RESUMO
The gamma-secretase complex processes substrate proteins within membranes and consists of four proteins: presenilin (PS), nicastrin, Aph-1 and Pen-2. PS harbours the enzymatic activity of the complex, and there are two mammalian PS homologues: PS1 and PS2. PS undergoes endoproteolysis, generating the N- and C-terminal fragments, NTF and CTF, which represent the active species of PS. To characterize the functional similarity between complexes of various PS composition, we analysed PS1, PS2, and chimeric PS composed of the NTF from PS1 and CTF from PS2, or vice versa, in assembly and function of the gamma-secretase complex. Chimeric PSs, like PS1 and PS2, undergo normal endoproteolysis when introduced into cells devoid of endogenous PS. Furthermore, PS2 CTF can, at least partially, restore processing in a truncated PS1, which cannot undergo endoproteolysis. All PS forms enable maturation of nicastrin and cleave full length Notch receptors, indicating that both PS1 and PS2 are present at the cell surface. Finally, when co-introduced as separate molecules, NTF and CTF of different PS origin reconstitute gamma-secretase activity. In conclusion, these data show that endoproteolysis, NTF-CTF interactions, and the assembly and activity of gamma-secretase complexes are very conserved between PS1 and PS2.
Assuntos
Endopeptidases/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Linhagem Celular , Endopeptidases/química , Endopeptidases/metabolismo , Ativação Enzimática/fisiologia , Regulação Enzimológica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Presenilina-1 , Presenilina-2 , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The activity of the gamma-secretase complex is critical for the processing of a number of transmembrane proteins, including Notch. Functional gamma-secretase activity can be reconstituted from four proteins--presenilin, nicastrin, Pen-2 and Aph-1--but the role of the individual proteins remains unclear. In this report we describe the cellular localization and protein interactions of Aph-1, with particular regard to Notch receptor processing. We found that Aph-1 is present at the cell surface, where it interacts with Pen-2, the mature forms of presenilin and nicastrin, and full-length Notch. Aph-1 also interacts with a truncated form of Notch, which is a direct substrate for gamma-secretase, but not with the Notch intracellular domain. Immunoprecipitation data for Notch and Aph-1 showed that the Notch-containing gamma-secretase complexes most likely form a small subset of the total number of gamma-secretase complexes. In conclusion, these data demonstrate that Aph-1 is present at the cell surface, presumably in active gamma-secretase complexes, and interacts with the Notch receptor, both before and after ligand activation.