Characterization/Crystallization of y-Secretase Complex

描述(申请人提供)：这项研究提案的长期目标是在原子水平上深入了解伽马分泌酶的功能活性。γ分泌酶是一种膜蛋白复合体，它能裂解淀粉样前体蛋白(APP)的C末端。APP的这种加工与其N-末端被β-分泌酶切割一起产生淀粉样蛋白？片段(A？？)，形成淀粉样斑块，典型地存在于阿尔茨海默病患者的大脑中。 早老素-1是伽玛分泌酶的一种成分，含有酶活性部位。早老素-1的突变与家族性阿尔茨海默病的病因有关；因此，早老素-1是治疗药物的潜在靶点。伽马分泌酶的原子结构有望为我们提供这种新型酶复合体的分子细节。这些结构信息与其突变形式一起，有望深入了解其在膜双层中的蛋白分解活性的机制，以及与阿尔茨海默病有关的伽马分泌酶突变形式中蛋白分解模式的变化。这种结构信息将有助于治疗药物的设计。这项建议的具体目的是提纯和结晶伽马分泌酶复合体，以获得适合于后续晶体结构测定的晶体。最初，我们将继续开发从HeLa细胞中纯化伽马分泌酶，并将利用最近开发的微流控技术或2D结晶试验进行3D结晶试验。或者，我们将使用杆状病毒过度表达系统来过度表达伽马分泌酶，以获得毫克量的蛋白质复合体，用于“传统的”3D结晶试验。杆状病毒过表达系统还将使我们能够生产用于生化和结构研究的突变蛋白质。

DESCRIPTION (provided by applicant): The long-range objective of this research proposal is to develop a thorough understanding of the functional activities of gamma secretase at the atomic level. Gamma Secretase is a membrane protease complex that cleaves the C-terminal end of the amyloid precursor protein (APP). This processing of APP together with its cleavage at the N-terminal end by ?-secretase produces amyloid ? fragments (A??), which form amyloid plaques that are characteristically present in the brain of Alzheimer's patients. Presenilin-1, a component of gamma secretase, contains the enzymatic active site. Mutations of presenilin-1 have been associated with the cause of familial Alzheimer's disease; presenilin-1 is therefore a potential target for therapeutic drugs. The atomic structure of gamma secretase can be expected to provide us with the molecular details of this novel enzyme complex. This structural information together with those of its mutant forms can be expected to yield insights into the mechanism of its proteolytic activity within the membrane bilayer and the change in the proteolytic pattern found in mutant forms of gamma secretase that are linked to Alzheimer's disease. Such structural information will help in the design of therapeutic drugs. The specific aim of this proposal is to purify and crystallize gamma secretase complex in order to obtain crystals suitable for subsequent crystallographic structure determination. Initially, we will continue to develop the purification of gamma secretase from HeLa cells and will perform 3D crystallization trials utilizing a recently developed microfluidic technology or 2D crystallization trials. Alternatively, we will overexpress gamma secretase using a baculovirus overexpression system in order to obtain milligram quantities of the protein complex for "conventional" 3D crystallization trails. The baculovirus overexpression system will also allow us to produce mutant proteins for biochemical and structural studies.