MEGAKARYOCYTE/PLATELET AMYLOID BETA-PROTEIN PRECURSOR

巨核细胞/血小板淀粉样β-蛋白前体

• 批准号: 3368690
• 负责人: RAYMOND R SCHLEEF
• 金额: $ 23.12万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1997-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3368690
• 关键词: Alzheimer's disease; amyloid proteins; endopeptidases; enzyme activity; granule; human tissue; immunoelectron microscopy; immunofluorescence technique; megakaryocytes; pathologic process; platelet aggregation; platelets; radioimmunoassay; western blottings

Alzheimer's disease (AD) is an idiopathic progressive dementia affecting a large proportion of our increasingly aged population. Although the pathogenesis of AD is not completely understood, present data suggest that it probably involves the abnormal proteolytic cleavage of a large precursor protein to yield about 42 residue protein, referred to as amyloid Beta protein or A4, which subsequently polymerizes and forms the amyloid deposits characteristic of this disease. The precursor of the amyloid Beta-protein (i.e., amyloid Beta-protein precursor; APP) is encoded by at least three mRNAs that arise through alternative splicing of two exons. The two most abundantly expressed forms contain a 56 residue insert, which has significant homology with the Kunitz-type protease inhibitors (KPI). Platelet coagulation Factor Xla-inhibitor was recently identified to be a form of APP that contains the KPI domain. Presently, little information exists on the origin of APP in the platelet or its processing during the maturation of megakaryocytes/platelets. The long-term objective of the work described in this application is to provide a detailed understanding of the production and processing of APP associated with megakaryocytes/platelets. Immunologic probes will be developed against specific regions of the APP molecule and utilized in immunofluorescent and immunoelectron microscopic techniques to define the cellular distribution of APP in (i) intact platelets, (ii) platelets during adhesion/aggregation reactions, (iii) megakaryocytic cell lines, and (iv) freshly isolated and cultured bone marrow megakaryocytes as they mature. The forms of APP present in intact platelets, as well as the processing of APP that occurs during platelet adhesion/aggregation reactions, will be analyzed utilizing immunoblotting protocols and radioimmunoassays. These studies will form the framework for analyzing the location and forms of APP associated with abnormal platelets that are present in the blood of AD patients. The origin of platelet APP will then be defined by utilizing immunologic assays and metabolic labeling/immunoprecipitation experiments to analyze the production of APP during the differentiation of megakaryocytic cell lines and bone marrow-derived cells in vitro. These studies will be complemented by characterizing the protease activity(ies) responsible for the processing of megakaryocyte/platelet APP. Furthermore, the structural domain(s) on APP that are critical for its targeting into platelet alpha-granules will be defined by the use of expression vector/APP constructs in cell lines that exhibit a storage secretory pathway. These constructs will include deletion mutants of APP, site-directed mutants of APP, and hybrid APP molecules. Because platelets have been proposed as a peripheral model for neurons, it is expected that information on the mechanisms by which megakaryocytes/platelets process APP will likely provide an insight into the expression and cleavage of this protein by neurons.

阿尔茨海默病（AD）是一种特发性进行性痴呆， 在我们日益老龄化的人口中占很大比例。 虽然 目前的数据表明，AD的发病机制尚未完全清楚 它可能涉及一个大的蛋白质的异常蛋白水解切割， 前体蛋白，以产生约42个残基的蛋白质，称为 淀粉样β蛋白或A4，其随后聚合并形成 这种疾病的特征性淀粉样沉积物。 的前体 淀粉样β-蛋白（即，淀粉样β蛋白前体; APP）是 由至少三种通过选择性剪接产生的mRNA编码 两个exons。 两种表达最丰富的形式含有56 残基插入片段，与Kunitz型具有显著的同源性 蛋白酶抑制剂（KPI）。 血小板凝血因子XIa抑制剂 最近被确定为包含KPI的APP形式 域 目前，关于APP起源的信息很少 血小板或其在成熟过程中的加工 巨核细胞/血小板。 所述工作的长期目标 在本申请中的目的是提供对 APP的生产和加工， 巨核细胞/血小板。 免疫探针将针对 APP分子的特定区域，并用于免疫荧光 和免疫电镜技术来确定细胞 APP在（i）完整血小板，（ii）血小板中的分布 粘附/聚集反应，（iii）巨核细胞系，和 (iv)新鲜分离和培养的骨髓巨核细胞， 成熟 APP的形式存在于完整的血小板，以及 血小板粘附/聚集过程中发生的APP加工 将使用免疫印迹方案分析反应， 放射免疫测定 这些研究将构成分析的框架 与异常血小板相关的APP的位置和形式， 存在于AD患者的血液中。 血小板APP的起源 然后将通过利用免疫测定和代谢测定来确定 标记/免疫沉淀实验，以分析 APP在巨核细胞系和骨分化过程中的作用 骨髓来源的细胞在体外。 这些研究将得到以下方面的补充： 表征负责加工的蛋白酶活性 此外，巨核细胞/血小板APP上的结构域 APP对于其靶向血小板α颗粒至关重要 将通过在细胞中使用表达载体/APP构建体来定义 表现出储存分泌途径的细胞系。 这些结构将 包括APP缺失突变体、APP定点突变体， 杂合APP分子。 因为血小板被认为是 对于神经元的外周模型，预计关于神经元的信息将被提供。 巨核细胞/血小板处理APP的机制可能 提供了一个深入了解这种蛋白质的表达和切割， 神经元