title: Rapid purification of active gamma-secretase, an intramembrane protease implicated in Alzheimer's disease.
abstract: Gamma-secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid-beta precursor protein generates the amyloid-beta protein (Abeta) of Alzheimer's disease, partially inhibiting gamma-secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of gamma-secretase, but at low resolution (15 A). The limited amount of purified gamma-secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X-ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S-20) that overexpresses strikingly high levels of all four gamma-secretase components (presenilin, nicastrin, Aph-1 and Pen-2). We then used these cells to develop a rapid protocol for the high-grade purification of proteolytically active gamma-secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme.
authors.affiliation: Brain Mind Institute and School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Cacquevel
authors.forename: Lorene
authors.initials: L
authors.lastname: Aeschbach
authors.forename: Pamela
authors.initials: P
authors.lastname: Osenkowski
authors.forename: Dongyang
authors.initials: D
authors.lastname: Li
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Ye
authors.forename: Michael S
authors.initials: MS
authors.lastname: Wolfe
authors.forename: Huilin
authors.initials: H
authors.lastname: Li
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Selkoe
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Fraering
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology* / Q000648:ultrastructure; D016229:Amyloid beta-Peptides / Q000378:metabolism; D000818:Animals; D002461:Cell Line, Transformed; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D046529:Microscopy, Electron, Transmission / Q000379:methods
chemical_list: D016229:Amyloid beta-Peptides; D053829:Amyloid Precursor Protein Secretases