RESUMEN
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.
Asunto(s)
Ceramidas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Humanos , Células PC12 , ARN Mensajero/genética , Ratas , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
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.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Línea Celular , Cisteína Endopeptidasas/química , ADN Complementario/metabolismo , Humanos , Inmunoprecipitación , Modelos Genéticos , Complejos Multienzimáticos/química , Mutación , Células PC12 , Plásmidos/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Transducción de Señal , Técnicas del Sistema de Dos Híbridos , Ubiquitina/química , Ubiquitina-Proteína Ligasas/químicaRESUMEN
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.
Asunto(s)
Proteínas Portadoras/genética , Péptidos y Proteínas de Señalización Intracelular , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Ciclo Celular , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Proteínas de Unión al ADN , Expresión Génica , Prueba de Complementación Genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Factor de Crecimiento Nervioso , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/metabolismo , Análisis de Secuencia de ADN , Especificidad de la EspecieAsunto(s)
Aorta/anomalías , Cojera Animal , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/fisiología , Empalme Alternativo/genética , Animales , Aorta/embriología , Recuento de Células , Desarrollo Embrionario y Fetal , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Neuronas Aferentes/fisiología , Isoformas de Proteínas , Receptor de Factor de Crecimiento Nervioso , Células de Schwann/química , Células de Schwann/fisiologíaRESUMEN
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.
