Cell-free assays for gamma-secretase activity.
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.
McLendon
C
C
Department of Pharmacology, Mayo Clinic, Jacksonville, Florida 32224, USA.
Xin
T
T
Ziani-Cherif
C
C
Murphy
M P
MP
Findlay
K A
KA
Lewis
P A
PA
Pinnix
I
I
Sambamurti
K
K
Wang
R
R
Fauq
A
A
Golde
T E
TE
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
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D008565:Membrane Proteins; D009842:Oligopeptides; D011480:Protease Inhibitors; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases