PURIFICATION /RECONSTITUTION OF ACTIVE GAMMA SECRETASE

白粉病基因与表型关系的研究进展 家族性AD的早老性形式提供了一个独特的机会 阐明阿尔茨海默病中一个尚未解决的主要问题：伽马分泌酶 产生Abeta42肽，它在急性淋巴细胞白血病的早期积累 疾病的各种形式。PS1和PS2基因突变选择性增强Abeta42 在培养细胞、转基因小鼠和人脑组织中产生，以及 这种增加已经在症状前记录在血浆和原发性 突变基因携带者的细胞。我们最近发现PS1和PS2 在内质网和早期高尔基体中与APP形成稳定的复合体，该突变体 PS增加了这些站点的Abeta42。因此，我们的核心假设是 这个项目是PS突变在物理上改变了PS的相互作用 APP的一种方式允许通过一种 伽马-分泌酶在残基42处特异切割，并且 理解这种情况是如何发生的，将有助于揭示一个早期和不变的 常见的“散发性”AD的特征(Abeta42沉积)。至 针对这一假设，我们将实现4个目标：1)使用稳定的细胞 来自核心B和C的品系和PS转基因小鼠定义了 相互作用的APP和PS，突变如何改变相互作用，以及 更多的蛋白质(例如，Delta-catenin)参与复合体；2) 使用细胞生物学和药理学方法来定义所有的 Abeta40和Abeta42产生的主要亚细胞基因座及其PS 突变对这些基因有不同的影响；3)建立结构 APP中对伽马分泌酶切割的要求以及是否 在40℃(和42℃)时，蛋白水解酶通过直接膜内蛋白分解起作用。 通过与最近为SREBP描述的类似的“两次命中”机制；以及 4)确定42和40特异性的伽马分泌酶抑制剂，并使用它们来 鉴定并最终纯化认知蛋白水解酶(S)。所有四个目标 是基于强大的初步数据，它们将涉及广泛的 与本计划的其他3位PI合作。结果应该是 提供与蛋白质分解过程有关的一般信息 跨膜蛋白，特别是Abeta42的作用机制 一代人，阿尔茨海默病的新兴治疗靶点。

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.