ácido Cítrico/genética , Mitocôndrias/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
The Fe-S cluster of mitochondrial aconitase is rapidly and selectively inactivated by oxidants, yielding an inactive enzyme that can be reactivated by reductants and iron in vivo. In order to elucidate the metabolic impact of oxidant-dependent aconitase inhibition over the citric acid cycle, the respiratory chain reactions, and reactive species formation, we performed a metabolic analysis using isolated mitochondria from different rat tissues. Titrations with fluorocitrate showed IC50 for aconitase inhibition ranging from 7 to 24 µM. The aconitase inhibition threshold in mitochondrial oxygen consumption was determined to range from 63 to 98%. Of the tissues examined, brain and heart exhibited the highest values in the flux control coefficient (> 0.95). Aconitase-specific activity varied widely among tissues examined from ~60 mU/mg in liver to 321 mU/mg in kidney at 21% O2. In brain and heart, aconitase-specific activity increased by 42 and 12%, respectively, at 2% O2 reflecting aconitase inactivation by oxygen-derived oxidants at 21% O2. Both mitochondrial membrane potential and hydrogen peroxide production significantly decreased upon aconitase inhibition in heart and brain mitochondria. These results indicate that aconitase can exert control over respiration (with tissue specificity) and support the hypothesis that inactivation of aconitase may provide a control mechanism to prevent O2(â-) and H2O2 formation by the respiratory chain.
Assuntos
Aconitato Hidratase/biossíntese , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/enzimologia , Consumo de Oxigênio/fisiologia , Superóxidos/metabolismo , Aconitato Hidratase/antagonistas & inibidores , Animais , Encéfalo/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Transporte de Elétrons/fisiologia , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Miocárdio/metabolismo , Estresse Oxidativo , Ratos , Ratos WistarRESUMO
Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using (13)C-stable isotope resolved metabolomics and (2)H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid ß-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.
Assuntos
Aminoácidos/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Glucose/metabolismo , Leishmania mexicana/metabolismo , Leishmaniose Cutânea/metabolismo , Mitocôndrias/metabolismo , Aminoácidos/genética , Animais , Feminino , Glucose/genética , Leishmania mexicana/genética , Leishmaniose Cutânea/genética , Macrófagos/metabolismo , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/genéticaRESUMO
Recent years have seen considerable advances in our understanding of the particular physiological roles of the constituent enzymes of the tricarboxylic acid (TCA) cycle. Despite acquiring a fairly comprehensive overview of the functional importance of these proteins relatively little is known concerning how this important pathway is regulated. In this review we concentrate on the mitochondrial reactions since this organelle is the only one in which a full cycle can, at least theoretically, operate. We summarize what is known about the regulation of the enzymes of the pathway both from historical kinetic studies as well as discussing more recent transcriptional and proteomic studies and our enhanced understanding of subcellular compartmentation within the context of metabolic regulation.
Assuntos
Ciclo do Ácido Cítrico/fisiologia , Enzimas/metabolismo , Mitocôndrias/metabolismo , Cinética , Mitocôndrias/genética , Proteômica/métodos , TranscriptomaRESUMO
Studies have shown a relationship between energy metabolism and methylphenidate (MPH); however, there are no studies evaluating the effects of MPH in Krebs cycle. So, we investigated if MPH treatment could alter the activity of citrate synthase (CS), malate dehydrogenase (MD), and isocitrate dehydrogenase (ID) in the brain of young and adult Wistar rats. Our results showed that MPH (2 and 10 mg/kg) reduced CS in the striatum and prefrontal cortex (PF), with MPH at all doses in the cerebellum and hippocampus after chronic treatment in young rats. In adult rats the CS was reduced in the cerebellum after acute treatment with MPH at all doses, and after chronic treatment in the PF and cerebellum with MPH (10 mg/kg), and in the hippocampus with MPH (2 and 10 mg/kg). The ID decreased in the hippocampus and striatum with MPH (2 and 10 mg/kg), and in the cortex (10 mg/kg) after acute treatment in young rats. In adult rats acute treatment with MPH (2 and 10 mg/kg) reduced ID in the cerebellum, and with MPH (10 mg/kg) in the cortex; chronic treatment with MPH (10 mg/kg) decreased ID in the PF; with MPH (2 and 10 mg/kg) in the cerebellum, and with MPH at all doses in the hippocampus. The MD did not alter. In conclusion, our results suggest that MPH can alter enzymes of Krebs cycle in brain areas involved with circuits related with attention deficit hyperactivity disorder; however, such effects depend on age of animal and treatment regime.
Assuntos
Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Ciclo do Ácido Cítrico/efeitos dos fármacos , Metilfenidato/toxicidade , Fatores Etários , Animais , Ciclo do Ácido Cítrico/fisiologia , Ratos , Ratos WistarRESUMO
Studies have demonstrated that AMPHs produce long-term damage to the brain dopaminergic, serotoninergic and glutamatergic regions. Prefrontal cortex, amygdala, hippocampus and striatum appear to be involved in the toxicity and behavioral changes induced by AMPHs. A single dose of AMPH causes mitochondrial dysfunction and oxidative stress in rat brain. The goal of the present study was thus to investigate the potency of two amphetamines, dextroamphetamine (d-AMPH) and methamphetamine (m-AMPH), on the behavior and energetic dysfunction in the brain of rats. d-AMPH and m-AMPH increased the crossing and rearing behaviors. The numbers of visits to the center were increased by d-AMPH and m-AMPH only at 2mg/kg. Likewise, at a high dose (2 mg/kg), the injection of m-AMPH increased the amount of sniffing. The AMPHs significantly decreased the activities of Krebs cycle enzymes (citrate synthase and succinate dehydrogenase) and mitochondrial respiratory chain complexes (I-IV); nevertheless, this effect varied depending on the brain region evaluated. In summary, this study demonstrated that at high doses, m-AMPH, increased stereotyped (sniffing) behavior in rats, but d-AMPH did not. However, this study shows that d-AMPH and m-AMPH seem to have similar effects on the brains energetic metabolism.