Supported Biomembrane Microenvironments for the Protease Functional Analysis and Screening of Gamma-Secretase, the Notch Pathway Drug Target

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 biomembrane (proteolipobead) platform. Despite extensive studies, understanding of IMP regulation and catalysis has been hampered by membrane-associated enzymology. The precise reaction mechanisms of the intramembrane 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 gamma-secretase 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 screening 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 in native lipids. Due to these factors and the instability of these systems there are limitations in the scope of assays possible using solubilized systems and proteoliposomes in bulk solution. We have expanded in vitro models of gamma-secretase to include proteolipobeads (microsphere-supported biomembranes), using novel substrates such as Notch1-Sb1 (truncated Notch 1 with FLAG tag) to probe the enzyme.  Using confocal and super-resolution imaging in tandem with flow cytometry, we will present findings from i) direct measurements of substrate and gamma-secretase loading and in-membrane orientation within supported biomembranes, ii) the localization and quantification of enzyme:substrate and enzyme:inhibitor complexes within supported biomembranes, and iii) the introduction of new protein-orthogonal biomembrane tethering strategies that include membrane fusion.