title: Cell-free assays for gamma-secretase activity.
abstract: The amyloid b-protein (Ab) deposited in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid b-protein precursor (APP). Generation of Ab from the APP requires two sequential proteolytic events: an initial b-secretase cleavage at the amino terminus of the Ab sequence followed by g-secretase cleavage at the carboxyl terminus of Ab. We describe the development of a robust in vitro assay for g-secretase cleavage by showing de novo Ab production in vitro and establish that this assay monitors authentic gamma-secretase activity by documenting the production of a cognate g-CTF, confirming the size of the Ab produced by mass spectrometry, and inhibiting cleavage in this system with multiple inhibitors that alter g-secretase activity in living cells. Using this assay, we demonstrate that the g-secretase activity 1) is tightly associated with the membrane, 2) can be solubilized, 3) has a pH optimum of 6.8 but is active from pH 6.0 to pH >8.4, and 4) ascertain that activities of the g-40 and g-42 are indeed pharmacologically distinct. These studies should facilitate the purification of the protease or proteases that are responsible for this unusual activity, which is a major therapeutic target for the treatment of AD.
authors.affiliation: Department of Pharmacology, Mayo Clinic, Jacksonville, Florida 32224, USA.
authors.forename: C
authors.initials: C
authors.lastname: McLendon
authors.forename: T
authors.initials: T
authors.lastname: Xin
authors.forename: C
authors.initials: C
authors.lastname: Ziani-Cherif
authors.forename: M P
authors.initials: MP
authors.lastname: Murphy
authors.forename: K A
authors.initials: KA
authors.lastname: Findlay
authors.forename: P A
authors.initials: PA
authors.lastname: Lewis
authors.forename: I
authors.initials: I
authors.lastname: Pinnix
authors.forename: K
authors.initials: K
authors.lastname: Sambamurti
authors.forename: R
authors.initials: R
authors.lastname: Wang
authors.forename: A
authors.initials: A
authors.lastname: Fauq
authors.forename: T E
authors.initials: TE
authors.lastname: Golde
mesh_terms: D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002474:Cell-Free System; D006224:Cricetinae; D010450:Endopeptidases / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D009842:Oligopeptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D012995:Solubility
chemical_list: D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D008565:Membrane Proteins; D009842:Oligopeptides; D011480:Protease Inhibitors; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases