Gamma-Secretase: Structure of an Intramembrane Protease

描述（由申请人提供）：γ-分泌酶是一种前所未有的膜内切割乙酰基蛋白酶，是多细胞生物体中生命所必需的。它处理一系列广泛且不断增长的单跨膜蛋白，包括Notch、Delta/Jagged、CD 44、钙粘蛋白和某些钠通道亚基。γ-分泌酶最初被鉴定为影响APP最终断裂以释放淀粉样β-蛋白的蛋白酶，淀粉样β-蛋白与阿尔茨海默病（AD）有因果关系。早老素是导致早发性AD最常见和侵袭性形式的突变，其含有两个保守的膜内突变体，作为催化位点。γ-分泌酶复合物还含有3种其他的膜整合蛋白：Nicastrin、Aph-1和Pen-2。由于蛋白酶复合物含有19个跨膜结构域，在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。我们最近纯化的哺乳动物复合物基本上同质，并进行电子冷冻显微镜和单粒子图像分析，以获得第一个3D地图的膜内蛋白酶。我们现在建议通过显着提高结构的分辨率来建立这个初始模型。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的γ-分泌酶复合物上是否存在多个对接位点。这项工作应该产生更多的结构和机制的信息不寻常的膜内酶，这是需要在所有后生动物的细胞命运的决定。

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.