Gamma-Secretase: Structure of an Intramembrane Protease

描述（由申请人提供）：γ - secretase是多细胞生物生命所必需的一种前所未有的膜内切割天冬氨酸蛋白酶。它处理广泛且不断增长的单跨膜蛋白，包括Notch、Delta/Jagged、CD44、钙粘蛋白和某些钠通道亚基。γ -分泌酶最初被认为是一种影响APP最终断裂以释放淀粉样蛋白的蛋白酶，这与阿尔茨海默病（AD）有因果关系。早老素是引起最常见和最具侵袭性的早发性AD的突变，它含有两个保守的膜内天冬氨酸，作为催化位点。分泌酶复合物还含有3种其他完整膜蛋白：Nicastrin、Aph-1和Pen-2。由于蛋白酶复合物包含19个跨膜结构域，Nicastrin上有多个糖基化位点，并且在许多相对温和的洗涤剂中不稳定，因此对其结构的晶体学方法是困难和遥远的。我们最近纯化了哺乳动物复合物，基本达到均匀性，并进行了电子冷冻显微镜和单颗粒图像分析，获得了膜内蛋白酶的第一个3D图谱。我们现在建议在这个初始模型的基础上，显著提高结构的分辨率。我们将定位γ -分泌酶复合物的催化位点，绘制底物初始对接位点，并研究不同跨膜底物在γ -分泌酶复合物上是否存在多个对接位点。这项工作将产生更多关于这种不寻常的膜内酶的结构和机制信息，这是所有后生动物决定细胞命运所必需的。

DESCRIPTION (provided by applicant): gamma-Secretase is an unprecedented intramembrane-cleaving aspartyl protease necessary for life in multicellular organisms. It processes a broad and growing array of single-transmembrane proteins, including Notch, Delta/Jagged, CD44, cadherins and certain sodium channel subunits. gamma-Secretase was originally identified as the protease affecting the final scission of APP to release amyloid beta-protein, which is causally implicated in Alzheimer's disease (AD). Presenilin, mutations in which cause the most common and aggressive form of early-onset AD, contains two conserved intra-membrane aspartates that serve as the catalytic site. The gamma- secretase complex also contains 3 other integral membrane proteins: Nicastrin, Aph-1 and Pen-2. Because the protease complex contains 19 transmembrane domains, multiple glycosylation sites on Nicastrin, and are unstable in many relatively mild detergents, crystallographic approaches to its structure are difficult and remote. We recently purified the mammalian complex essentially to homogeneity and performed electron cryo- microscopy and single particle image analysis to obtain the first 3D map of the intramembrane protease. We now propose to build on this initial model by significantly improving the resolution of the structure. We will localize the catalytic site of the gamma-secretase complex, map the substrate initial docking site(s), and investigate if multiple docking sites exist on the gamma-secretase complex for different transmembrane substrates. This work should yield far more structural and mechanistic information on the unusual intramembrane enzyme, which is required for cell-fate decisions in all metazoans.