P3-330: Luciferase complementation imaging as a real-time reporter for the gamma-secretase cleavage of notch

As a key protease involved in the production of the amyloidogenic Aβ peptides, the gamma-secretase enzyme has been a prime therapeutic target for Alzheimer's disease. However, gamma-secretase cleaves other Type I membrane protein substrates, several of which play important roles during development and tissue renewal in adults, including the Notch receptors. Notch activation is triggered by ligand binding and depends on gamma-secretase-mediated release of the Notch intracellular domain (NICD), which translocates to the nucleus and associates with the DNA-binding protein CSL to activate gene expression. We report the use of luciferase complementation imaging (LCI) technology to monitor the interactions between the NICD and its nuclear cofactor CSL in real time. In this reporter system, complementary halves of firefly luciferase (NLuc and CLuc), which have no activity on their own, reconstitute luciferase activity when brought in close proximity by specific interactions between NICD and CSL. Our validation studies demonstrate LCI to be a specific, robust and sensitive system for imaging NICD/CSL interactions. Because the luciferase fragments contribute negligible binding energy and the luciferase fusion proteins constitute a complete assay system, they allow measurement of reconstituted luciferase activity in near real time, enabling kinetic analyses of gamma-secretase activity and of Notch inhibition in live cells, from which Aβ production can be simultaneously assessed. We have begun screening for molecules and gene products capable of modulating Notch cleavage and activation and are in the process of validating leads. The LCI system is versatile; it can be adapted for studying the pathway in different cell types and can be modified to monitor interactions of Notch with other proteins, including components of gamma-secretase. We are currently adapting the system to simultaneously monitor interactions of gamma-secretase with two substrates (Notch and APP) and assessing its utility as a screen for modifiers of substrate selection.