RESUMEN
Biogenesis of F1Fo ATP synthase, the key enzyme of mitochondrial energy provision, depends on transmembrane protein 70 (TMEM70), localized in the inner mitochondrial membrane of higher eukaryotes. TMEM70 absence causes severe ATP-synthase deficiency and leads to a neonatal mitochondrial encephalocardiomyopathy in humans. However, the exact biochemical function of TMEM70 remains unknown. Using TMEM70 conditional knockout in mice, we show that absence of TMEM70 impairs the early stage of enzyme biogenesis by preventing incorporation of hydrophobic subunit c into rotor structure of the enzyme. This results in the formation of an incomplete, pathologic enzyme complex consisting of F1 domain and peripheral stalk but lacking Fo proton channel composed of subunits c and a. We demonstrated direct interaction between TMEM70 and subunit c and showed that overexpression of subunit c in TMEM70-/- cells partially rescued TMEM70 defect. Accordingly, TMEM70 knockdown prevented subunit c accumulation otherwise observed in F1-deficient cells. Altogether, we identified TMEM70 as specific ancillary factor for subunit c. The biologic role of TMEM70 is to increase the low efficacy of spontaneous assembly of subunit c oligomer, the key and rate-limiting step of ATP-synthase biogenesis, and thus to reach an adequately high physiologic level of ATP synthase in mammalian tissues.-Kovalcíková, J., Vrbacký, M., Pecina, P., Tauchmannová, K., Nusková, H., Kaplanová, V., Brázdová, A., Alán, L., Eliás, J., Cunátová, K., Korínek, V., Sedlacek, R., Mrácek, T., Houstek, J. TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme.
Asunto(s)
Proteínas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Animales , Células Cultivadas , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes/métodos , Genotipo , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/genética , Proteolípidos/metabolismo , Tamoxifeno/farmacologíaRESUMEN
Metformin is widely prescribed as a first-choice antihyperglycemic drug for treatment of type 2 diabetes mellitus, and recent epidemiological studies showed its utility also in cancer therapy. Although it is in use since the 1970s, its molecular target, either for antihyperglycemic or antineoplastic action, remains elusive. However, the body of the research on metformin effect oscillates around mitochondrial metabolism, including the function of oxidative phosphorylation (OXPHOS) apparatus. In this study, we focused on direct inhibitory mechanism of biguanides (metformin and phenformin) on OXPHOS complexes and its functional impact, using the model of isolated brown adipose tissue mitochondria. We demonstrate that biguanides nonspecifically target the activities of all respiratory chain dehydrogenases (mitochondrial NADH, succinate, and glycerophosphate dehydrogenases), but only at very high concentrations (10-2-10-1 M) that highly exceed cellular concentrations observed during the treatment. In addition, these concentrations of biguanides also trigger burst of reactive oxygen species production which, in combination with pleiotropic OXPHOS inhibition, can be toxic for the organism. We conclude that the beneficial effect of biguanides should probably be associated with subtler mechanism, different from the generalized inhibition of the respiratory chain.
