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

在超出所提供的空间的情况下，淀粉样蛋白-L多肽(A[_)或特定亚型(AI342)的积累增加是导致所有形式的阿尔茨海默病(AD)神经变性的主要致病事件。在这个项目的前四年，我们重点研究了早老素(PS)FAD突变在细胞凋亡中的作用，以及细胞凋亡如何影响AI_2的产生。然而，越来越多的证据表明，虽然急性中枢神经系统损伤后可能发生细胞凋亡诱导的AI_3的产生，但由于APP和PS基因的家族性AD突变，A[3]的产生可能涉及其他致病机制。PS基因的克隆导致了被称为y-分泌酶的蛋白酶的初步鉴定，该酶在AI3的C末端裂解APP。Y-分泌酶是一种蛋白质的异构体复合体，到目前为止只发现了PS和尼古丁两种成分。PSI中的FAD突变增加了A[_az：alg40]的比例，并可能涉及一些尚未识别的蛋白在y-分泌酶/PS1中的作用。因此，在接下来的资助期，我们建议通过探索PS中的y-分泌酶复合体/PSI和FAD突变如何导致APP成熟和加工的改变，并影响AI3的产生，来扩展最初的应用程序的具体目标3。在我们的初步数据中，我们发现尼古丁和13个未知蛋白与来自碳酸钠洗涤的裂解物的PS1 C端和N端片段共免疫沉淀，从而代表了潜在的新的膜相关成分和/或7-分泌酶/PS1复合体的底物。我们还在含有该复合体的高尔基体/内体及其APP C末端底物中鉴定了一种亚细胞组分。我们已经初步确定了该复合体中的一条未知蛋白质带。为了进一步研究FAD早老素突变对γ-分泌酶活性的影响，我们建议鉴定和鉴定γ-分泌酶_S 1复合体的新成分，特别是那些调节AI3合成和A1342/A13tot的成分。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，以确定与AD的家族相关性。我们计划确定γ-分泌酶/PS1复合体的亚细胞定位并阐明其生理功能。最后，我们进行了体外γ-分泌酶测定，并将分离的复合体重组为单层脂质体，以研究其体外活性。这些研究的总体目标是确定PS中100多个FAD突变影响AT3代的致病机制，并最终确定减少AD中A[3代]的潜在靶点。PLHEORMANCE SITE(S)(州奥伽马扎顿)&GT；马萨诸塞州查尔斯敦16街114号马萨诸塞州综合医院遗传学和老龄研究单位大楼02129 Key Personnel========================================Section End===========================================

EXCEED THE SPACE PROVIDED, 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. PLHEORMANCE SITE(S) (orgamzatton, ctty, state) > Massachusetts General Hospital Genetics and Aging Research Unit Building 114, 16th Street Charlestown, MA 02129 KEY PERSONNEL ========================================Section End===========================================