AMYLOID PRECURSOR PROTEIN

淀粉样前体蛋白

• 批准号: 2259864
• 负责人: HENRY W QUERFURTH
• 金额: $ 9.34万
• 依托单位: STEWARD ST. ELIZABETH'S MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-03 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2259864
• 关键词: Alzheimer's disease; Downs syndrome; SDS polyacrylamide gel electrophoresis; amyloid proteins; cellular pathology; chromosomes; clathrin; complementary DNA; cytoskeleton; family genetics; gene dosage; genetic promoter element; genetic transcription; glycoprotein biosynthesis; human subject; immunoprecipitation; integrins; messenger RNA; point mutation; reporter genes; spectrin; structural genes; tissue /cell culture; western blottings

The mechanism of amyloid beta peptide (beta/A4) accumulation in familial Alzheimers disease (FAD) and Down's syndrome is unknown and probably heterogeneous. The discovery of point mutations in exon 17 of betaPP770 in affected members of several families with AD is evidence that an abnormal beta amyloid precursor protein (betaAPP) can cause the disease in some cases. No genetic defect has been reported in the large Canadian and Italian FAD pedigrees which have known linkage to chromosome 21. Gene duplication is implicated in Down's syndrome. Our goal to elucidate the cause of beta/A4 accumulation in each of these conditions -will be addressed by the following specific experiments: 1) To examine the possibility of altered transcription of the betaAPP gene between affected and unaffected members of the Canadian and Italian families. We ask whether differences in the levels betaAPP MRNA or protein in FAD occur and if so, whether this reflects altered functioning of the promotor region or changes in MRNA stability. Total endogenous MRNA in dividing and stressed fibroblast cell lines from family members will be correlated with betaAPP protein levels by immunoblot. The same cell lines will also be transiently transfected with a full length betaApp promotor-reporter gene construct. 2) To examine the question, of how betaAPP levels are regulated as a function of gene dosage, we will address the unmet need to analyze rigorously the level of betaAPP transcripts in cells from living patients with trisomy 21 and controls. Total betaAPP MRNA and protein levels in lymphocyte lysates will be quantitated and compound to other gene products on or off chromosome 21. 30 To determine whether betaAPP exon 17 mutations a) favor an alternative intracellular cleavage event that generates carboxyl terminal fragment(s) containing the intact betaA4 or b) cause the appearance of such fragments only under special cellular conditions (e.g. stress). Pulse chase experiments and immunoprecipitations with various betaAPP antibodies of cells transfected with the mutant betaAPP CDNA'S will be conducted. 40 To examine the hypothesis that a) the extracellular portion of the integral membrane glycoprotein, betaAPP, functions as a substrate adhesion molecule receptor, similar to the Integrins; and b) the intracellular portion binds to the cytoskeleton. Cellular ligands that bind to betaAPP under normal conditions will be identified as co-precipitating proteins on SDS- PAGE after cells in culture are metabolic labeled with 35 S methionine. The effect of the exon 17 mutations on transfected cell adhesion to the substratum will also be studied . An understanding of betaAPP gene transcription, MRNA stability, protein degradation and its normal function is critical to the prevention of beta/A4 accumulation.

淀粉样β肽（β/A4）在家族性中积累的机理 阿尔茨海默氏病（FAD）和唐综合症尚不清楚，可能 异质。 在Betapp770的外显子17中发现点突变 在受影响的几个有广告家庭的成员中，有证据表明 异常的β淀粉样蛋白前体蛋白（betaapp）可能引起该疾病 在某些情况下。 在大型加拿大人中尚无遗传缺陷 和意大利时尚的血统，它们已知与21染色体的联系。 基因复制与唐氏综合症有关。 我们阐明的目标 在每种情况下β/A4积累的原因 - 将是 通过以下特定实验解决：1）检查 受影响的betaapp基因转录转录的可能性 以及未受影响的加拿大和意大利家庭的成员。 我们问 FAD中的betaapp mRNA或蛋白质的差异是否发生 如果是这样，这是否反映了启动子的功能改变 区域或mRNA稳定性变化。 总内源mRNA分裂 来自家庭成员的压力成纤维细胞系将相关 通过免疫印迹与BetaApp蛋白水平。 相同的单元线也将 用全长的betaapp启动子重新转染瞬时转染 基因构建体。 2）检查betaapp级别的问题 根据基因剂量的函数，我们将满足未满足的需求 严格分析细胞中BetaApp转录本的水平 三体三体患者21和对照。 总Betaapp mRNA和 淋巴细胞裂解物中的蛋白质水平将被定量，并化合 其他基因产物在21。30染色体上或关闭染色体以确定是否是 betaapp外显子17突变a）偏爱替代细胞内裂解 生成包含完整的羧基末端片段的事件 betaa4或b）仅在特殊下出现此类片段 细胞条件（例如压力）。 脉冲追逐实验和 带有各种betaApp抗体的细胞转染的免疫沉淀 随着突变体betaapp cDNA的进行。 40检查 假设a）整体膜的细胞外部分 糖蛋白，βApp充当底物粘附分子 受体，类似于整联蛋白； b）细胞内部分 结合细胞骨架。 与betaApp结合的细胞配体 正常情况将被确定为SDS-上的共沉淀蛋白 培养细胞后，用35 s蛋氨酸将其代谢标记。 外显子17突变对转染的细胞粘附在 也将研究底层。 对betaapp基因的理解 转录，mRNA稳定性，蛋白质降解及其正常 功能对于预防β/A4积累至关重要。