RESUMO
Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Caenorhabditis elegans/isolamento & purificação , Membrana Celular/metabolismo , Endopeptidases/metabolismo , Proteínas de Homeodomínio/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Clonagem Molecular , Proteínas de Drosophila , Drosophila melanogaster , Elementos Facilitadores Genéticos/genética , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon , Proteínas de Helminto/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação/genética , Fragmentos de Peptídeos/metabolismo , Presenilina-1 , Precursores de Proteínas/metabolismo , Receptores Notch , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais/genéticaRESUMO
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.