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. 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 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. Thus, for instance, if a candidate peptide substrate with an altered sequence does not show cleavage activity in bulk assay, this question arises: did the sequence change affect delivery to the cell membrane fragments that contain gamma-secretase or was the peptide truly catalytically inactive? This situation is amplified if the substrate candidate is a larger protein of greater hydrophobicity and more prone to aggregation. Furthermore, 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), enabling: 1) characterization and verification of biomembrane loading of substrates, inhibitors and protein effectors, 2) studies of co-localization, interactions and lateral mobility of membrane-bound assay constituents, 3) the development of flow cytometry-based assays of cleavage, and 4) stability–enhanced microenvironments. 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 supported assays to existing systems.
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 with HTPS with this platform.