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).

Project Narrative Alzheimer’s disease (AD) is one of the most serious challenges to public health in the United States, and is characterized by an accumulation of amyloid plaque in the brain. We will use single molecule techniques to study the detailed mechanism of how amyloid-b peptide (Ab) is generated by a complex enzymatic process. Our results will be the first single molecule view of Ab production by g-secretase in real time, providing novel insights into the mechanisms of intramembrane proteolysis, amyloid production, and for AD drug discovery.