Probing Regulatory Mechanism of Human γ-secretase via Membrane Cholesterol Using Cryogenic Electron Microscopy

Neurodegenerative disorders are a collective term referring to diseases caused by the progressive loss of neurons resulting in improper function of the human brain. γ-secretase is the critical protein involved in amyloid precursor protein cleavage, resulting in amyloid-beta peptide production, whose accumulation is a hallmark feature of neurodegenerative disorders. γ-secretase is also known as the "proteasome of the membrane" due to its ability to cleave over 90 type I transmembrane proteins with minimal sequence conservation. However, the mechanisms of its catalysis and regulation remain unclear.The cleavage activity of γ-secretase can be regulated by the interactions between protein subunits and between protein and membrane lipids, particularly cholesterol. Previous studies have shown that its activity is associated with membrane cholesterol levels. The removal of membrane cholesterol results in a significant decrease in γ-secretase activity, and replenishing cholesterol levels restores its activity. In addition, cholesterol has been implicated in the hydrophobic mismatching hypothesis to modulate the bilayer thickness, thereby inducing protein conformational change. Therefore, it is hypothesized that local cholesterol concentration modulates γ-secretase cleavage activity via hydrophobic mismatching. This study aims at studying the conformational changes of γ-secretase induced by membrane cholesterol using lipid nanodisc and cryo-EM imaging. The structures of the γ-secretase with various cholesterol levels will be critical to the understanding of its activation mechanism. Empty lipid nanodisc reconstitution under varying concentrations of cholesterol is first done to investigate the interaction and distribution of cholesterol and POPC in the lipid system. An increase in cholesterol level resulted in a splitting of peaks in FPLC profile, with each peak subsequently analyzed using HPLC to determine its composition. The data indicated the possibility of uneven cholesterol distribution within the lipid system as cholesterol levels increase, which could potentially facilitate the activation of γ-secretase. Following this, γ-secretase was reconstituted into cholesterol-containing lipid nanodiscs, optimizing the protein-to-lipid-to-MSP ratio, reaction temperature, and detergent elimination speed. Initial data from negative staining 2D classes showed promising reconstitution, but further data is required to fully support these findings.