Role of the g-secretase/PS1 complex in APP processing

淀粉样蛋白-L多肽(A[_)或特定异构体(AI342)积聚增加是主要致病因素 所有形式的阿尔茨海默病(AD)中潜在的神经变性事件。在这头四年里 项目中，我们重点研究了早老素(PS)FAD突变在细胞凋亡中的作用，以及细胞凋亡是如何 影响AI_Products。然而，越来越多的证据表明，当细胞凋亡诱导AI_ 可能发生在急性中枢神经系统损伤后，其他致病机制可能涉及 A[3由于APP和PS基因的家族性AD突变所致。PS基因的克隆导致了 在AI3的C末端裂解APP的被称为y-分泌酶的蛋白酶的初步特征。 Y-分泌酶是一种蛋白质的异构体复合体，其中只有两个组分被鉴定为 DATE、PS和NICASTIN。PSI中的FAD突变增加了A[_az：alg40]的比例，并可能涉及 Y-分泌酶/PS1中尚未确定的蛋白质的AS数目。因此，在即将到来的供资期间，我们 建议通过探索y-分泌酶如何在原始应用程序的特定目标3上进行扩展 PS的Complex/PSI和FAD突变导致APP和APP的成熟和加工的改变 影响AI3的生产。在我们的初步数据中，我们发现尼古丁和13种未知蛋白质共同作用于 用来自碳酸钠洗涤的裂解物的PS1C-和N-末端片段进行免疫共沉淀，从而 代表潜在的新的膜相关成分和/或7-分泌酶/PS1的底物 很复杂。我们还在高尔基体/内体中发现了含有该复合体的亚细胞部分， 以及它的APP C终端基板。我们已经初步确定了其中一个未知的 复合体中的蛋白质条带。跟进这些发现，并扩大我们对FAD影响的研究 早老素突变对γ-分泌酶活性的影响，我们建议鉴定和表征新的成分 γ-分泌酶_S 1复合体，特别是调节AI3合成的复合体和A1342/A13tot复合体。我们 还将测试编码y-分泌酶/PS1复合体新成分的基因的多态， 以家庭为基础与AD的关系。我们计划确定亚细胞定位并阐明 γ-分泌酶/PS1复合体的生理功能。最后，我们正在进行体外γ-分泌酶分析。 将分离得到的复合体重组为单层脂质体，研究其体外活性。整体而言 这些研究的目标是确定PS中100多个FAD突变的致病机制 影响AT3代，并最终确定减少AD中A[3代]的潜在目标。

Increased accumulation of the amyloid-l_ peptide (A[_) or specific isoforms (AI342) is a major pathogenic event underlying neurodegeneration in all forms of Alzheimer's disease (AD). In the first four years of this project, we have focused on the role of presenilin (PS) FAD mutations in apoptosis, and how apoptosis influences AI_ production. However, evidence has mounted to suggest that while apoptosis-induced AI_ generation may occur following acute injury to the CNS, other pathogenic mechanisms are likely involved in A[3 production owing to familial AD mutations in APP and the PS genes. Cloning of the PS genes has led to the initial characterization of the protease called y-secretase that cleaves APP at the C-terminal end of AI3. y-secretase is a heteromeric complex of proteins, in which only two components have been identified to date, PS and nicastrin. FAD mutations in PSI increase the ratio of A[_az:Alg40 and are likely to involve a number of as of yet unidentified proteins in the y-secretase/PS1. Thus, in the coming funding period, we propose to expand upon specific aim 3 of the original application, by exploring how the y-secretase complex/PSI and FAD mutations in PS lead to alterations in the maturation and processing of APP and affect AI3 production. In our preliminary data, we show that nicastrin and thirteen unknown proteins co- immunoprecipitate with PS1 C- and N-terminal fragments from a sodium carbonate-washed lysate, thereby representing potentially novel membrane-associated components and/or substrates of the 7-secretase/PS1 complex. We have also identified a subcellular fraction in the Golgi/endosomes harboring the complex, together with its APP C-terminal substrates. We have tentatively already identified one of the unknown protein bands in the complex. To follow up on these findings and extend our studies of the effect of FAD presenilin mutations on y-secretase activity, we propose to identify and characterize novel components of the y-secretase_S 1 complex, especially those that modulate AI3 production and the A1342/A13tot,riatio. We will also test polymorphisms in genes that encode novel components of the y-secretase/PS1 complex, for family-based association with AD. We plan to determine the subcellular localization and elucidate the physiological functions of the y-secretase/PS1 complex. Finally, we are performing in vitro y-secretase assays and reconstituting the isolated complex into unilamellar liposomes to study its activity in vitro. The overall goal of these studies is to define the pathogenetic mechanism by which more than 100 FAD mutations in PS affect At3 generation, and to ultimately identify potential targets for reducing A[3 generation in AD.