RESUMO
Presenilin 1 or presenilin 2, nicastrin, APH-1, and PEN-2 form high molecular weight complexes that play a pivotal role in the cleavage of various Type I transmembrane proteins, including the beta-amyloid precursor protein. The specific function of PEN-2 is unclear. To explore its function and intermolecular interactions, we conducted deletion and mutagenesis studies on a series of conserved residues at the C terminus of PEN-2. These studies suggest that: 1) both the presence and amino acid sequence of the conserved DYLSF domain at the C terminus of PEN-2 (residues 90-94) is critical for binding PEN-2 to other components in the presenilin complex and 2) the overall length of the exposed C terminus is critical for functional gamma-secretase activity.
Assuntos
Proteínas de Membrana/química , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/química , Animais , Encéfalo/metabolismo , Linhagem Celular , Centrifugação com Gradiente de Concentração , Cicloeximida/farmacologia , DNA Complementar/metabolismo , Deleção de Genes , Biblioteca Gênica , Humanos , Imunoprecipitação , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNARESUMO
Several lines of evidence have indicated that the presenilin proteins function within macromolecular complexes and are necessary for the regulated intramembranous proteolysis of certain type 1 transmembrane proteins, including the amyloid precursor protein, Notch, and p75. Data from multiple complementary experiments now suggest that there may be several distinct presenilin complexes. We show here that presenilin mutations and certain detergents affect the abundance and componentry of the presenilin complexes, and these structural effects correlate with their effects on gamma-secretase activity. Our data suggest that there are at least three complexes, including a approximately 150-kDa nicastrin-aph-1 complex (which is likely to be a precursor complex). There is a stable and abundant intermediate complex of approximately 440 kDa, which contains aph-1, pen-2, nicastrin, and PS1. However, it is the very low abundance, high mass (>/=670 kDa) heteromeric complexes that are associated with the highest gamma-secretase-specific activity.