Substrate recruitment and cleavage by γ-secretase

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gamma-Secretase is a pivotal intramembrane protease and major Alzheimer disease (AD) drug target. Besides the AD-associated beta-amyloid precursor protein (APP), currently in the range of hundred substrates are known to be cleaved by gamma-secretase. How these are recognized and selected is only poorly understood. Despite recent structural information of gamma-secretase, the substrate-binding site(s) of the enzyme are not yet defined. Moreover, it is not known which substrate features make a gamma-secretase substrate a good (i.e. efficiently cleavable) or a bad (i.e. poorly cleavable) one, respectively. In this project proposal, we will identify the substrate-binding sites of gamma-secretase using engineered substrates carrying photocrosslinkable amino acids at defined positions. Thus, upon UV irradiation, the subunits, which directly bind substrate will be unambiguously identified. Using this approach, which will be introduced to our research field for the first time, we will initially start to map the binding sites of APP and then extend this analysis to the crucial gamma-secretase substrate Notch1 and other substrates of interest (goal 1). By mutational analysis, we will additionally identify and define sequence determinants that govern the efficiency of substrate cleavage in proteolysis assays (goal 2). Finally, we will analyze how the lipid environment of the protease will modulate the substrate recognition process (goal 3). Our studies will provide fundamental insights into how intramembrane proteases recruit and cleave its substrates.