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: 6579262
• 负责人: RUDOLPH Emile TANZI
• 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/6579262
• 关键词: Alzheimer's disease; amyloid proteins; endopeptidases; enzyme activity; enzyme complex; family genetics; gene mutation; genetic polymorphism; glycoproteins; immunoprecipitation; liposomes; membrane proteins; presenilin; protein localization; protein metabolism; protein structure function; tissue /cell culture

DESCRIPTION (provided by applicant): Increased accumulation of the amyloid-Beta peptide (ABeta) or specific isoforms (ABeta42) 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 ABeta production. However, evidence has mounted to suggest that while apoptosis-induced ABeta generation may occur following acute injury to the CNS, other pathogenic mechanisms are likely involved in ABeta 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 gamma-secretase that cleaves APP at the C-terminal end of ABeta. Gamma-secretase is a heteromeric complex of proteins, in which only two components have been identified to date, PS and nicastrin. FAD mutations in PS1 increase the ratio of ABeta 42:ABeta40 and are likely to involve a number of as of yet unidentified proteins in the gamma-secretase/PS1. Thus, in the coming funding period, we propose to expand upon Specific Aim 3 of the original application, by exploring how the gamma-secretase complex/PS1 and FAD mutations in PS lead to alterations in the maturation and processing of APP and affect ABeta production. In our preliminary data, we show that nicastrin and thirteen unknown proteins coimmunoprecipitate 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 gamma-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 gamma-secretase activity, we propose to identify and characterize novel components of the gamma-secretase/PS1 complex, especially those that modulate ABeta production and the ABeta42/ABetatotal ratio. We will also test polymorphisms in genes that encode novel components of the gamma-secretase/PS1 complex, for family-based association with Alzheimer's disease. We plan to determine the subcellular localization and elucidate the physiological functions of the gamma-secretase/PS1 complex. Finally, we are performing in vitro gamma-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 ABeta generation, and to ultimately identify potential targets for reducing ABeta generation in Alzheimer's disease.

描述（由申请人提供）：淀粉样β肽（ABeta）或特定亚型（ABeta 42）的积累增加是所有形式阿尔茨海默病（AD）中神经变性的主要致病事件。在这个项目的前四年，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ABeta的生产。然而，越来越多的证据表明，虽然阿尔茨海默病诱导的ABeta产生可能发生在急性损伤的中枢神经系统，其他致病机制可能涉及ABeta生产由于APP和PS基因的家族性AD突变。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ABeta的C-末端切割APP。γ-分泌酶是一种异聚体蛋白复合物，其中迄今为止仅鉴定出两种组分，PS和nicastrin。PS1中的FAD突变增加了ABeta 42：ABeta 40的比例，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γ-分泌酶复合物/PS 1和PS中的FAD突变如何导致APP成熟和加工的改变并影响ABeta的产生来扩展原始申请的特定目标3。在我们的初步数据中，我们表明，nicastrin和13个未知的蛋白质coimmunoprecipitate与PS1的C-和N-末端片段从碳酸钠洗涤裂解液，从而代表潜在的新的膜相关的组件和/或基板的γ-分泌酶/PS1复合物。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。为了跟进这些发现，并扩展我们的研究FAD早老素突变对γ-分泌酶活性的影响，我们建议确定和表征γ-分泌酶/PS1复合物的新成分，特别是那些调节ABeta生产和ABeta 42/ABetatotal比率的成分。我们还将测试编码γ-分泌酶/PS1复合物新组分的基因多态性，以研究与阿尔茨海默病的家族关联。我们计划确定的亚细胞定位和阐明的γ-分泌酶/PS1复合物的生理功能。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ABeta生成的发病机制，并最终确定减少阿尔茨海默病中ABeta生成的潜在靶点。