Biochemical characterization of the gamma-secretase activity that producesbeta-amyloid peptides

Recent studies of gamma -secretase have pointed out that it may be comprise d of a multisubunit complex with presenilin 1 and presenilin 2 as central c omponents. Elucidation of the biochemical mechanism of this enzymatic activ ity will provide important information for developing gamma -secretase inhi bitors in Alzheimer's disease therapy. Here we describe the biochemical cha racterization of gamma -secretase activities using a sensitive, membrane ba sed assay system, Membranes were isolated from 293 cells expressing C99, th e substrate of gamma -secretase, Upon incubation at 37 degreesC, C99 is cle aved by the endogenous gamma -secretase, and AP peptides are liberated. A b eta 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 s ingle enzyme is involved in both A beta 40 and A beta 42 production. Pepsta tin A inhibited A beta 40 and A beta 42 gamma -secretase activities with si milar potency, Peptide difluoroketone and peptide aldehyde inhibitors inhib ited A beta 40 production in a dose-dependent fashion, enhanced A beta 42 p roduction at low concentrations, and inhibited A beta 42 production at high concentrations. Although the selective increase of A beta 42 by low concen trations of peptide difluoroketone and peptide aldehyde inhibitors has been reported in intact cells, the finding that this phenomenon occurs in a mem brane-based assay system suggests that these compounds increase A beta 42 b y a direct effect on gamma -secretase. The ability of these compounds to in crease A beta 42 production may reflect allosteric modulation of the gamma -secretase complex by a mechanism related to that responsible for the incre ase of A beta 42 production by mutations in presenilins.