AMYLOID DEPOSITION IN AGING AND ALZHEIMER'S DISEASE

衰老和阿尔茨海默病中的淀粉样蛋白沉积

• 批准号: 3119484
• 负责人: Huntington Potter
• 金额: $ 16.23万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-02-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3119484
• 关键词: Alzheimer's disease; Downs syndrome; SDS polyacrylamide gel electrophoresis; aging; amyloid proteins; chemical binding; chymotrypsin inhibitor; clone cells; endopeptidases; gene expression; high performance liquid chromatography; human genetic material tag; human tissue; immunoelectron microscopy; in situ hybridization; molecular cloning; northern blottings; nucleic acid sequence; paired helical filament; polymerase chain reaction; protein purification; protein sequence; protein structure; proteolysis; western blottings

Alzheimer's disease is a neurodegenerative disorder of the central nervous system resulting in a progressive loss of memory and other intellectual functions beginning in middle to late life. The disease is characterized by the accumulation of certain neuropathological lesions in the brains of affected individuals. The identification of alpha1-antichymotrypsin (ACT) as a component of Alzheimer amyloid filaments and the ? that the beta- protein-the major amyloid protein-is a proteolytic fragment of a larger precursor protein, ? the subject of antiproteases and proteases into the biochemical discussion of Alzheimer's disease. The discovery of a Kunitz inhibitor domain in the beta-protein precursor, and our finding that the beta-protein itself ? a striking resemblance to the active site of serine proteases and can stably bind to ACT via a protease-antiprotease interaction further underscores the need to understand the biochemistry and physiology of protease and antiprotease function in Alzheimer brain. This application proposes to continue our study of two ? of the role of proteases and their inhibitors in Alzheimer's disease-in the structure of the amyloid ? and in the proteolytic processing of the beta-protein from its precursor. With respect to the structure of Alzheimer amyloid filaments, we will determine the specific amino acids in the beta-protein and in the active ? of ACT necessary for their interaction in vitro. We will also design peptides that should bind ACT better in the beta-protein and might serve to inhibit ACT-beta-protein binding in vitro and potentially in vivo. We have discovered two chymotrypsin-like proteases (CLIPs) with the substrate specificity to cleave the terminus of the beta- protein from its precursor. One (clipsin) has been purified to homogeneity. The enzyme ? been partially sequenced and is likely identical to rat mast cell protease I. It preferentially degrades the protein precursor in brain membrane extracts, is inhibited by ACT, and can make the correct cleavage in ? peptide substrates. We will establish whether clipsin can cleave the beta-protein from its native precursor and are in the process of cloning the clipsin gene from rat and human brain cDNA. While clipsin/RMCP I is the best beta-protein-generating candidate protease now known, we are using PCR identify and clone other brain CLIPs. These will be expressed in a baculovirus system and their encoded ? tested for their ability to cleave the beta-protein N-terminus. We will also test the level and distribution their expression in normal, Alzheimer, and Down syndrome brain, to determine why the beta-protein is differentially generated in the disease process.

阿尔茨海默氏病是一种中枢神经系统的神经退行性疾病 导致记忆和其他智力逐渐丧失的系统 开始于生命的中后期。 该疾病的特征 由于某些神经病理学病变在大脑中的积累， 受影响的个人。 α 1-抗糜蛋白酶（ACT）的鉴定 作为阿尔茨海默氏症淀粉样纤维的一种成分，测试版 蛋白质-主要的淀粉样蛋白-是一个较大的蛋白质的蛋白水解片段， 前体蛋白，？抗蛋白酶和蛋白酶的主题 阿尔茨海默病的生化讨论。 库尼茨人的发现 抑制剂结构域的β蛋白前体，我们的发现， β-蛋白本身？与丝氨酸的活性部位惊人的相似 蛋白酶，并可通过蛋白酶-抗蛋白酶稳定结合ACT 相互作用进一步强调了了解生物化学和 阿尔茨海默病脑中蛋白酶和抗蛋白酶功能生理学。 这 申请建议继续我们的研究两个？的作用 蛋白酶及其抑制剂在阿尔茨海默氏病中的作用 淀粉样蛋白以及在β-蛋白质的蛋白水解加工中， 它的前身。 关于阿尔茨海默氏症淀粉样蛋白的结构 纤维，我们将确定β蛋白中的特定氨基酸 在积极？的ACT必要的相互作用在体外。 我们 还将设计能更好地结合ACT β蛋白的肽 并可能在体外抑制ACT-β-蛋白结合， 可能在体内。 我们已经发现了两种胰凝乳蛋白酶样蛋白酶 （CLIP）具有底物特异性，可切割β- 蛋白质从它的前体。 一个（clipsin）已被纯化， 同质性 酶？已经部分测序， 大鼠肥大细胞蛋白酶I 它优先降解蛋白质 前体在脑细胞膜提取物中，被ACT抑制， 正确的乳沟？肽底物。 我们将确定 夹蛋白酶可以将β-蛋白从其天然前体上切割下来， 从大鼠和人脑cDNA中克隆clipsin基因的过程。 而clipsin/RMCP I是产生β蛋白的最佳候选蛋白酶 目前，我们正在使用PCR鉴定和克隆其他脑CLIP。 这些 将在杆状病毒系统中表达并编码？测试 它们切割β蛋白N末端的能力。 我们还将测试 在正常人、阿尔茨海默病患者和唐氏症患者中表达水平和分布 综合征的大脑，以确定为什么β-蛋白质是不同的 疾病过程中产生的。