Biochemical characterization of the γ-secretase activity that produces β-amyloid peptides

Recent studies of gamma -secretase have pointed out that it may be comprised of a multisubunit complex with presenilin 1 and presenilin 2 as central components. Elucidation of the biochemical mechanism of this enzymatic activity will provide important information for developing gamma -secretase inhibitors in Alzheimer's disease therapy. Here we describe the biochemical characterization of gamma -secretase activities using a sensitive, membrane based assay system, Membranes were isolated from 293 cells expressing C99, the substrate of gamma -secretase, Upon incubation at 37 degreesC, C99 is cleaved by the endogenous gamma -secretase, and AP peptides are liberated. A beta 40 and A beta 42 gamma -secretase activities are very similar in terms of their kinetic profiles and pH dependence, supporting the notion that a single enzyme is involved in both A beta 40 and A beta 42 production. Pepstatin A inhibited A beta 40 and A beta 42 gamma -secretase activities with similar potency, Peptide difluoroketone and peptide aldehyde inhibitors inhibited A beta 40 production in a dose-dependent fashion, enhanced A beta 42 production at low concentrations, and inhibited A beta 42 production at high concentrations. Although the selective increase of A beta 42 by low concentrations of peptide difluoroketone and peptide aldehyde inhibitors has been reported in intact cells, the finding that this phenomenon occurs in a membrane-based assay system suggests that these compounds increase A beta 42 by a direct effect on gamma -secretase. The ability of these compounds to increase A beta 42 production may reflect allosteric modulation of the gamma -secretase complex by a mechanism related to that responsible for the increase of A beta 42 production by mutations in presenilins.