Isolation of functionally active gamma-secretase protein complex and methods for detection of activity and inhibitors thereof

摘要

The present invention provides an isolated, functionally-active protein that catalyzes cleavage of a gamma-secretase substrate. The functional activity of the isolated protein suggests that the isolated protein includes gamma-secretase. In one embodiment, the isolated gamma-secretase protein is associated with PS1. The present invention also relates to homogeneous methods for monitoring cleavage of beta-amyloid precursor protein (betaAPP) by gamma-secretase, wherein the steps of of isolating and retrieving cleavage products have been eliminated. Cleavage can be detected by binding a pair of fluorescent adducts to the gamma-cleaved betaAPP fragment. Preferably, a first fluorescent adduct binds to the carboxy-terminal end of the gamma-cleaved betaAPP fragment, with substantially no cross-reactivity to uncleaved betaAPP or to other types of gamma-cleaved betaAPP fragments, while a second fluorescent adduct binds to a portion within the amino-terminal region on the gamma-cleaved betaAPP fragment. Detection of binding to the gamma-cleaved betaAPP fragment is determined by monitoring the fluorescent energy transfer between the adducts.