RESUMO
Loss-of-function mutations in the parkin gene (PARK2) and PINK1 gene (PARK6) are associated with autosomal recessive parkinsonism. PINK1 deficiency was recently linked to mitochondrial pathology in human cells and Drosophila melanogaster, which can be rescued by parkin, suggesting that both genes play a role in maintaining mitochondrial integrity. Here we demonstrate that an acute down-regulation of parkin in human SH-SY5Y cells severely affects mitochondrial morphology and function, a phenotype comparable with that induced by PINK1 deficiency. Alterations in both mitochondrial morphology and ATP production caused by either parkin or PINK1 loss of function could be rescued by the mitochondrial fusion proteins Mfn2 and OPA1 or by a dominant negative mutant of the fission protein Drp1. Both parkin and PINK1 were able to suppress mitochondrial fragmentation induced by Drp1. Moreover, in Drp1-deficient cells the parkin/PINK1 knockdown phenotype did not occur, indicating that mitochondrial alterations observed in parkin- or PINK1-deficient cells are associated with an increase in mitochondrial fission. Notably, mitochondrial fragmentation is an early phenomenon upon PINK1/parkin silencing that also occurs in primary mouse neurons and Drosophila S2 cells. We propose that the discrepant findings in adult flies can be explained by the time of phenotype analysis and suggest that in mammals different strategies may have evolved to cope with dysfunctional mitochondria.
Assuntos
Proteínas de Drosophila/fisiologia , Mitocôndrias/metabolismo , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Linhagem Celular , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitina-Proteína Ligases/genéticaRESUMO
Mutations in the parkin gene are a major cause of autosomal recessive Parkinson's disease. Here we show that the E3 ubiquitin ligase parkin activates signaling through the IkappaB kinase (IKK)/nuclear factor kappaB (NF-kappaB) pathway. Our analysis revealed that activation of this signaling cascade is causally linked to the neuroprotective potential of parkin. Inhibition of NF-kappaB activation by an IkappaB super-repressor or a kinase-inactive IKKbeta interferes with the neuroprotective activity of parkin. Furthermore, pathogenic parkin mutants with an impaired neuroprotective capacity show a reduced ability to stimulate NF-kappaB-dependent transcription. Finally, we present evidence that parkin interacts with and promotes degradation-independent ubiquitylation of IKKgamma/NEMO (NF-kappaB essential modifier) and TRAF2 [TNF (tumor necrosis factor) receptor-associated factor 2], two critical components of the NF-kappaB pathway. Thus, our results support a direct link between the neuroprotective activity of parkin and ubiquitin signaling in the IKK/NF-kappaB pathway.
Assuntos
Citoproteção/fisiologia , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Ativação Enzimática/fisiologia , Humanos , Mutação , Ratos , Estresse Fisiológico/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transfecção , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/farmacologiaRESUMO
The genetic hallmark of Ewing's sarcoma family of tumours (ET) is the presence of the translocation t(11;22)(q24;q12), which creates the ET fusion gene, leading to cellular transformation. Five human gamma-glutamyl transpeptidase (gamma-GT) genes are located near the chromosomal translocation in ET. gamma-GT is a major enzyme involved in glutathione homoeostasis. Five human cell lines representative of primary or metastatic tumours were investigated to study whether gamma-GT alterations could occur at the chromosomal breaks and rearrangements in ET. As shown by enzymic assays and FACS analyses, all ET cell lines consistently expressed a functional gamma-GT which however did not discriminate steps of ET progression. As shown previously [Sancéau, Hiscott, Delattre and Wietzerbin (2000) Oncogene 19, 3372-3383], ET cells respond to the antiproliferative effects of interferons (IFNs) type I (alpha and beta) and to a much less degree to IFN type II (gamma). IFN-alpha and -beta arrested cells in the S-phase of the cell cycle. We found an enhancement of gamma-GT mRNA species with IFN-alpha and -beta by reverse transcriptase-PCR analyses. This is reflected by up-regulation of gamma-GT protein, which coincides with the increase in gamma-GT-specific enzymic activity. Similarly, IFNs up-regulate the levels of gamma-GT in another IFN-responsive B cell line. Whether this up-regulation of gamma-GT by IFNs is of physiological relevance to cell behaviour remains to be studied.