(494d) The Dependence of Gamma-Secretase Substrate Cleavage on Lipid Microdomains in the Context of Spatial Diffusion-Reaction Models

In these studies, the influence of cholesterol-rich lipid domain structures on the enzymology of the intramembrane protease γ−secretase is probed using microsphere-supported biomembranes.  This important protein is a particularly complex enzymatic system composed of four subunits that catalyzes the cleavage of over 80 known transmembrane protein substrates. Despite extensive studies and decisive roles in both the Notch signaling pathway and in amyloid precursor protein (APP) degradation and processing in Alzheimer’s disease, deciphering γ−secretase regulation and catalysis has been hampered by membrane-associated enzymology. Multiple lines of evidence in cell-based systems has implicated cholesterol and cholesterol-rich membrane microdomains, termed lipid rafts, in the regulation of substrate cleavage of γ-secretase relating to Alzheimer’s disease pathology.  In  this work we have incorporated controlled cholesterol-rich domain formation into our recently developed method to investigate and directly visualize active γ-secretase and its substrates in microsphere-supported biomembranes. This enables us to directly probe enzyme and substrate mobility, localization and cleavage in phase-separated domains to investigate the cholesterol dependence of γ-secretase. 

In this work we have characterized the structures, phase colocalization and compositions of gamma-secretase proteolipobead systems by employing correlative optical and cryoelectron microscopy from the micro- to the nanoscale. We have probed the formation of L_o domains in situ, monitored using 3D FRET phase detection.   We have probed the phase partitioning of enzyme, substrates and cleavage products in PLB systems. A combination of biomembrane mobility methods are being used to probe the diffusivities and mobile fractions of lipid, substrates and gamma-secretase.  The results of phase partioning and diffusivity measurements are being integrated into bottom-up spatial diffusion-reaction models to correlate microenvironment with enzyme function.