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 coinfection with baculoviruses carrying the PS1, nicastrin, pen-2, and aph-1a 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 PS1 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.
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