Purification, Pharmacological Modulation, and Biochemical Characterization of Interactors of Endogenous Human γ-Secretase†

γ-Secretase is a unique intramembrane-cleaving protease complex, which cleaves the Alzheimer′s disease-associated β-amyloid precursor protein (APP) and a number of other type I membrane proteins. Human γ-secretase consists of the catalytic subunit presenilin (PS) (PS1 or PS2), the substrate receptor nicastrin, APH-1 (APH-1a or APH-1b), and PEN-2. To facilitate in-depth biochemical analysis of γ-secretase, we developed a fast and convenient multistep purification procedure for the endogenous enzyme. The enzyme was purified from HEK293 cells in an active form and had a molecular mass of ∼500 kDa. Purified γ-secretase was capable of producing the major amyloid-β peptide (Aβ) species, such as Aβ40 and Aβ42, from a recombinant APP substrate in physiological ratios. Aβ generation could be modulated by pharmacological γ-secretase modulators. Moreover, the Aβ42/Aβ40 ratio was strongly increased by purified PS1 L166P, an aggressive familial Alzheimer’s disease mutant. Tandem mass spectrometry analysis revealed the...