281854 Stabilized Proteolipobead Microenvironments for the Functional Analysis and Drug Screening of the Cancer and Alzheimer's Disease Drug Target Gamma-Secretase
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 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 HTPS 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. 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 confocal imaging in tandem with flow cytometry, we will present findings from i) direct measurements of substrate and gamma-secretase loading within supported biomembranes, ii) the localization and quantification of enzyme:substrate and enzyme:inhibitor complexes within supported biomembranes, and iii) comparisons of stability of tether-supported assays to existing systems. We have also initiated studies of fused biomembrane systems make new in vitro models of gamma secretase catalysis and regulation.
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