RESUMEN
Mitochondria are essential organelles that generate energy via oxidative phosphorylation. Plant mitochondrial genome encodes some of the respiratory complex subunits, and these transcripts require accurate processing, including C-to-U RNA editing and intron splicing. Pentatricopeptide repeats (PPR) proteins are involved in various organellar RNA processing events. PPR596, a P-type PPR protein, was previously identified to function in the C-to-U editing of mitochondrial rps3 transcripts in Arabidopsis. Here, we demonstrate that PPR596 functions in the cis-splicing of nad2 intron 3 in mitochondria. Loss of the PPR596 function affects the editing at rps3eU1344SS, impairs nad2 intron 3 splicing and reduces the mitochondrial complex I's assembly and activity, while inducing alternative oxidase (AOX) gene expression. This defect in nad2 intron splicing provides a plausible explanation for the slow growth of the ppr595 mutants. Although a few P-type PPR proteins are involved in RNA C-to-U editing, our results suggest that the primary function of PPR596 is intron splicing.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Regulación de la Expresión Génica de las Plantas , Intrones/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Plantas/genética , Empalme del ARNRESUMEN
NADH:ubiquinone oxidoreductase, more commonly referred to as mitochondrial complex I (CI), is the largest discrete enzyme of the oxidative phosphorylation system (OXPHOS). It is localized to the mitochondrial inner membrane. CI oxidizes NADH generated from the tricarboxylic acid cycle to NAD+, in a series of redox reactions that culminates in the reduction of ubiquinone, and the transport of protons from the matrix across the inner membrane to the intermembrane space. The resulting proton-motive force is consumed by ATP synthase to generate ATP, or harnessed to transport ions, metabolites and proteins into the mitochondrion. CI is also a major source of reactive oxygen species. Accordingly, impaired CI function has been associated with a host of chronic metabolic and degenerative disorders such as diabetes, cardiomyopathy, Parkinson's disease (PD) and Leigh syndrome. Studies on Drosophila have contributed to our understanding of the multiple roles of CI in bioenergetics and organismal physiology. Here, we explore and discuss some of the studies on Drosophila that have informed our understanding of this complex and conclude with some of the open questions about CI that can be resolved by studies on Drosophila.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Mitocondrias/metabolismo , Animales , Fosforilación Oxidativa , Subunidades de Proteína/metabolismoRESUMEN
Fatty acid ß-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are mitochondrial redox processes that generate ATP. The biogenesis of the respiratory Complex I, a 1â MDa multiprotein complex that is responsible for initiating OXPHOS, is mediated by assembly factors including the mitochondrial complex I assembly (MCIA) complex. However, the organisation and the role of the MCIA complex are still unclear. Here we show that ECSIT functions as the bridging node of the MCIA core complex. Furthermore, cryo-electron microscopy together with biochemical and biophysical experiments reveal that the C-terminal domain of ECSIT directly binds to the vestigial dehydrogenase domain of the FAO enzyme ACAD9 and induces its deflavination, switching ACAD9 from its role in FAO to an MCIA factor. These findings provide the structural basis for the MCIA complex architecture and suggest a unique molecular mechanism for coordinating the regulation of the FAO and OXPHOS pathways to ensure an efficient energy production.
Asunto(s)
Complejo I de Transporte de Electrón/química , Flavina-Adenina Dinucleótido/metabolismo , Mitocondrias/metabolismo , Acil-CoA Deshidrogenasas/genética , Acil-CoA Deshidrogenasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Microscopía por Crioelectrón , Complejo I de Transporte de Electrón/metabolismo , Metabolismo Energético , Flavina-Adenina Dinucleótido/química , Humanos , Fosforilación Oxidativa , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
NUBPL (Nucleotide-binding protein like) protein encodes a member of the Mrp/NBP35 ATP-binding proteins family, deemed to be involved in mammalian complex I (CI) assembly process. Exome sequencing of a patient presenting with infantile-onset hepatopathy, renal tubular acidosis, developmental delay, short stature, leukoencephalopathy with minimal cerebellar involvement and multiple OXPHOS deficiencies revealed the presence of two novel pathogenic compound heterozygous variants in NUBPL (p.Phe242Leu/p.Leu104Pro). We investigated patient's and control immortalised fibroblasts and demonstrated that both the peripheral and the membrane arms of complex I are undetectable in mutant NUBPL cells, resulting in virtually absent CI holocomplex and loss of enzyme activity. In addition, complex III stability was moderately affected as well. Lentiviral-mediated expression of the wild-type NUBPL cDNA rescued both CI and CIII assembly defects, confirming the pathogenicity of the variants. In conclusion, this is the first report describing a complex multisystemic disorder due to NUBPL defect. In addition, we confirmed the role of NUBPL in Complex I assembly associated with secondary effect on Complex III stability and we demonstrated a defect of mtDNA-related translation which suggests a potential additional role of NUBPL in mtDNA expression.
