RESUMO
Mutations in the parkin gene cause autosomal-recessive early-onset parkinsonism as a result of the degeneration of mesencephalic dopaminergic neurons. In cell culture models, parkin expression has been shown to protect against cell death mediated by the sphingolipid ceramide. To determine whether the antiapoptotic effect of parkin involves changes in gene expression, we used Affymetrix oligonucleotide microarrays to analyse gene expression in stably transfected PC12 cells which conditionally overexpress parkin, that were treated or not with C2-ceramide. Overexpression of parkin and ceramide treatment both modulated gene expression. A number of the genes upregulated in the presence of ceramide, and modulated by parkin, were associated with apoptosis or cellular stress reactions. We validated the upregulation of four such genes (CHK, EIF4EBP1, GADD45A and PTPN-5) by real-time PCR after 3, 6, 9 and 12 h of ceramide treatment in cells that overexpressed parkin or not. All were upregulated 2 to 11-fold, 3 and 6 h after application of ceramide. Parkin overexpression reduced the upregulation of EIF4EBP1, GADD45A and PTPN-5, but only at 6 h. These results suggest that, in this assay, the cytoprotective effect of parkin might result not only from its E3-ligase activity, but also from direct or indirect modulation of gene expression in a time-dependent manner.
Assuntos
Ceramidas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Animais , Humanos , Células PC12 , RNA Mensageiro/genética , Ratos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Mutations in the parkin gene encoding an E3 ligase are responsible for autosomal recessive Parkinson's disease. Putative parkin substrates and interacting partners have been identified, but the molecular mechanism underlying parkin-related neurodegeneration is still unclear. We have identified the 20S proteasomal subunit alpha4 (synonyms: PSMA7, XAPC7, subunit alpha type 7) as a new interacting partner of parkin. The C-terminal IBR-RING domain of parkin and the C-terminal part of alpha4 were essential for the interaction. Biochemical studies revealed that alpha4 was not a substrate for parkin-dependent ubiquitylation. Putative functions of the interaction might therefore be substrate presentation to the proteasome or regulation of proteasomal activity. Full-length parkin and parkin lacking the N-terminal ubiquitin-like domain slightly increased the proteasomal activity in HEK 293T cells, in line with the latter hypothesis.
Assuntos
Cisteína Endopeptidases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Cisteína Endopeptidases/química , DNA Complementar/metabolismo , Humanos , Imunoprecipitação , Modelos Genéticos , Complexos Multienzimáticos/química , Mutação , Células PC12 , Plasmídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/química , Ubiquitina-Proteína Ligases/químicaRESUMO
The zinc finger protein NRIF (neurotrophin receptor interacting factor) was originally identified by virtue of its interaction with the neurotrophin receptor p75NTR and its participation in embryonic apoptosis. Targeted deletion of the nrif gene in mice is embryonically lethal in the C57BL6 genetic background, where it blocks cell cycle progression, but not in the Sv129 strain. We have now identified a second, highly homologous nrif gene, designated nrif2, encoding a protein with similar structural and biochemical properties as well as subcellular distribution as NRIF1, and whose over-expression in transfected fibroblasts also correlates with impaired BrdU incorporation. Unexpectedly, the nrif2 transcript becomes significantly upregulated in nrif1-/- mice only in Sv129, the genetic background where the mutants are viable, suggesting that the functional complementation of the two nrif genes may be strain-specific.
Assuntos
Proteínas de Transporte/genética , Peptídeos e Proteínas de Sinalização Intracelular , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Ciclo Celular , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Proteínas de Ligação a DNA , Expressão Gênica , Teste de Complementação Genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Análise de Sequência de DNA , Especificidade da EspécieAssuntos
Aorta/anormalidades , Coxeadura Animal , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/fisiologia , Processamento Alternativo/genética , Animais , Aorta/embriologia , Contagem de Células , Desenvolvimento Embrionário e Fetal , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Neurônios Aferentes/fisiologia , Isoformas de Proteínas , Receptor de Fator de Crescimento Neural , Células de Schwann/química , Células de Schwann/fisiologiaRESUMO
NRIF (neurotrophin receptor interacting factor) is a ubiquitously expressed zinc finger protein of the Krüppel family which interacts with the neurotrophin receptor p75(NTR). The interaction was first detected in yeast and then biochemically confirmed using recombinant GST-NRIF fusions and p75(NTR) expressed by eukaryotic cells. Transgenic mice carrying a deletion in the exon encoding the p75(NTR)-binding domain of NRIF display a phenotype which is strongly dependent upon genetic background. While at the F(2 )generation there is only limited (20%) embryonic lethality, in a congenic BL6 strain nrif(-/-) mice cannot survive beyond E12, but are viable and healthy to adulthood in the Sv129 background. The involvement of NRIF in p75(NTR)/NGF-mediated developmental cell death was examined in the mouse embryonic neural retina. Disruption of the nrif gene leads to a reduction in cell death which is quantitatively indistinguishable from that observed in p75(NTR)(-/-) and ngf(-/-) mice. These results indicate that NRIF is an intracellular p75(NTR)-binding protein transducing cell death signals during development.
