Characterization of the reconstituted γ-secretase complex from Sf9 cells co-expressing presenilin 1, nacastrin, aph-1a, and pen-2

gamma-Secretase catalyzes the proteolytic processing of a number of integral membrane proteins, including amyloid precursor protein (APP) and Notch. The native gamma-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 gamma-secretase complex in SO cells by coinfection with baculoviruses carrying the PS 1, nicastrin, pen-2, and aph-1a genes. The reconstituted enzyme processes C99 and the Notch-like substrate N160 and displays the characteristic features of gamma-secretase in terms of sensitivity to a gamma-secretase inhibitor, upregulation of A beta 42 production by a familial Alzheimer's disease (FAD) mutation in the APP gene, and downregulation of Notch processing by PSI FAD mutations. However, the ratio of A beta 42:A beta 40 production by the reconstituted gamma-secretase is significantly higher than that of the native enzyme from 293 cells. Unlike in mammalian cells where PSI FAD mutations cause an increase in A beta 42 production, PS 1 FAD missense mutations in the reconstitution system alter the cleavage sites in the C99 substrate without changing the A beta 42:A beta 40 ratio. In addition, PS1 Delta E9 is a loss-of-function mutation in both C99 and N160 processing. Reconstitution of gamma-secretase provides a homogeneous system for studying the individual gamma-secretase complexes and their roles in A beta production, Notch processing and AD pathogenesis. These studies may provide important insight into the development of a new generation of selective gamma-secretase inhibitors with an improved side effect profile.