RESUMEN
A decline in mitochondrial respiration represents the root cause of a large number of inborn errors of metabolism. It is also associated with common age-associated diseases and the aging process. To gain insight into the systemic, biochemical consequences of respiratory chain dysfunction, we performed a case-control, prospective metabolic profiling study in a genetically homogenous cohort of patients with Leigh syndrome French Canadian variant, a mitochondrial respiratory chain disease due to loss-of-function mutations in LRPPRC. We discovered 45 plasma and urinary analytes discriminating patients from controls, including classic markers of mitochondrial metabolic dysfunction (lactate and acylcarnitines), as well as unexpected markers of cardiometabolic risk (insulin and adiponectin), amino acid catabolism linked to NADH status (α-hydroxybutyrate), and NAD(+) biosynthesis (kynurenine and 3-hydroxyanthranilic acid). Our study identifies systemic, metabolic pathway derangements that can lie downstream of primary mitochondrial lesions, with implications for understanding how the organelle contributes to rare and common diseases.
Asunto(s)
Enfermedad de Leigh/metabolismo , Metaboloma , Mitocondrias/metabolismo , Adiponectina/sangre , Adolescente , Adulto , Aminas/metabolismo , Biomarcadores/sangre , Biomarcadores/orina , Estudios de Casos y Controles , Niño , Femenino , Humanos , Insulina/sangre , Enfermedad de Leigh/sangre , Enfermedad de Leigh/genética , Enfermedad de Leigh/orina , Metabolismo de los Lípidos , Masculino , NAD/metabolismo , Proteínas de Neoplasias/genéticaRESUMEN
Folate metabolism is central to cell proliferation and a target of commonly used cancer chemotherapeutics. In particular, the mitochondrial folate-coupled metabolism is thought to be important for proliferating cancer cells. The enzyme MTHFD2 in this pathway is highly expressed in human tumors and broadly required for survival of cancer cells. Although the enzymatic activity of the MTHFD2 protein is well understood, little is known about its larger role in cancer cell biology. We here report that MTHFD2 is co-expressed with two distinct gene sets, representing amino acid metabolism and cell proliferation, respectively. Consistent with a role for MTHFD2 in cell proliferation, MTHFD2 expression was repressed in cells rendered quiescent by deprivation of growth signals (serum) and rapidly re-induced by serum stimulation. Overexpression of MTHFD2 alone was sufficient to promote cell proliferation independent of its dehydrogenase activity, even during growth restriction. In addition to its known mitochondrial localization, we found MTHFD2 to have a nuclear localization and co-localize with DNA replication sites. These findings suggest a previously unknown role for MTHFD2 in cancer cell proliferation, adding to its known function in mitochondrial folate metabolism.
Asunto(s)
N-Acetiltransferasa de Aminoácidos/metabolismo , Núcleo Celular/enzimología , Ácido Fólico/metabolismo , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Neoplasias Experimentales/enzimología , Neoplasias Experimentales/patología , Animales , Línea Celular Tumoral , Proliferación Celular , Células HeLa , Humanos , Ratones , Mitocondrias/metabolismo , Proteínas Nucleares/metabolismo , Ratas , Especificidad de la EspecieRESUMEN
Metabolic remodeling is now widely regarded as a hallmark of cancer, but it is not clear whether individual metabolic strategies are frequently exploited by many tumours. Here we compare messenger RNA profiles of 1,454 metabolic enzymes across 1,981 tumours spanning 19 cancer types to identify enzymes that are consistently differentially expressed. Our meta-analysis recovers established targets of some of the most widely used chemotherapeutics, including dihydrofolate reductase, thymidylate synthase and ribonucleotide reductase, while also spotlighting new enzymes, such as the mitochondrial proline biosynthetic enzyme PYCR1. The highest scoring pathway is mitochondrial one-carbon metabolism and is centred on MTHFD2. MTHFD2 RNA and protein are markedly elevated in many cancers and correlated with poor survival in breast cancer. MTHFD2 is expressed in the developing embryo, but is absent in most healthy adult tissues, even those that are proliferating. Our study highlights the importance of mitochondrial compartmentalization of one-carbon metabolism in cancer and raises important therapeutic hypotheses.
Asunto(s)
Aminohidrolasas/genética , Ácido Fólico/metabolismo , Redes y Vías Metabólicas/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Mitocondrias/metabolismo , Complejos Multienzimáticos/genética , Neoplasias/enzimología , Neoplasias/genética , Aminohidrolasas/metabolismo , Muerte Celular , Línea Celular Transformada , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Complejos Multienzimáticos/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
CLYBL is a human mitochondrial enzyme of unknown function that is found in multiple eukaryotic taxa and conserved to bacteria. The protein is expressed in the mitochondria of all mammalian organs, with highest expression in brown fat and kidney. Approximately 5% of all humans harbor a premature stop polymorphism in CLYBL that has been associated with reduced levels of circulating vitamin B12. Using comparative genomics, we now show that CLYBL is strongly co-expressed with and co-evolved specifically with other components of the mitochondrial B12 pathway. We confirm that the premature stop polymorphism in CLYBL leads to a loss of protein expression. To elucidate the molecular function of CLYBL, we used comparative operon analysis, structural modeling and enzyme kinetics. We report that CLYBL encodes a malate/ß-methylmalate synthase, converting glyoxylate and acetyl-CoA to malate, or glyoxylate and propionyl-CoA to ß-methylmalate. Malate synthases are best known for their established role in the glyoxylate shunt of plants and lower organisms and are traditionally described as not occurring in humans. The broader role of a malate/ß-methylmalate synthase in human physiology and its mechanistic link to vitamin B12 metabolism remain unknown.
