Supported Biomembrane Microenvironments of Controlled Composition for Gamma-Secretase Substrate Cleavage Assays

The intramembrane protease gamma-secretase is a current target of therapeutic intervention, with pivotal pathological functions within Alzheimer’s disease and cancer. Our primary objective is to expand methods for in vitro studies of the intramembrane proteases (IMPs) with the development of a microsphere-supported (proteolipobead) and planar biomembrane platforms. IMPs are found throughout all branches of life and their functions are extremely broad. Despite extensive studies, understanding of IMP regulation and catalysis has been hampered by membrane-associated enzymology. Rhomboids and Secretases are polytopic membrane proteases that are widely conserved in all organisms. The precise reaction mechanisms of the intermembrane proteases remain to be elucidated and furthermore, rigorous analysis of the kinetics of interfacial catalysis in these systems has not yet been undertaken.

High throughput of screening of drug candidates has been carried out in bulk assay systems using cell membrane fragments, solubilized enzymes and is underway in proteoliposomes. A critical barrier to further progress in the study and HTS of gamma-secretase is that such bulk systems do not allow for the direct in situ quantification of enzyme, substrates, or inhibitors or their relative distributions within the structures under assay. We have expanded in vitro models of gamma-secretase to include supported biomembranes, enabling: 1) characterization and verification of biomembrane loading of substrates, inhibitors and protein effectors, 2) studies of co-localization, phase-partitioning and lateral mobility of membrane-bound assay constituents, and 3) the development of flow cytometry-based assays of cleavage. In this work we have characterized the structures, phase localization and compositions of gamma-secretase proteolipobead and planar systems by employing correlative optical and surface microscopy from the micro- to the nanoscale. We have probed the formation of Lo domains in situ, monitored using 3D FRET phase detection. We have probed the phase partitioning of enzyme, substrates and cleavage products in PLB systems with superresolution microscopy. 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. As new drug targets in cancer and infectious disease involved in regulated intramembrane proteolysis (RIP) based cell signaling are uncovered, it is expected that other IMP enzymes can be subsequently studied and probed at high throughput with this platform.