PURIFICATION /RECONSTITUTION OF ACTIVE GAMMA SECRETASE

基因型与表型关系的研究进展 家族性AD的早老素形式提供了一个独特的机会， 阐明AD中一个主要的未解决的问题：γ-分泌酶如何 产生Abeta 42肽，其在所有 疾病的形式。PS1和PS2中的突变选择性增强Abeta 42 在培养细胞、转基因小鼠和人脑组织中的生产，以及 这种增加在症状前的血浆和原发性 突变基因携带者的细胞。我们最近发现PS1和PS2 在ER和早期高尔基体中与APP形成稳定的复合物， PS增加这些位点中的Abeta 42。因此，我们的中心假设 这个项目是PS突变在物理上改变PS的相互作用， 与APP以一种方式，允许增加后者的蛋白水解， γ-分泌酶特异性地在残基42处裂解，并且 了解这是如何发生的，将有助于了解早期和不变的 AD的常见“散发性”形式的特征（A β 42沉积）。到 针对这一假设，我们将实现4个目标：1）使用稳定的细胞 品系和来自核心B和C的PS转基因小鼠，定义了 APP和PS相互作用，突变如何改变相互作用以及是否 另外的蛋白质（例如，δ-连环蛋白）参与复合物; 2） 使用细胞生物学和药理学方法来定义所有的 A β 40和A β 42产生的主要亚细胞位点以及PS 突变差异影响这些; 3）建立结构 APP内γ分泌酶裂解的要求以及 蛋白酶通过在40（和42）处的直接膜内蛋白水解作用起作用，或 通过类似于最近描述的SREBP的“2次命中”机制;以及 4)鉴定42-和40-特异性γ-分泌酶抑制剂， 表征并最终纯化认知蛋白酶。所有4个目标 是基于强有力的初步数据，他们将涉及广泛的 与该计划的其他3个PI合作。结果应 提供一般与蛋白水解加工相关的信息， 跨膜蛋白，特别是Abeta 42的机制 一代，阿尔茨海默病的一个新兴的治疗靶点。

Progress in deciphering the genotype to phenotype relationship of the presenilin form of the familial AD provides a unique opportunity to elucidate a major unresolved issue in AD in general: how gamma-secretase generates the Abeta42 peptide which accumulates early in the course of all forms of he disease. Mutations in PS1 and PS2 selective enhance Abeta42 production in cultured cells, transgenic mice and human brain tissue, and this increase has been documented presymptomatically in plasma and primary cells from mutant gene carriers. We recently found that both PS1 and PS2 form stable complexes with APP in the ER and early Golgi and that mutant PS increases Abeta42 in these sites. Therefore, our central hypothesis of this project is that PS mutations physically alter the interaction of PS with APP in a way that allows increased proteolysis of the latter by a gamma-secretase cleaving specifically at residue 42, and that understanding how this occurs will shed light on an early and invariant feature (Abeta42 deposition) of the common "sporadic" form of AD. To address this hypothesis, we will carry out 4 Aims: 1) using stable cell lines and PS transgenic mice from Cores B and C, define the regions of APP and PS that interact, how mutations change the interaction and whether additional proteins (e.g., delta-catenin) participate in the complexes; 2) use cell biological and pharmacological methods to define all of the principal subcellular loci for Abeta40 and Abeta42 production and how PS mutations differentially effect these; 3) establish the structural requirements within APP for the gamma secretase cleavages and whether the proteases act by direct intramembranous endoproteolysis at 40 (and 42) or by a "2 hit" mechanism analogous to that recently described for SREBP; and 4) identify 42- and 40-specific gamma-secretase inhibitors and use them to characterize and ultimately purify the cognitive protease(s). All 4 Aims are based on strong preliminary data, and they will involve extensive collaborations with the other 3 PI's of this Program. The results should provide information relevant in general to the proteolytic processing of transmembrane proteins and in particular to the mechanism of Abeta42 generation, an emerging therapeutic target in Alzheimer's disease.