Characterization of the reconstituted gamma-secretase complex from Sf9 cells co-expressing presenilin 1, nicastrin [correction of nacastrin], aph-1a, and pen-2

γ-Secretase catalyzes the proteolytic processing of a number of integral membrane proteins, including amyloid precursor protein (APP) and Notch. The native γ-secretase is a heterogeneous population of large membrane protein complexes containing presenilin 1 (PS1) or presenilin 2 (PS2), aph-1a or aph-1b, nicastrin, and pen-2. Here we report the reconstitution of a γ-secretase complex in Sf9 cells by co-infection with baculoviruses carrying the PS1, nicastrin, pen-2, and aph-la genes. The reconstituted enzyme processes C99 and the Notch-like substrate N160 and displays the characteristic features of γ-secretase in terms of sensitivity to a γ-secretase inhibitor, upregulation of Aβ42 production by a familial Alzheimer's disease (FAD) mutation in the APP gene, and downregulation of Notch processing by PS1 FAD mutations. However, the ratio of Aβ42:Aβ40 production by the reconstituted γ-secretase is significantly higher than that of the native enzyme from 293 cells. Unlike in mammalian cells where PS 1 FAD mutations cause an increase in Aβ42 production, PS1 FAD missense mutations in the reconstitution system alter the cleavage sites in the C99 substrate without changing the Aβ42:Aβ40 ratio. In addition, PS1ΔE9 is a loss-of-function mutation in both C99 and N160 processing. Reconstitution of γ-secretase provides a homogeneous system for studying the individual γ-secretase complexes and their roles in Aβ production, Notch processing and AD pathogenesis. These studies may provide important insight into the development of a new generation of selective γ-secretase inhibitors with an improved side effect profile.