RESUMO
Optic Atrophy 1 (OPA1) gene mutations cause diseases ranging from isolated dominant optic atrophy (DOA) to various multisystemic disorders. OPA1, a large GTPase belonging to the dynamin family, is involved in mitochondrial network dynamics. The majority of OPA1 mutations encodes truncated forms of the protein and causes DOA through haploinsufficiency, whereas missense OPA1 mutations are predicted to cause disease through deleterious dominant-negative mechanisms. We used 3D imaging and biochemical analysis to explore autophagy and mitophagy in fibroblasts from seven patients harbouring OPA1 mutations. We report new genotype-phenotype correlations between various types of OPA1 mutation and mitophagy. Fibroblasts bearing dominant-negative OPA1 mutations showed increased autophagy and mitophagy in response to uncoupled oxidative phosphorylation. In contrast, OPA1 haploinsufficiency was correlated with a substantial reduction in mitochondrial turnover and autophagy, unless subjected to experimental mitochondrial injury. Our results indicate distinct alterations of mitochondrial physiology and turnover in cells with OPA1 mutations, suggesting that the level and profile of OPA1 may regulate the rate of mitophagy.
Assuntos
Autofagia/genética , GTP Fosfo-Hidrolases/genética , Mutação , Atrofia Óptica Autossômica Dominante/genética , Adolescente , Adulto , Células Cultivadas , Pré-Escolar , Feminino , Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Mitofagia/genéticaRESUMO
The 22 members of the NUDIX (NUcleoside DIphosphate linked to another moiety, X) hydrolase superfamily can hydrolyze a variety of phosphorylated molecules including (d)NTPs and their oxidized forms, nucleotide sugars, capped mRNAs and dinucleotide coenzymes such as NADH and FADH. Beside this broad range of enzymatic substrates, the NUDIX proteins can also be found in different cellular compartments, mainly in the nucleus and in the cytosol, but also in the peroxisome and in the mitochondria. Here we studied two members of the family, NUDT6 and NUDT9. We showed that NUDT6 is expressed in human cells and localizes exclusively to mitochondria and we confirmed that NUDT9 has a mitochondrial localization. To elucidate their potential role within this organelle, we investigated the functional consequences at the mitochondrial level of NUDT6- and NUDT9-deficiency and found that the depletion of either of the two proteins results in an increased activity of the respiratory chain and an alteration of the mitochondrial respiratory chain complexes expression. We demonstrated that NUDT6 and NUDT9 have distinct substrate specificity in vitro, which is dependent on the cofactor used. They can both hydrolyze a large range of low molecular weight compounds such as NAD+(H), FAD and ADPR, but NUDT6 is mainly active towards NADH, while NUDT9 displays a higher activity towards ADPR.
Assuntos
NAD , Pirofosfatases , Humanos , Hidrólise , Mitocôndrias/metabolismo , Pirofosfatases/genética , Pirofosfatases/química , Pirofosfatases/metabolismoRESUMO
Mitochondrial transcription factor A (TFAM) is essential for the maintenance, expression, and packaging of mitochondrial DNA (mtDNA). Recently, a pathogenic homozygous variant in TFAM (P178L) has been associated with a severe mtDNA depletion syndrome leading to neonatal liver failure and early death. We have performed a biochemical characterization of the TFAM variant P178L in order to understand the molecular basis for the pathogenicity of this mutation. We observe no effects on DNA binding, and compaction of DNA is only mildly affected by the P178L amino acid change. Instead, the mutation severely impairs mtDNA transcription initiation at the mitochondrial heavy and light strand promoters. Molecular modeling suggests that the P178L mutation affects promoter sequence recognition and the interaction between TFAM and the tether helix of POLRMT, thus explaining transcription initiation deficiency.
Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Mitocondriais , Mutação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Mitochondrial DNA (mtDNA) can undergo double-strand breaks (DSBs), caused by defective replication, or by various endogenous or exogenous sources, such as reactive oxygen species, chemotherapeutic agents or ionizing radiations. MtDNA encodes for proteins involved in ATP production, and maintenance of genome integrity following DSBs is thus of crucial importance. However, the mechanisms involved in mtDNA maintenance after DSBs remain unknown. In this study, we investigated the consequences of the production of mtDNA DSBs using a human inducible cell system expressing the restriction enzyme PstI targeted to mitochondria. Using this system, we could not find any support for DSB repair of mtDNA. Instead we observed a loss of the damaged mtDNA molecules and a severe decrease in mtDNA content. We demonstrate that none of the known mitochondrial nucleases are involved in the mtDNA degradation and that the DNA loss is not due to autophagy, mitophagy or apoptosis. Our study suggests that a still uncharacterized pathway for the targeted degradation of damaged mtDNA in a mitophagy/autophagy-independent manner is present in mitochondria, and might provide the main mechanism used by the cells to deal with DSBs.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Mitocondrial , Southern Blotting , Western Blotting , Ciclo-Oxigenase 1/genética , Reparo do DNA , Endonucleases/metabolismo , Exonucleases/metabolismo , Citometria de Fluxo , Células HEK293 , Humanos , Cinética , Mitocôndrias/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência , TransfecçãoRESUMO
BACKGROUND: Poly-ADP ribosylation (PARylation) is a post translational modification, catalyzed by Poly(ADP-ribose)polymerase (PARP) family. In Drosophila, PARP-I (human PARP-1 ortholog) is considered to be the only enzymatically active isoform. PARylation is involved in various cellular processes such as DNA repair in case of base excision and strand-breaks. OBSERVATIONS: Strand-breaks (SSB and DSB) are detrimental to cell viability and, in Drosophila, that has a unique PARP family organization, little is known on PARP involvement in the control of strand-breaks repair process. In our study, strands-breaks (SSB and DSB) are chemically induced in S2 Drosophila cells using bleomycin. These breaks are efficiently repaired in S2 cells. During the bleomycin treatment, changes in PARylation levels are only detectable in a few cells, and an increase in PARP-I and PARP-II mRNAs is only observed during the recovery period. These results differ strongly from those obtained with Human cells, where PARylation is strongly activating when DNA breaks are generated. Finally, in PARP knock-down cells, DNA stability is altered but no change in strand-breaks repair can be observed. CONCLUSIONS: PARP responses in DNA strands-breaks context are functional in Drosophila model as demonstrated by PARP-I and PARP-II mRNA increases. However, no modification of the global PARylation profile is observed during strand-breaks generation, only changes at cellular levels are detectable. Taking together, these results demonstrate that PARylation process in Drosophila is tightly regulated in the context of strands-breaks repair and that PARP is essential during the maintenance of DNA integrity but dispensable in the DNA repair process.
Assuntos
Reparo do DNA , Proteínas de Drosophila/metabolismo , Macrófagos/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Animais , Bleomicina/farmacologia , Linhagem Celular , DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
DNA repair mechanisms are key components for the maintenance of the essential mitochondrial genome. Among them, base excision repair (BER) processes, dedicated in part to oxidative DNA damage, are individually well known in mitochondria. However, no large view of these systems in differential physiological conditions is available yet. Combining the use of pure mitochondrial fractions and a multiplexed oligonucleotide cleavage assay on a microarray, we demonstrated that a large range of glycosylase activities were present in Drosophila mitochondria. Most of them were quantitatively different from their nuclear counterpart. Moreover, these activities were modified during aging.