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: 7173808
• 负责人: DORA M KOVACS
• 金额: $ 38.3万
• 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 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7173808
• 关键词: Acute; Address; Affect; Aging; Alzheimer' s Disease; Amyloid; Apoptosis; Axon; Biochemical Genetics; Biological Assay; C-terminal; Cell Line; Chicago; Cleaved cell; Cloning; Complex; Data; Doctor of Medicine; Doctor of Philosophy; Endopeptidases; Endosomes; Event; Face; Family; Funding; General Hospitals; Generations; Genes; Genetic; Genetic Polymorphism; Goals; Golgi Apparatus; Hippocampus (Brain); Human Resources; In Vitro; Injury; Instruction; Integral Membrane Protein; Laboratories; Last Name; Lead; Lipids; Liposomes; Location; Massachusetts; Membrane; Mutation; N-terminal; Names; Nerve Degeneration; Neurons; Numbers; Peptide Hydrolases; Peptides; Physiological; Play; Principal Investigator; Printing; Production; Progress Reports; Protein Isoforms; Protein Structure Initiative; Proteins; Research; Research Personnel; Research Project Grants; Role; Site; Subcellular Fractions; Testing; Transmembrane Domain; Universities; Vesicle; age related; amyloid precursor protein processing; base; density; familial Alzheimer disease; follow-up; in vivo; medical schools; nicastrin protein; notch protein; novel; novel therapeutics; peptide A; plexin; presenilin; presenilin-1; programs; receptor; reconstitution; secretase; size; sodium carbonate; therapeutic target; trafficking

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.

淀粉样蛋白-L_肽（A[_]）或特定亚型（AI 342）的积累增加是一种主要的致病因素， 在所有形式的阿尔茨海默病（AD）中，神经变性是潜在的事件。在这个项目的头四年里， 项目中，我们重点关注早老素（PS）FAD突变在细胞凋亡中的作用，以及细胞凋亡是如何发生的。 影响AI生产。然而，越来越多的证据表明，虽然糖尿病诱导的AI_ 可能发生在CNS急性损伤后，其他致病机制可能参与 由于APP和PS基因中的家族性AD突变而产生A[3]。PS基因的克隆导致了 称为γ-分泌酶的蛋白酶的初始表征，其在A13的C-末端切割APP。 γ-分泌酶是一种异聚体蛋白复合物，其中只有两种组分被鉴定为 枣子，PS和尼克酸。PSI中的FAD突变增加了A[_az]：Alg 40的比例，并且可能涉及到一种新的细胞因子。 γ-分泌酶/PS1中尚未鉴定的蛋白质的数量。因此，在未来的资助期内，我们 我建议通过探索γ-分泌酶如何 PS中的复合物/PSI和FAD突变导致APP成熟和加工的改变， 影响AI 3的生产。在我们的初步数据中，我们表明nicastrin和13种未知蛋白质共同作用， 用来自碳酸钠洗涤裂解物的PS1 C-和N-末端片段免疫沉淀，从而 代表7-分泌酶/PS1的潜在新的膜相关组分和/或底物 复杂.我们还在高尔基体/核内体中鉴定了一个亚细胞组分， 连同其APP C-末端底物。我们已经初步确定了一个未知的 复合物中的蛋白质条带。跟进这些发现，并扩展我们对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 γ-分泌酶_S1复合物，特别是调节A1342/A13 totratio的γ-分泌酶_S1复合物。我们 还将检测编码γ-分泌酶/PS1复合物新组分的基因的多态性， 以家庭为基础的协会与AD。我们计划确定亚细胞定位并阐明 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构为单层脂质体以研究其体外活性。整体 这些研究的目的是确定PS中100多个FAD突变的发病机制， 影响Δ t3生成，并最终确定减少AD中Δ t3生成的潜在靶点。