γ-Secretase, Evidence for Multiple Proteolytic Activities and Influence of Membrane Positioning of Substrate on Generation of Amyloid β Peptides of Varying Length*

γ-Secretase activity is the final cleavage event that releases the amyloid β peptide (Aβ) from the β-secretase cleaved carboxyl-terminal fragment of the amyloid β protein precursor (APP). No protease responsible for this highly unusual, purportedly intramembranous, cleavage has been definitively identified. We examined the substrate specificity of γ-secretase by mutating various residues within or adjacent to the transmembrane domain of the APP and then analyzing Aβ production from cells transfected with these mutant APPs by enzyme-linked immunosorbent assay and mass spectrometry. Aβ production was also analyzed from a subset of transmembrane domain APP mutants that showed dramatic shifts in γ-secretase cleavage in the presence or absence of pepstatin, an inhibitor of γ-secretase activity. These studies demonstrate that γ-secretase's cleavage specificity is primarily determined by location of the γ-secretase cleavage site of APP with respect to the membrane, and that γ-secretase activity is due to the action of multiple proteases exhibiting both a pepstatin- sensitive activity and a pepstatin-insensitive activity. Given that γ-secretase is a major therapeutic target in Alzheimer's disease these studies provide important information with respect to the mechanism of Aβ production that will direct efforts to isolate the γ-secretases and potentially to develop effective therapeutic inhibitors of pathologically relevant γ-secretase activities.