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Purification and Characterization of the Human γ-Secretase Complex

2004; American Chemical Society; Volume: 43; Issue: 30 Linguagem: Inglês

10.1021/bi0494976

1943-295X

Patrick C. Fraering, Wenjuan Ye, Jean‐Marc Strub, Georgia Dolios, Matthew J. LaVoie, Beth L. Ostaszewski, Alain Van Dorsselaer, Rong Wang, Dennis J. Selkoe, Michael S. Wolfe,

Glycosylation and Glycoproteins Research

γ-Secretase is a member of an unusual class of proteases with intramembrane catalytic sites. This enzyme cleaves many type I membrane proteins, including the amyloid β-protein (Aβ) precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of γ-secretase: heterodimeric presenilin (PS) composed of its N- and C-terminal fragments (PS-NTF/CTF), a mature glycosylated form of nicastrin (NCT), Aph-1, and Pen-2. Recent data from studies in Drosophila, mammalian, and yeast cells suggest that PS, NCT, Aph-1, and Pen-2 are necessary and sufficient to reconstitute γ-secretase activity. However, many unresolved issues, in particular the possibility of other structural or regulatory components, would be resolved by actually purifying the enzyme. Here, we report a detailed, multistep purification procedure for active γ-secretase and an initial characterization of the purified protease. Extensive mass spectrometry of the purified proteins strongly suggests that PS-NTF/CTF, mNCT, Aph-1, and Pen-2 are the components of active γ-secretase. Using the purified γ-secretase, we describe factors that modulate the production of specific Aβ species: (1) phosphatidylcholine and sphingomyelin dramatically improve activity without changing cleavage specificity within an APP substrate; (2) increasing CHAPSO concentrations from 0.1 to 0.25% yields a ∼100% increase in Aβ42 production; (3) exposure of an APP-based recombinant substrate to 0.5% SDS modulates cleavage specificity from a disease-mimicking pattern (high Aβ42/43) to a physiological pattern (high Aβ40); and (4) sulindac sulfide directly and preferentially decreases Aβ42 cleavage within the purified complex. Taken together, our results define a procedure for purifying active γ-secretase and suggest that the lipid-mediated conformation of both enzyme and substrate regulate the production of the potentially neurotoxic Aβ42 and Aβ43 peptides.