AMYLOID PRECURSOR PROTEIN

淀粉样前体蛋白

• 批准号: 2259865
• 负责人: HENRY W QUERFURTH
• 金额: $ 9.33万
• 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/2259865
• 关键词: Alzheimer's disease; Downs syndrome; SDS polyacrylamide gel electrophoresis; amyloid proteins; cellular pathology; chromosome 21; 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.

家族性淀粉样β蛋白(β/A4)蓄积机制的研究 阿尔茨海默病(FAD)和唐氏综合症是未知的，很可能 异质的。β-PP770基因第17外显子点突变的发现 在几个阿尔茨海默病家庭的受影响成员中，有证据表明 异常的β-淀粉样前体蛋白(β-APP)可导致这种疾病 在某些情况下。目前还没有关于大型加拿大人的基因缺陷的报道 以及已知与21号染色体有关联的意大利时尚家系。 唐氏综合症与基因复制有关。我们的目标是澄清 在上述每种情况下，β/A4累积的原因是 通过以下具体实验解决：1)检查 β-APP基因在受感染人群中转录改变的可能性 以及加拿大和意大利家庭中未受影响的成员。我们问 FAD患者APP基因和蛋白水平是否存在差异 如果是，这是否反映了发起人的功能发生了变化 区域或mRNA稳定性的变化。总内源mRNA在分裂中的作用 来自家庭成员的应激成纤维细胞系将被关联 免疫印迹法检测β-APP蛋白水平。同样的细胞系也会 瞬时转染全长BetaApp启动子--记者 基因构建。2)为了研究这个问题，BetaAPP水平是多少 作为基因剂量的函数，我们将解决未得到满足的需求 严格分析细胞中β-APP转录本的水平 存活的21三体患者和对照组。总的βAPP基因和 淋巴细胞裂解产物中的蛋白质水平将被定量并合成到 21号染色体上或染色体外的其他基因产物。30以确定是否 BetaAPP外显子17突变a)有利于另一种细胞内切割 生成包含完整的羧基末端片段的事件(S BetaA4或b)仅在特殊情况下才会导致此类碎片的出现 细胞状态(如应激)。脉冲追逐实验和 不同β-APP抗体对转基因细胞的免疫沉淀作用 与突变体BetaAPP的CDNA将进行。40以检查 假设a)完整膜的胞外部分 糖蛋白，β-APP，起底物黏附分子的作用。 受体，类似于整合素；和b)细胞内部分 结合到细胞骨架上。与β-APP结合的细胞配体 正常情况下将被鉴定为共沉淀蛋白质的十二烷基硫酸钠- 培养的细胞经PAGE后代谢标记35 S蛋氨酸。 外显子17突变对转基因细胞黏附能力的影响 还将对基质进行研究。对BetaAPP基因的认识 转录、信使核糖核酸稳定性、蛋白质降解及其正常 功能对防止β/A4蓄积至关重要。