(598f) Confocal Imaging and Flow Cytometry-Based Platform for Intramembrane Protease Analysis and Drug Discovery

Our primary objective is to develop a new platform for assaying the function and inhibition of membrane-bound Intramembrane Protease (IMP) signal transduction systems. IMPs have emerged as a new class of membrane proteins, in which the catalytic residues of the protease reside within predicted transmembrane domains. Moreover, the scissile bond of substrates appears to be situated within a transmembrane domain as well. IMPs are found throughout all branches of life and their functions are extremely broad. These include cellular signaling, stem cell differentiation, transcriptional control, and parasite invasion. 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 remains to be elucidated and furthermore, rigorous analysis of the kinetics of interfacial catalysis in these systems has not yet been undertaken.

The functional immobilization of intramembrane proteases in supported membranes will facilitate the biochemical analysis of these molecules and lead to a new platform for functional analysis and ultimately intramembrane protease drug discovery. To our knowledge, thus far, none of these IMP enzymes have been studied in solid-supported lipid bilayer microenvironments. These systems could provide a new means to elicit control over intermembrane concentrations of enzymes, substrates and inhibitors that is not possible with proteoliposome-based bulk assay systems. We have constructed IMP solid-supported membranes on microspheres, enabling in situ confocal imaging and mobility measurements and also new flow cytometry-based assays. This is the first step toward a solid phase assay system that would be of practical use in detecting and/or analyzing the functional effects of presumed effectors and inhibitors and lead the way into drug discovery applications.