RESUMEN
Nicastrin is a component of the gamma-secretase complex that has been shown to adhere to presenilin-1 (PS1), Notch, and APP. Here we demonstrate that Nicastrin-deficient mice showed a phenotype that is indistinguishable from PS1/PS2 double knock-out mice, whereas heterozygotes were healthy and viable. Fibroblasts derived from Nicastrin-deficient embryos were unable to generate amyloid beta-peptide and failed to release the intracellular domain of APP- or Notch1-Gal4-VP16 fusion proteins. Additionally, C- and N-terminal fragments of PS1 and the C-terminal fragments of PS2 were not detectable in Nicastrin-null fibroblasts, whereas full-length PS1 accumulated in null fibroblasts, indicating that Nicastrin is required for the endoproteolytic processing of presenilins. Interestingly, cells derived from Nicastrin heterozygotes produced relatively higher levels of amyloid beta-peptide whether the source was endogenous mouse or transfected human APP. These data demonstrate that Nicastrin is essential for the gamma-secretase cleavage of APP and Notch in mammalian cells and that Nicastrin has both positive and negative functions in the regulation of gamma-secretase activity.
Asunto(s)
Endopeptidasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Adenoviridae/genética , Alelos , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Membrana Celular/metabolismo , Medios de Cultivo Condicionados/farmacología , ADN Complementario/metabolismo , Fibroblastos/metabolismo , Genes Reporteros , Genotipo , Proteínas Fluorescentes Verdes , Heterocigoto , Humanos , Luciferasas/metabolismo , Proteínas Luminiscentes/metabolismo , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/fisiología , Ratones , Ratones Noqueados , Péptidos/química , Fenotipo , Presenilina-1 , Presenilina-2 , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Factores de TiempoRESUMEN
Mutations in the human presenilin genes (PS1 or PS2) have been linked to autosomal dominant, early onset Alzheimer's disease (AD). Presenilins, probably as an essential part of gamma-secretase, modulate gamma-cleavage of the amyloid protein precursor (APP) to the amyloid beta-peptide (Abeta). Mutations in sel-12, a Caenorhabditis elegans presenilin homologue, cause a defect in egg laying that can be suppressed by loss of function mutations in a second gene, SEL-10. SEL-10 protein is a homologue of yeast Cdc4, a member of the SCF (Skp1-Cdc53/CUL1-F-box protein) E2-E3 ubiquitin ligase family. In this study, we show that human SEL-10 interacts with PS1 and enhances PS1 ubiquitination, thus altering cellular levels of unprocessed PS1 and its N- and C-terminal fragments. Co-transfection of sel-10 and APP cDNAs in HEK293 cells leads to an alteration in the metabolism of APP and to an increase in the production of amyloid beta-peptide, the principal component of amyloid plaque in Alzheimer's disease.
