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

g-分泌酶/PS1复合物在APP加工中的作用

• 批准号: 6823235
• 负责人: DORA M KOVACS
• 金额: $ 40.39万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-15 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6823235
• 关键词: Role; secretase; PS1; complex; APP

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===========================================

淀粉样蛋白-L肽（A[_]）或特定亚型（AI 342）的积累增加是所有形式的阿尔茨海默病（AD）中神经变性的主要致病事件。在这个项目的前四年里，我们集中在早老素（PS）FAD突变在细胞凋亡中的作用，以及细胞凋亡如何影响AI_的产生。然而，越来越多的证据表明，虽然阿尔茨海默病诱导的A13产生可能发生在CNS急性损伤后，但由于APP和PS基因中的家族性AD突变，其他致病机制可能参与A13产生。PS基因的克隆导致了称为γ-分泌酶的蛋白酶的初步表征，该酶在AI 3的C-末端切割APP。γ-分泌酶是蛋白质的异聚体复合物，其中迄今为止仅鉴定出两种组分，PS和nicastrin。PSI中的FAD突变增加了A[az]：Alg 40的比率，并且可能涉及γ-分泌酶/PS1中许多尚未鉴定的蛋白质。因此，在即将到来的资助期内，我们建议通过探索PS中的γ-分泌酶复合物/PSI和FAD突变如何导致APP成熟和加工的改变并影响AI 3的产生来扩展原始申请的具体目标3。在我们的初步数据中，我们表明nicastrin和13种未知蛋白质与来自碳酸钠洗涤的裂解物的PS1 C-和N-末端片段共免疫沉淀，从而代表潜在的新型膜相关组分和/或7-分泌酶/PS1复合物的底物。我们还确定了一个亚细胞级分在高尔基体/内涵体窝藏的复杂，连同其APP C-末端底物。我们已经初步鉴定了复合物中的一条未知蛋白带。为了进一步研究FAD早老素突变对γ-分泌酶活性的影响，我们提出鉴定和表征γ-分泌酶_S1复合物的新组分，特别是那些调节A1342/A13 totatio的组分。我们还将测试基因的多态性，编码新的成分的γ-分泌酶/PS1复合物，为基础的家庭与AD的关联。我们计划确定的亚细胞定位和阐明的生理功能的γ-分泌酶/PS1复合物。最后，我们正在进行体外γ-分泌酶测定，并将分离的复合物重组到单层脂质体中以研究其体外活性。这些研究的总体目标是确定PS中超过100种FAD突变影响Δ t3生成的发病机制，并最终确定减少AD中Δ t3生成的潜在靶点。多个部位（orgamzatton，ctty，state）>马萨诸塞州总医院遗传学和衰老研究中心查尔斯敦第16街114号楼，MA 02129关键人员=