Asunto(s)
Variación Genética , Heterocigoto , Leucoencefalopatías/genética , Proteínas Mitocondriales/genética , Adolescente , Adulto , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Niño , ADN Mitocondrial , Femenino , Humanos , Lactante , Recién Nacido , Leucoencefalopatías/diagnóstico , Imagen por Resonancia Magnética , Masculino , Mitocondrias/patología , Mutación , Adulto JovenRESUMEN
Disorders of the white matter are genetically very heterogeneous including several genes involved in mitochondrial bioenergetics. Diagnosis of the underlying cause is aided by pattern recognition on neuroimaging and by next-generation sequencing. Recently, genetic changes in the complex I assembly factor NUBPL have been characterized by a consistent recognizable pattern of leukoencephalopathy affecting deep white matter including the corpus callosum and cerebellum. Here, we report twin boys with biallelic variants in NUBPL, an unreported c.351 G > A; p.(Met117Ile) and a previously reported pathological variant c. 693 + 1 G > A. Brain magnetic resonance imaging showed abnormal T2 hyperintense signal involving the periventricular white matter, external capsule, corpus callosum, and, prominently, the bilateral thalami. The neuroimaging pattern evolved over 18 months with marked diffuse white matter signal abnormality, volume loss, and new areas of signal abnormality in the cerebellar folia and vermis. Magnetic resonance spectroscopy showed elevated lactate. Functional studies in cultured fibroblasts confirmed pathogenicity of the genetic variants. Complex I activity of the respiratory chain was deficient spectrophotometrically and on blue native gel with in-gel activity staining. There was absent assembly and loss of proteins of the matrix arm of complex I when traced with an antibody to NDUFS2, and incomplete assembly of the membrane arm when traced with an NDUFB6 antibody. There was decreased NUBPL protein on Western blot in patient fibroblasts compared to controls. Compromised NUBPL activity impairs assembly of the matrix arm of complex I and produces a severe, rapidly-progressive leukoencephalopathy with thalamic involvement on MRI, further expanding the neuroimaging phenotype.
Asunto(s)
Enfermedades en Gemelos/genética , Complejo I de Transporte de Electrón/metabolismo , Leucoencefalopatías/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Tálamo/diagnóstico por imagen , Línea Celular , Cuerpo Calloso/diagnóstico por imagen , Cuerpo Calloso/patología , Enfermedades en Gemelos/diagnóstico por imagen , Enfermedades en Gemelos/metabolismo , Enfermedades en Gemelos/fisiopatología , Complejo I de Transporte de Electrón/deficiencia , Complejo I de Transporte de Electrón/genética , Cápsula Externa/diagnóstico por imagen , Cápsula Externa/patología , Ojo/fisiopatología , Fibroblastos/metabolismo , Humanos , Lactante , Ácido Láctico/metabolismo , Leucoencefalopatías/diagnóstico por imagen , Leucoencefalopatías/metabolismo , Leucoencefalopatías/fisiopatología , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Masculino , Mitocondrias/genética , Proteínas Mitocondriales/metabolismo , Mutación , NADH Deshidrogenasa/metabolismo , Gemelos Monocigóticos/genética , Sustancia Blanca/diagnóstico por imagen , Sustancia Blanca/patología , Secuenciación del ExomaRESUMEN
Ketogenic diet (KD) which combined carbohydrate restriction and the addition of ketone bodies has emerged as an alternative metabolic intervention used as an anticonvulsant therapy or to treat different types of neurological or mitochondrial disorders including MELAS syndrome. MELAS syndrome is a severe mitochondrial disease mainly due to the m.3243Aâ¯>â¯G mitochondrial DNA mutation. The broad success of KD is due to multiple beneficial mechanisms with distinct effects of very low carbohydrates and ketones. To evaluate the metabolic part of carbohydrate restriction, transmitochondrial neuronal-like cybrid cells carrying the m.3243Aâ¯>â¯G