S2-03-04: Structure and function of gamma-secretase

Abstract

Gamma–Secretase belongs to an atypical class of aspartic protease involved in intramembrane proteolysis of a set of type I single span membrane proteins including APP and Notch. Presenilins (PS) comprise the catalytic center of gamma–secretase, and missense mutations of PS genes cause familial Alzheimer's disease by altering the preferred gamma–secretase cleavage sites and causing increased production of Abeta42, the latter being more prone to form amyloid fibrils. Gamma–Secretase is composed of four membrane proteins, i.e. PS, nicastrin, Aph–1 and Pen–2. We have shown that nicastrin/Aph–1 subcomplex binds to PS and forms a stable tripartite complex, to which Pen–2 is bound and renders the tetrapartite complex into an active form. We further located the binding site of Pen–2 at the 4th transmembrane domain of PS1. We have recently applied cysteine scanning to the structural analysis of PS1 and obtained data supporting the 9th membrane spanning model of PS1. We further expressed human gamma–secretase in baculoviral membranes and applied the purified enzymes to electron microscopic analysis. Structural information derived by molecular biological and morphological analyses will be critical to the understanding of the function of gamma–secretase. We further set out to the screening of genes and small molecules that modulate gamma–secretase activities. RNAi screening of Drosophila genes using a gamma–cleavage–dependent reporter analysis in S2 cells identified several genes that modify gamma–secretase activities. Gamma–Secretase inhibitors provide clues to the understanding of the mechanism of intramembrane cleavage. We identified PS1 CTF as the binding site of a dipeptidic–type gamma–secretase inhibitor, DAPT, using a photocrosslinking strategy. We further identified a couple of novel gamma–secretase inhibitors by a compound library screening using an in vitro gamma–secretase assay. Unifying molecular cell biological and chemical biological approaches will facilitate the understanding of the mechanism of intramembrane cleavage as well as the development of inhibitors/modulators that selectively inhibit Abeta production and are applicable to the disease–modifying therapy of Alzheimer's disease.