Single molecule kinetic studies of gamma-secretase/substrate interaction and the effects of AD-causing mutations

Project Summary Amyloid plaque, composed of amyloid-b peptide (Ab), is a pathological hallmark of Alzheimer’s disease (AD). g-secretase is responsible for the cleavage of C99, the C-terminal fragment of 99 residues of amyloid precursor protein (APP), to generate Ab. Previous kinetics studies of g-

secretase measured the final production of APP intracellular domain (AICD) and/or Ab. However, the kinetics rates for individual steps of the generation of Ab from C99 are lacking. Here we will use single molecule fluorescence studies to observe enzyme/substrate molecules in real time, and measure the kinetics of enzyme/substrate association and cleavage in g-

secretase-mediated intramembrane proteolysis to generate Ab (Aim1), and determine how familial AD (FAD) mutations alter the kinetics of enzyme/substrate association and cleavage in AD (Aim2).