Asunto(s)
Enzimas/metabolismo , Malato Sintasa/metabolismo , Oxo-Ácido-Liasas/metabolismo , Acetilcoenzima A/metabolismo , Acilcoenzima A/metabolismo , Glioxilatos/metabolismo , Humanos , Malatos/metabolismo , Especificidad por SustratoRESUMEN
Mitochondrial calcium uptake is present in nearly all vertebrate tissues and is believed to be critical in shaping calcium signaling, regulating ATP synthesis and controlling cell death. Calcium uptake occurs through a channel called the uniporter that resides in the inner mitochondrial membrane. Recently, we used comparative genomics to identify MICU1 and MCU as the key regulatory and putative pore-forming subunits of this channel, respectively. Using bioinformatics, we now report that the human genome encodes two additional paralogs of MICU1, which we call MICU2 and MICU3, each of which likely arose by gene duplication and exhibits distinct patterns of organ expression. We demonstrate that MICU1 and MICU2 are expressed in HeLa and HEK293T cells, and provide multiple lines of biochemical evidence that MCU, MICU1 and MICU2 reside within a complex and cross-stabilize each other's protein expression in a cell-type dependent manner. Using in vivo RNAi technology to silence MICU1, MICU2 or both proteins in mouse liver, we observe an additive impairment in calcium handling without adversely impacting mitochondrial respiration or membrane potential. The results identify MICU2 as a new component of the uniporter complex that may contribute to the tissue-specific regulation of this channel.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Mitocondrias/metabolismo , Complejos Multiproteicos/metabolismo , Secuencia de Aminoácidos , Animales , Canales de Calcio/química , Canales de Calcio/genética , Señalización del Calcio , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Respiración de la Célula/genética , Células HEK293 , Células HeLa , Humanos , Hígado/metabolismo , Potencial de la Membrana Mitocondrial/genética , Ratones , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Familia de Multigenes , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , Interferencia de ARN , Alineación de SecuenciaRESUMEN
Pantothenate kinase-associated neurodegeneration (PKAN) is a rare, inborn error of metabolism characterized by iron accumulation in the basal ganglia and by the presence of dystonia, dysarthria, and retinal degeneration. Mutations in pantothenate kinase 2 (PANK2), the rate-limiting enzyme in mitochondrial coenzyme A biosynthesis, represent the most common genetic cause of this disorder. How mutations in this core metabolic enzyme give rise to such a broad clinical spectrum of pathology remains a mystery. To systematically explore its pathogenesis, we performed global metabolic profiling on plasma from a cohort of 14 genetically defined patients and 18 controls. Notably, lactate is elevated in PKAN patients, suggesting dysfunctional mitochondrial metabolism. As predicted, but never previously reported, pantothenate levels are higher in patients with premature stop mutations in PANK2. Global metabolic profiling and follow-up studies in patient-derived fibroblasts also reveal defects in bile acid conjugation and lipid metabolism, pathways that require coenzyme A. These findings raise a novel therapeutic hypothesis, namely, that dietary fats and bile acid supplements may hold potential as disease-modifying interventions. Our study illustrates the value of metabolic profiling as a tool for systematically exploring the biochemical basis of inherited metabolic diseases.
Asunto(s)
Coenzima A/deficiencia , Mitocondrias/enzimología , Distrofias Neuroaxonales/metabolismo , Neurodegeneración Asociada a Pantotenato Quinasa/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Adolescente , Adulto , Ácidos y Sales Biliares/metabolismo , Niño , Preescolar , Codón sin Sentido , Coenzima A/biosíntesis , Coenzima A/genética , Estudios de Cohortes , Femenino , Humanos , Trastornos del Metabolismo del Hierro , Ácido Láctico/sangre , Metabolismo de los Lípidos/genética , Trastornos del Metabolismo de los Lípidos/genética , Trastornos del Metabolismo de los Lípidos/metabolismo , Masculino , Metaboloma , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Distrofias Neuroaxonales/diagnóstico , Distrofias Neuroaxonales/enzimología , Neurodegeneración Asociada a Pantotenato Quinasa/enzimología , Neurodegeneración Asociada a Pantotenato Quinasa/genética , Ácido Pantoténico/sangre , Esfingomielinas/sangre , Adulto JovenRESUMEN
Mitochondria from diverse organisms are capable of transporting large amounts of Ca(2+) via a ruthenium-red-sensitive, membrane-potential-dependent mechanism called the uniporter. Although the uniporter's biophysical properties have been studied extensively, its molecular composition remains elusive. We recently used comparative proteomics to identify MICU1 (also known as CBARA1), an EF-hand-containing protein that serves as a putative regulator of the uniporter. Here, we use whole-genome phylogenetic profiling, genome-wide RNA co-expression analysis and organelle-wide protein coexpression analysis to predict proteins functionally related to MICU1. All three methods converge on a novel predicted transmembrane protein, CCDC109A, that we now call 'mitochondrial calcium uniporter' (MCU). MCU forms oligomers in the mitochondrial inner membrane, physically interacts with MICU1, and resides within a large molecular weight complex. Silencing MCU in cultured cells or in vivo in mouse liver severely abrogates mitochondrial Ca(2+) uptake, whereas mitochondrial respiration and membrane potential remain fully intact. MCU has two predicted transmembrane helices, which are separated by a highly conserved linker facing the intermembrane space. Acidic residues in this linker are required for its full activity. However, an S259A point mutation retains function but confers resistance to Ru360, the most potent inhibitor of the uniporter. Our genomic, physiological, biochemical and pharmacological data firmly establish MCU as an essential component of the mitochondrial Ca(2+) uniporter.