Asunto(s)
Biguanidas/farmacología , Hipoglucemiantes/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Tejido Adiposo Pardo/citología , Animales , Glicerolfosfato Deshidrogenasa/metabolismo , Peróxido de Hidrógeno/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Metformina/farmacología , Oxidación-Reducción/efectos de los fármacos , Fenformina/farmacología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Ácido Succínico/metabolismoRESUMEN
AIMS: Iron replacement improves clinical status in iron-deficient patients with heart failure (HF), but the pathophysiology is poorly understood. Iron is essential not only for erythropoiesis, but also for cellular bioenergetics. The impact of myocardial iron deficiency (MID) on mitochondrial function, measured directly in the failing human heart, is unknown. METHODS AND RESULTS: Left ventricular samples were obtained from 91 consecutive HF patients undergoing transplantation and 38 HF-free organ donors (controls). Total myocardial iron content, mitochondrial respiration, citric acid cycle and respiratory chain enzyme activities, respiratory chain components (complex I-V), and protein content of reactive oxygen species (ROS)-protective enzymes were measured in tissue homogenates to quantify mitochondrial function. Myocardial iron content was lower in HF compared with controls (156 ± 41 vs. 200 ± 38 µg·g-1 dry weight, P < 0.001), independently of anaemia. MID (the lowest iron tercile in HF) was associated with more extensive coronary disease and less beta-blocker usage compared with non-MID HF patients. Compared with controls, HF patients displayed reduced myocardial oxygen2 respiration and reduced activity of all examined mitochondrial enzymes (all P < 0.001). MID in HF was associated with preserved activity of respiratory chain enzymes but reduced activity of aconitase and citrate synthase (by -26% and -15%, P < 0.05) and reduced expression of catalase, glutathione peroxidase, and superoxide dismutase 2. CONCLUSION: Myocardial iron content is decreased and mitochondrial functions are impaired in advanced HF. MID in HF is associated with diminished citric acid cycle enzyme activities and decreased ROS-protecting enzymes. MID may contribute to altered myocardial substrate use and to worsening of mitochondrial dysfunction that exists in HF.
Asunto(s)
Insuficiencia Cardíaca/metabolismo , Hierro/metabolismo , Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/cirugía , Trasplante de Corazón , Humanos , Masculino , Persona de Mediana EdadRESUMEN
TMEM70, a 21-kDa protein localized in the inner mitochondrial membrane, has been shown to facilitate the biogenesis of mammalian F1Fo ATP synthase. Mutations of the TMEM70 gene represent the most frequent cause of isolated ATP synthase deficiency resulting in a severe mitochondrial disease presenting as neonatal encephalo-cardiomyopathy (OMIM 604273). To better understand the biological role of this factor, we generated Tmem70-deficient mice and found that the homozygous Tmem70-/- knockouts exhibited profound growth retardation and embryonic lethality at â¼9.5 days post coitum. Blue-Native electrophoresis demonstrated an isolated deficiency in fully assembled ATP synthase in the Tmem70-/- embryos (80% decrease) and a marked accumulation of F1 complexes indicative of impairment in ATP synthase biogenesis that was stalled at the early stage, following the formation of F1 oligomer. Consequently, a decrease in ADP-stimulated State 3 respiration, respiratory control ratio and ATP/ADP ratios, indicated compromised mitochondrial ATP production. Tmem70-/- embryos exhibited delayed development of the cardiovascular system and a disturbed heart mitochondrial ultrastructure, with concentric or irregular cristae structures. Tmem70+/- heterozygous mice were fully viable and displayed normal postnatal growth and development of the mitochondrial oxidative phosphorylation system. Nevertheless, they presented with mild deterioration of heart function. Our results demonstrated that Tmem70 knockout in the mouse results in embryonic lethality due to the lack of ATP synthase and impairment of mitochondrial energy provision. This is analogous to TMEM70 dysfunction in humans and verifies the crucial role of this factor in the biosynthesis and assembly of mammalian ATP synthase.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/genética , Adenosina Trifosfato/metabolismo , Animales , Cardiomiopatías/metabolismo , Femenino , Homocigoto , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Errores Innatos del Metabolismo/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/biosíntesis , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Mutación , Fosforilación Oxidativa , EmbarazoRESUMEN
Mitochondrial ATP synthase, ADP/ATP translocase (ANT), and inorganic phosphate carrier (PiC) are supposed to form a supercomplex called ATP synthasome. Our protein and transcript analysis of rat tissues indicates that the expression of ANT and PiC is transcriptionally controlled in accordance with the biogenesis of ATP synthase. In contrast, the content of ANT and PiC is increased in ATP synthase deficient patients' fibroblasts, likely due to a post-transcriptional adaptive mechanism. A structural analysis of rat heart mitochondria by immunoprecipitation, blue native/SDS electrophoresis, immunodetection and MS analysis revealed the presence of ATP synthasome. However, the majority of PiC and especially ANT did not associate with ATP synthase, suggesting that most of PiC, ANT and ATP synthase exist as separate entities.