MEGAKARYOCYTE/PLATELET AMYLOID BETA PROTEIN PRECURSOR

巨核细胞/血小板淀粉样β蛋白前体

基本信息

批准号：
2028837
负责人：
RAYMOND R SCHLEEF
金额：
$ 26.49万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-09-30 至 1998-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2028837
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

项目摘要

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.

阿尔茨海默氏病（AD）是一种特发性进步痴呆，影响我们越来越老化的人口中很大一部分。虽然目前的数据表明，AD的发病机理尚不完全了解它可能涉及大的蛋白水解裂解前体蛋白产生约42种残基蛋白，称为淀粉样β蛋白或A4，随后聚合并形成淀粉样蛋白沉积的特征。是淀粉样β-蛋白质（即淀粉样蛋白β-蛋白前体; App）是通过替代剪接产生的至少三个mRNA编码两个外显子。两种表达最丰富的形式包含56 残留插入物，与kunitz型具有重要同源性蛋白酶抑制剂（KPI）。血小板凝血因子XLA抑制剂最近被确定为包含KPI的应用程序形式领域。目前，几乎没有关于应用程序来源的信息血小板或其在成熟期间的处理巨核细胞/血小板。描述的工作的长期目标在此应用中，是为了提供对与应用相关的应用程序的生产和处理巨核细胞/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子显微镜技术来定义细胞在（i）完整血小板中分布应用程序，（ii）血小板粘附/聚集反应，（iii）巨核细胞系和（iv）新鲜隔离和培养的骨髓巨核细胞时成熟。完整血小板中存在的应用程序形式以及在血小板粘附/聚集期间发生的应用程序处理反应将通过使用免疫印迹方案进行分析和放射免疫测定。这些研究将构成分析的框架与异常血小板相关的应用的位置和形式存在于AD患者的血液中。血小板应用的起源然后将通过使用免疫学测定和代谢来定义标记/免疫沉淀实验，以分析生产在巨核细胞系和骨骼的分化过程中应用骨髓衍生的细胞体外。这些研究将得到补充表征负责处理的蛋白酶活动（IES）巨核细胞/血小板应用。此外，结构域应用对于将其靶向血小板α颗粒至关重要的应用程序将通过在单元格中使用表达向量/应用构建体来定义表现出存储分泌途径的线。这些结构将包括应用程序的删除突变体，APP的站点定向突变体，以及混合应用分子。因为已经提出了血小板神经元的外围模型，可以预期巨核细胞/血小板过程应用程序可能会使用机制通过深入了解该蛋白的表达和裂解神经元。