RESUMEN
Emerging gene therapy approaches that aim to eliminate pathogenic mutations of mitochondrial DNA (mtDNA) rely on efficient degradation of linearized mtDNA, but the enzymatic machinery performing this task is presently unknown. Here, we show that, in cellular models of restriction endonuclease-induced mtDNA double-strand breaks, linear mtDNA is eliminated within hours by exonucleolytic activities. Inactivation of the mitochondrial 5'-3'exonuclease MGME1, elimination of the 3'-5'exonuclease activity of the mitochondrial DNA polymerase POLG by introducing the p.D274A mutation, or knockdown of the mitochondrial DNA helicase TWNK leads to severe impediment of mtDNA degradation. We do not observe similar effects when inactivating other known mitochondrial nucleases (EXOG, APEX2, ENDOG, FEN1, DNA2, MRE11, or RBBP8). Our data suggest that rapid degradation of linearized mtDNA is performed by the same machinery that is responsible for mtDNA replication, thus proposing novel roles for the participating enzymes POLG, TWNK, and MGME1.
Asunto(s)
División del ADN , Replicación del ADN , ADN Mitocondrial/genética , Edición Génica/métodos , Mitocondrias/genética , Secuencia de Bases , Sistemas CRISPR-Cas , Roturas del ADN de Doble Cadena , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Polimerasa gamma/genética , ADN Polimerasa gamma/metabolismo , ADN Mitocondrial/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Terapia Genética , Células HEK293 , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Mitochondrial protein synthesis requires charging mt-tRNAs with their cognate amino acids by mitochondrial aminoacyl-tRNA synthetases, with the exception of glutaminyl mt-tRNA (mt-tRNAGln). mt-tRNAGln is indirectly charged by a transamidation reaction involving the GatCAB aminoacyl-tRNA amidotransferase complex. Defects involving the mitochondrial protein synthesis machinery cause a broad spectrum of disorders, with often fatal outcome. Here, we describe nine patients from five families with genetic defects in a GatCAB complex subunit, including QRSL1, GATB, and GATC, each showing a lethal metabolic cardiomyopathy syndrome. Functional studies reveal combined respiratory chain enzyme deficiencies and mitochondrial dysfunction. Aminoacylation of mt-tRNAGln and mitochondrial protein translation are deficient in patients' fibroblasts cultured in the absence of glutamine but restore in high glutamine. Lentiviral rescue experiments and modeling in S. cerevisiae homologs confirm pathogenicity. Our study completes a decade of investigations on mitochondrial aminoacylation disorders, starting with DARS2 and ending with the GatCAB complex.
Asunto(s)
Cardiomiopatías/enzimología , Cardiomiopatías/genética , Enfermedades Mitocondriales/enzimología , Enfermedades Mitocondriales/genética , Mutación/genética , Transferasas de Grupos Nitrogenados/genética , Subunidades de Proteína/genética , Secuencia de Aminoácidos , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Lactante , Recién Nacido , Lentivirus/metabolismo , Masculino , Modelos Moleculares , Miocardio/patología , Miocardio/ultraestructura , Transferasas de Grupos Nitrogenados/química , Transferasas de Grupos Nitrogenados/metabolismo , Fosforilación Oxidativa , Linaje , Biosíntesis de Proteínas , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Mammalian mitochondria can be transferred between cells both in culture and in vivo. There is evidence that isolated mitochondria enter cells by endocytosis, but the mechanism has not been fully characterised. We investigated the entry mechanism of isolated mitochondria into human osteosarcoma (HOS) cells. Initially we confirmed that respiratory-competent cells can be produced following incubation of HOS cells lacking mitochondrial DNA (mtDNA) with functional exogenous mitochondria and selection in a restrictive medium. Treatment of HOS cells with inhibitors of different endocytic pathways suggest that uptake of EGFP-labelled mitochondria occurs via an actin-dependent endocytic pathway which is consistent with macropinocytosis. We later utilised time-lapse microscopy to show that internalised mitochondria were found in large, motile cellular vesicles. Finally, we used confocal imaging to show that EGFP-labelled mitochondria colocalise with a macropinocytic cargo molecule during internalisation, HOS cells produce membrane ruffles interacting with external mitochondria during uptake and EGFP-labelled mitochondria are found within early macropinosomes inside cells. In conclusion our results are consistent with isolated mitochondria being internalised by macropinocytosis in HOS cells.