Tristan Sprague

Familial Alzheimer’s disease is an aggressive disease that begins before age 65, quickly progresses, and is passed down from parents to children. γ-secretase is a membrane protease that cleaves the transmembrane domain of many single-pass membrane proteins. Mutations in the active subunit of γ-secretase, presenilin, or one of its substrates, amyloid precursor protein, results in familial Alzheimer’s disease (FAD). We hope that understanding γ-secretase biochemistry will help us understand how Alzheimer’s disease starts. Most FAD mutations reduce γ-secretase’s enzymatic efficiency, but the structure-function relationship resulting in this reduction is not understood. My project uses chemical biology to investigate how mutation changes γ-secretase functionality and how γ-secretase interacts with its membrane environment. Currently, our lab hypothesizes that many FAD mutations stall the enzyme-substrate complex; that is, the mutant enzyme can still bind substrate but fails to proteolyze it. To test this hypothesis, I am designing single-molecule fluorescence experiments to observe the enzyme at work. By labeling both ends of a transmembrane substrate, it should be possible to observe the time substrate is bound and the time to proteolysis of the substrate C-terminal. I’m also studying the relationship between γ-secretase and cholesterol, which can make up to 45% of a natural membrane. I plan on using stereoisomers of cholesterol, as well as diazirine-alkyne derivatives, to probe what characteristics of cholesterol-containing membranes affect γ-secretase and to search for potential cholesterol binding sites on the enzyme. Knowledge of the mutations’ effect on the enzyme’s function and of any allosteric sites could be key to rational, structure-based drug design for Alzheimer's.