mutation, shown to be associated with a severe complex I deficiency was exposed during 3â¯weeks to glucose restriction. Mitochondrial enzyme defects were combined with an accumulation of complex I (CI) matrix intermediates in the untreated mutant cells, leading to a drastic reduction in CI driven respiration. The severe reduction of CI was also paralleled in post-mortem brain tissue of a MELAS patient carrying high mutant load. Importantly, lowering significantly glucose concentration in cell culture improved CI assembly with a significant reduction of matrix assembly intermediates and respiration capacities were restored in a sequential manner. In addition, OXPHOS protein expression and mitochondrial DNA copy number were significantly increased in mutant cells exposed to glucose restriction. The accumulation of CI matrix intermediates appeared as a hallmark of MELAS pathophysiology highlighting a critical pathophysiological mechanism involving CI disassembly, which can be alleviated by lowering glucose fuelling and the induction of mitochondrial biogenesis, emphasizing the usefulness of metabolic interventions in MELAS syndrome.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Glucosa/metabolismo , Síndrome MELAS/enzimología , Mitocondrias/enzimología , Neuronas/enzimología , Mutación Puntual , Línea Celular Tumoral , Complejo I de Transporte de Electrón/genética , Femenino , Humanos , Síndrome MELAS/genética , Síndrome MELAS/patología , Masculino , Mitocondrias/genética , Mitocondrias/patología , Neuronas/patología , Fosforilación OxidativaRESUMEN
Mitochondrial complex I is an intricate 1MDa membrane protein complex with a central role in aerobic energy metabolism. The minimal form of complex I consists of fourteen central subunits that are conserved from bacteria to man. In addition, eukaryotic complex I comprises some 30 accessory subunits of largely unknown function. The gene for the accessory NDUFS4 subunit of human complex I is a hot spot for fatal pathogenic mutations in humans. We have deleted the gene for the orthologous NUYM subunit in the aerobic yeast Yarrowia lipolytica, an established model system to study eukaryotic complex I and complex I linked diseases. We observed assembly of complex I which lacked only subunit NUYM and retained weak interaction with assembly factor N7BML (human NDUFAF2). Absence of NUYM caused distortion of iron sulfur clusters of the electron input domain leading to decreased complex I activity and increased release of reactive oxygen species. We conclude that NUYM has an important stabilizing function for the electron input module of complex I and is essential for proper complex I function.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , NADH Deshidrogenasa/metabolismo , Yarrowia/metabolismo , Electrones , Metabolismo Energético/fisiología , Proteínas Fúngicas/metabolismo , Humanos , Mitocondrias/metabolismo , Subunidades de Proteína/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Mammalian complex I is composed of 44 different subunits and its assembly requires at least 13 specific assembly factors. Proper function of the mitochondrial respiratory chain enzyme is of crucial importance for cell survival due to its major participation in energy production and cell signaling. Complex I assembly depends on the coordination of several crucial processes that need to be tightly interconnected and orchestrated by a number of assembly factors. The understanding of complex I assembly evolved from simple sequential concept to the more sophisticated modular assembly model describing a convoluted process. According to this model, the different modules assemble independently and associate afterwards with each other to form the final enzyme. In this review, we aim to unravel the complexity of complex I assembly and provide the latest insights in this fundamental and fascinating process. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.
