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Characterization/Crystallization of y-Secretase Complex

γ-分泌酶复合物的表征/结晶

基本信息

批准号：
6892888
负责人：
Bing K. Jap
金额：
$ 24.52万
依托单位：
UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-06-01 至 2006-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6892888
关键词：
Alzheimer&apos s disease Baculoviridae HeLa cells aspartic endopeptidases crystallization electron microscopy enzyme activity fluid flow gene mutation microorganism mass culture posttranslational modifications protein purification protein quantitation /detection protein structure function

项目摘要

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.

描述（由申请人提供）：本研究提案的长期目标是在原子水平上深入了解γ分泌酶的功能活性。γ分泌酶是一种膜蛋白酶复合物，其切割淀粉样前体蛋白（APP）的C末端。APP的这种加工及其在N-末端的切割是由？分泌酶产生淀粉样蛋白？片段（A？？），形成淀粉样斑块，这是阿尔茨海默氏症患者大脑中的特征。早老素-1是γ分泌酶的一个组分，含有酶活性位点。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。γ分泌酶的原子结构有望为我们提供这种新型酶复合物的分子细节。这种结构信息连同其突变形式的那些可以预期产生深入了解其蛋白水解活性在膜双分子层内的机制以及在与阿尔茨海默病相关的γ分泌酶的突变形式中发现的蛋白水解模式的变化。这些结构信息将有助于治疗药物的设计。本提案的具体目的是纯化和结晶γ分泌酶复合物，以获得适用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γ分泌酶，并将利用最近开发的微流体技术或2D结晶试验进行3D结晶试验。或者，我们将使用杆状病毒过表达系统过表达γ分泌酶，以获得毫克量的蛋白质复合物用于“常规”3D结晶试验。杆状病毒过表达系统也将使我们能够产生突变蛋白，用于生化和结构研究。