(158e) Characterizing the Role of Biomembrane Phase Separation in the Function and Regulation of the Drug Target Gamma-Secretase

The primary goal of this work to investigate the influence of biomembrane liquid-liquid phase behavior on the function of gamma-secretase. This enzyme is composed of four essential proteins and cleaves over 90 substrates within transmembrane domains and is a prominent drug target in both Cancer and Alzheimer’s disease. Together with rhomboids, signal peptide peptidases (SPP) and site 2 proteases (S2P), it is the most intensely investigated protease involved in regulated intramembrane proteolysis (RIP). This proteolysis is an evolutionary conserved process that is required for signal transduction and yet also for the degradation of transmembrane protein fragments, depending on the cellular context. Deciphering gamma-secretase regulation and catalysis has been hampered by the complexities of membrane-associated enzymology.

Multiple lines of evidence, from the organismal scale to the the molecular level, have implicated changes in cholesterol and cholesterol-rich membrane microdomains, termed lipid rafts, in the regulation of substrate cleavage by gamma-secretase. In essence, the cell may regulate enzyme function through thermodynamic influence imposed by cholesterol-rich microdomains on 1) MP partitioning, 2) membrane thickness effects on structure and activity, and 3) MP diffusivity differences in ordered versus disordered phases in lipid bilayers. The indirect biochemical approaches used to examine the biomembrane distribution of gamma-secretase, its substrates, and cleavage products have been applied to 1) cellular membrane microdomains, 2) bulk proteoliposome cleavage assays in vitro, and 3) multiple studies in neuronal cell culture. These indirect methods do not allow for the in situ quantification of enzyme and substrate partitioning, activity, and diffusivity in the context of intact biomembranes with cholesterol-containing phase-separated lipid domains. Our aim is to incorporate controlled cholesterol-rich domain formation commonly used to study giant unilamellar vesicles (GUVs) into our new method to investigate gamma-secretase in supported biomembranes.

We have characterized the structures, phase colocalization, membrane protein (MP) orientations and compositions by employing correlative optical and atomic force microscopy from the micro- to the nanoscale in spherical proteolipobead and planar formats. This allowed direct visualization of the phase partitioning and diffusivity of active gamma-secretase and its substrates in micron-scale domains along with averaged measurements when observing systems containing (invisible) nanoscopic domains. We have probed the dependence of secretase substrate cleavage and inhibition on phase composition in the context of spatial diffusion-reaction models. The macroscopic cleavage results have been correlated to "bottom-up" built spatial diffusion-reaction models utilizing experimental diffusivities and phase partitioning results. The modeling was carried out using previous kinetic rate data in proteoliposomes, with relevant spatial catalytic scenarios pertaining to configurations of lipid phase separation, including cholesterol depletion controls. The modeling allows us to examine the potential effects of substrate feedback and the presence of immobile populations of active enzyme.