Asunto(s)
Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/ultraestructura , Proteínas Mitocondriales/química , Proteínas Mitocondriales/ultraestructura , Modelos Químicos , Simulación de Dinámica Molecular , Animales , Sitios de Unión , Activación Enzimática , Humanos , Unión Proteica , Conformación ProteicaRESUMEN
Ketogenic Diet used to treat refractory epilepsy for almost a century may represent a treatment option for mitochondrial disorders for which effective treatments are still lacking. Mitochondrial complex I deficiencies are involved in a broad spectrum of inherited diseases including Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes syndrome leading to recurrent cerebral insults resembling strokes and associated with a severe complex I deficiency caused by mitochondrial DNA (mtDNA) mutations. The analysis of MELAS neuronal cybrid cells carrying the almost homoplasmic m.3243A>G mutation revealed a metabolic switch towards glycolysis with the production of lactic acid, severe defects in respiratory chain activity and complex I disassembly with an accumulation of assembly intermediates. Metabolites, NADH/NAD+ ratio, mitochondrial enzyme activities, oxygen consumption and BN-PAGE analysis were evaluated in mutant compared to control cells. A severe complex I enzymatic deficiency was identified associated with a major complex I disassembly with an accumulation of assembly intermediates of 400kDa. We showed that Ketone Bodies (KB) exposure for 4weeks associated with glucose deprivation significantly restored complex I stability and activity, increased ATP synthesis and reduced the NADH/NAD+ ratio, a key component of mitochondrial metabolism. In addition, without changing the mutant load, mtDNA copy number was significantly increased with KB, indicating that the absolute amount of wild type mtDNA copy number was higher in treated mutant cells. Therefore KB may constitute an alternative and promising therapy for MELAS syndrome, and could be beneficial for other mitochondrial diseases caused by complex I deficiency.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Cuerpos Cetónicos/farmacología , Síndrome MELAS/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Línea Celular , Respiración de la Célula/efectos de los fármacos , Variaciones en el Número de Copia de ADN/efectos de los fármacos , ADN Mitocondrial/genética , Dieta Cetogénica , Complejo I de Transporte de Electrón/deficiencia , Humanos , Síndrome MELAS/genética , Síndrome MELAS/metabolismo , Síndrome MELAS/patología , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Mitocondriales/complicaciones , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
Group II introns are common in the mitochondrial genome of higher plant species. The splicing of these introns is a complex process involving the synergistic action of multiple factors. However, few of these factors have been characterized in maize. In this study, we found that the Empty pericarp11 (Emp11) gene, which encodes a P-type pentatricopeptide repeat (PPR) protein, is required for the development of maize seeds. The loss of Emp11 function seriously impairs embryo and endosperm development, resulting in empty pericarp seeds in maize, and alteration in Emp11 expression leads to quantitative variation in kernel size and weight. We found that the emp11 mutants showed a failure in nad1 intron splicing, exhibited a severe reduction in complex I assembly and activity, mitochondrial structure disturbances, and an increase in alternative oxidase AOX2 and AOX3 levels. Interestingly, the emp11 phenotype was very severe in the W22 inbred line but could be partially recovered in B73 BC2F2 segregating ears. These results suggest that EMP11 serves as a factor for the splicing of mitochondrial nad1 introns and is required for mitochondrial function to ensure proper seed development in maize.
Asunto(s)
Mitocondrias/genética , Proteínas de Plantas/metabolismo , Semillas/crecimiento & desarrollo , Zea mays/genética , Regulación de la Expresión Génica de las Plantas , Intrones , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación , NADH Deshidrogenasa/genética , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Empalme del ARN , Semillas/genética , Zea mays/crecimiento & desarrolloRESUMEN
L-Galactono-1,4-lactone dehydrogenase (GLDH) catalyses the last enzymatic step of the ascorbate biosynthetic pathway in plants. GLDH is localised to mitochondria and several reports have shown that GLDH is associated with complex I of the respiratory chain. In a gldh knock-out mutant, complex I is not detectable, suggesting that GLDH is essential for complex I assembly or stability. GLDH has not been identified as a genuine complex I subunit, instead, it is present in a smaller, lowly abundant version of complex I called complex I*. In addition, GLDH activity has also been detected in smaller protein complexes within mitochondria membranes. Here, we investigated the role of GLDH during complex I assembly. We identified GLDH in complexes co-localising with some complex I assembly intermediates. Using a mutant that accumulates complex I assembly intermediates, we confirmed that GLDH is associated with the complex I assembly intermediates of 400 and 450 kDa. In addition, we detected accumulation of the 200 kDa complex I assembly intermediate in the gldh mutant. Taken together, our data suggest that GLDH is an assembly factor of the membrane arm of complex I. This function appears to be independent of the role of GLDH in ascorbate synthesis, as evidenced by the ascorbate-deficient mutant vtc2-1 accumulating wild-type levels of complex I. Therefore, we propose that GLDH is a dual-function protein that has a second, non-enzymatic function in complex I assembly as a plant-specific assembly factor. We propose an updated model for complex I assembly that includes complex I* as an assembly intermediate.
