Substrate recruitment and cleavage by γ-secretase

γ-分泌酶是一种关键的膜内蛋白水解酶，也是阿尔茨海默病(AD)的主要药物靶点。除了AD相关的β-淀粉样前体蛋白(APP)外，目前已知有数百种底物被伽马分泌酶切割。这些是如何被识别和选择的，人们还知之甚少。尽管最近有伽马分泌酶的结构信息，但该酶的底物结合位点(S)尚未确定。此外，还不知道哪些底物特性使伽马分泌酶底物分别是好的(即可有效切割的)或坏的(即较差的)底物。在这个项目提案中，我们将使用在特定位置携带可光交联性氨基酸的工程底物来鉴定伽马分泌酶的底物结合部位。因此，在紫外线照射下，直接与底物结合的亚基将被明确识别。使用这种将首次引入我们的研究领域的方法，我们将首先开始绘制APP的结合位点图，然后将这种分析扩展到关键的伽马分泌酶底物Notch1和其他感兴趣的底物(目标1)。通过突变分析，我们还将确定和定义在蛋白质分解分析中控制底物切割效率的序列决定因素(目标2)。最后，我们将分析蛋白酶的脂环境如何调节底物识别过程(目标3)。我们的研究将为膜内蛋白酶如何招募和裂解其底物提供基本的见解。

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.