P2‐270: Effect of the components of gamma‐secretase on activity and specificity

The cause and progression of Alzheimer's disease (AD) is not well understood. However one of the hallmarks of AD is the development of amyloid plaques in the diseased brain. These plaques are predominantly composed of amyloid-beta fragments which are generated by gamma-secretase mediated cleavage of Amyloid Precursor Protein (APP). Understanding how gamma-secretase is regulated would give new therapeutic targets for AD research. Gamma-secretase is a multi-subunit protease that cleaves various substrates within the hydrophobic environment of the membrane. Gamma-secretase requires at least four components to function, all of which are transmembrane proteins: Presenilin, Nicastrin, Aph-1 and Pen-2. We have characterized four cells lines which overexpress different components of gamma-secretase in HEK293 cells: SW3, the parent cell line, does not overexpress any gamma secretase component; PP22 overexpresses PS1 and Pen-2; ANP24 expresses Aph-1, Nicastrin and PS1; and ANPP8 has all four components overexpressed. We have characterized these cell lines by Western blot analysis, APP and Notch-based in vitro gamma-secretase activity assays, and by pull down of active complexes via an inhibitor-based photolabeling approach. After determining the activity of APP and Notch cleavage and the amount of photolabeled species, we found that the total activity and the specific activity of the enzyme are not always correlated. This data suggests that other factors besides the four aforementioned components are necessary for a fully functional gamma secretase complex, and other regulatory mechanisms may be at work. These studies offer critical insight into the role of the individual components of gamma-secretase and their relation to the pathogenesis of Alzheimer's disease.