Asunto(s)
Arabidopsis/enzimología , Ácido Ascórbico/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Arabidopsis/genética , Transporte de Electrón , Complejo I de Transporte de Electrón/genética , Membranas Intracelulares/enzimología , Mitocondrias/enzimología , Modelos Biológicos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genéticaRESUMEN
Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the first component of the mitochondrial respiratory chain. In recent years, high-resolution cryo-EM studies of complex I from various species have greatly enhanced the understanding of the structure and function of this important membrane-protein complex. Less well studied is the structural basis of complex I biogenesis. The assembly of this complex of more than 40 subunits, encoded by nuclear or mitochondrial DNA, is an intricate process that requires at least 20 different assembly factors in humans. These are proteins that are transiently associated with building blocks of the complex and are involved in the assembly process, but are not part of mature complex I. Although the assembly pathways have been studied extensively, there is limited information on the structure and molecular function of the assembly factors. Here, the insights that have been gained into the assembly process using cryo-EM are reviewed.
Asunto(s)
Complejo I de Transporte de Electrón , Mitocondrias , Humanos , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Microscopía por Crioelectrón , Mitocondrias/metabolismoRESUMEN
The formation of fruiting bodies is a highly regulated process that requires the coordinated formation of different cell types. By analyzing developmental mutants, many developmental factors have already been identified. Yet, a complete understanding of fruiting body formation is still lacking. In this study, we analyzed developmental mutant pro34 of the filamentous ascomycete Sordaria macrospora. Genome sequencing revealed a deletion in the pro34 gene encoding a putative mitochondrial complex I assembly factor homologous to Neurospora crassa CIA84. We show that PRO34 is required for fast vegetative growth, fruiting body and ascospore formation. The pro34 transcript undergoes adenosine to inosine editing, a process correlated with sexual development in fruiting body-forming ascomycetes. Fluorescence microscopy and western blot analysis showed that PRO34 is a mitochondrial protein, and blue-native PAGE revealed that the pro34 mutant lacks mitochondrial complex I. Inhibitor experiments revealed that pro34 respires via complexes III and IV, but also shows induction of alternative oxidase, a shunt pathway to bypass complexes III and IV. We discuss the hypothesis that alternative oxidase is induced to prevent retrograde electron transport to complex I intermediates, thereby protecting from oxidative stress.
RESUMEN
Complex I is the first enzyme involved in the mitochondrial electron transport chain. With >40 subunits of dual genetic origin, the biogenesis of complex I is highly intricate and poorly understood. We used Chlamydomonas reinhardtii as a model system to reveal factors involved in complex I biogenesis. Two insertional mutants, displaying a complex I assembly defect characterized by the accumulation of a 700 kDa subcomplex, were analyzed. Genetic analyses showed these mutations were allelic and mapped to the gene AMC1 (Cre16.g688900) encoding a low-complexity protein of unknown function. The complex I assembly and activity in the mutant was restored by complementation with the wild-type gene, confirming AMC1 is required for complex I biogenesis. The N terminus of AMC1 targets a reporter protein to yeast mitochondria, implying that AMC1 resides and functions in the Chlamydomonas mitochondria. Accordingly, in both mutants, loss of AMC1 function results in decreased abundance of the mitochondrial nd4 transcript, which encodes the ND4 membrane subunit of complex I. Loss of ND4 in a mitochondrial nd4 mutant is characterized by a membrane arm assembly defect, similar to that exhibited by loss of AMC1. These results suggest AMC1 is required for the production of mitochondrially-encoded complex I subunits, specifically ND4. We discuss the possible modes of action of AMC1 in mitochondrial gene expression and complex I biogenesis.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Plantas/metabolismo , Sitios de Unión , Chlamydomonas reinhardtii/genética , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/genética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Unión Proteica , Multimerización de ProteínaRESUMEN
Complex I is the largest member of the electron transport chain in human mitochondria. It comprises 45 subunits and requires at least 15 assembly factors. The subunits can be divided into 14 "core" subunits that carry out oxidation-reduction reactions and proton translocation, as well as 31 additional supernumerary (or accessory) subunits whose functions are less well known. Diminished levels of complex I activity are seen in many mitochondrial disease states. This review seeks to tabulate mutations in the supernumerary subunits of humans that appear to cause disease. Mutations in 20 of the supernumerary subunits have been identified. The mutations were analyzed in light of the tertiary and quaternary structure of human complex I (PDB id = 5xtd). Mutations were found that might disrupt the folding of that subunit or that would weaken binding to another subunit. In some cases, it appeared that no protein was made or, at least, could not be detected. A very common outcome is the lack of assembly of complex I when supernumerary subunits are mutated or missing. We suggest that poor assembly is the result of disrupting the large network of subunit interactions that the supernumerary subunits typically engage in.
RESUMEN
Due to its pivotal role in NADH oxidation and ATP synthesis, mitochondrial complex I (CI) emerged as a crucial regulator of cellular metabolism. A functional CI relies on the sequential assembly of nuclear- and mtDNA-encoded subunits; however, whether CI assembly status is involved in the metabolic adaptations in CI deficiency still remains largely unknown. Here, we investigated the relationship between CI functions, its structure and the cellular metabolism in 29 patient fibroblasts representative of most CI mitochondrial diseases. Our results show that, contrary to the generally accepted view, a complex I deficiency does not necessarily lead to a glycolytic switch, i.e. the so-called Warburg effect, but that this particular metabolic adaptation is a feature of CI assembly defect. By contrast, a CI functional defect without disassembly induces a higher catabolism to sustain the oxidative metabolism. Mechanistically, we demonstrate that reactive oxygen species overproduction by CI assembly intermediates and subsequent AMPK-dependent Pyruvate Dehydrogenase inactivation are key players of this metabolic reprogramming. Thus, this study provides a two-way-model of metabolic responses to CI deficiencies that are central not only in defining therapeutic strategies for mitochondrial diseases, but also in all pathophysiological conditions involving a CI deficiency.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Mitocondrias/metabolismo , Ciclo del Ácido Cítrico , Fibroblastos/citología , Fibroblastos/metabolismo , Glucólisis , Humanos , Ingeniería Metabólica , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , NADH Deshidrogenasa/antagonistas & inhibidores , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Análisis de Componente Principal , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Assembly of complex I of the mitochondrial respiratory chain (MRC) requires not only structural subunits for electron transport, but also assembly factors. In the nematode Caenorhabditis elegans, NUAF-1 and NUAF-3 are the only two assembly factors that have been characterized. In this study, we identify ACDH-12 as an assembly factor of the respiratory complex I. We demonstrate for the first time that a deficiency of ACDH-12 affects the formation and function of complex I. RNAi knockdown of acdh-12 also shortens lifespan and decreases fecundity. Although ACDH-12 has long been recognized as a very long-chain acyl-CoA dehydrogenase (VLCAD), the knockdown nematodes did not exhibit any change in body fat content. We suggested that in Caenorhabditis elegans, ACDH-12 is required for the assembly of the respiratory complex I, but may not be crucial to fatty acid oxidation. Interestingly, sequence analysis shows high homology between ACDH-12 and the human ACAD9, a protein that has initially been identified as a VLCAD, but later found to also be involved in the assembly of complex I in human.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Caenorhabditis elegans/fisiología , Fertilidad , Técnicas de Silenciamiento del Gen , Longevidad , Multimerización de ProteínaRESUMEN
The human protein arginine methyltransferase NDUFAF7 controls the assembly of the â¼1-MDa mitochondrial complex I (CI; the NADH ubiquinone oxidoreductase) by methylating its subunit NDUFS2. We determined crystal structures of MidA, the Dictyostelium ortholog of NDUFAF7. The MidA catalytic core domain resembles other eukaryotic methyltransferases. However, three large core loops assemble into a regulatory domain that is likely to control ligand selection. Binding of MidA to NDUFS2 is weakened by methylation, suggesting a mechanism for methylation-controlled substrate release. Structural and bioinformatic analyses support that MidA and NDUFAF7 and their role in CI assembly are conserved from bacteria to humans, implying that protein methylation already existed in proteobacteria. In vivo studies confirmed the critical role of the MidA methyltransferase activity for CI assembly, growth, and phototaxis of Dictyostelium. Collectively, our data elucidate the origin of protein arginine methylation and its use by MidA/NDUFAF7 to regulate CI assembly.