SIGNAL TRANSDUCTION AND ALZHEIMER'S DISEASE--MOLECULAR BIOLOGICAL STUDIES

信号转导与阿尔茨海默病--分子生物学研究

• 批准号: 3746067
• 负责人: ERIC SCHAEFFER
• 金额: --
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3746067
• 关键词: Alzheimer's disease; CD4 molecule; CD8 molecule; Drosophilidae; amyloid proteins; biological signal transduction; calmodulin dependent protein kinase; cell adhesion molecules; chimeric proteins; gene expression; genetic transcription; genetically modified animals; growth factor; growth factor receptors; laboratory mouse; messenger RNA; molecular biology; molecular pathology; neurons; neurotransmitters; phosphoprotein phosphatase; phosphorylation; protein kinase; protein kinase A; protein kinase C; protein tyrosine kinase; second messengers; tissue /cell culture; transfection

General goals of the Program include the characterization of signal transduction pathways which regulate the biology of the Alzheimer amyloid percussor protein (APP), its Drosophila homologue, APPL, and the APP promoter-binding homeoproteins. Other Projects have focussed on the regulation and biological consequences of the phosphorylation of these substrates. Project 4 proposes to study in detail the regulation by protein phosphorylation of the transcription of APP and APPL mRNA, using several approaches. The first approach is to use specific treatments which activate protein phosphorylation and analyze for their effects on APP transcription. This will be performed in intact cells, using biochemical and molecular biological techniques. First messengers (neurotransmitters, neuropeptides, growth factors), and membrane permeable second messengers and direct enzyme activating agents will be applied to cells and RNA extracted from those cells will be assayed for APP expression. This approach, though convenient, is limited by the availability of relatively inaccessible to stimulation by permeable agents. In order to cause stimulated activity of isolated pathways, cDNAs for individual enzymes will be transfected into cells and the effect on APP mRNA expression will be measured. In related experiments, homeoproteins and homeoprotein kinases or phosphatases (identified by Project 1) will be cotransfected and effects on APP expression will be compared with that observed in cells transfected with either alone. In order to study APPL, flies will be transformed with protein kinases, including protein kinase C and calcium/calmodulin-dependent protein kinase II. In addition to morphologic examination for phenotypic abnormalities, these flies will be studied for expression of APPL. Primary cultures, derived from these flies, will also be studied: this Project will study APPL expression while Project 1 will study APPL phosphorylation. Because of its predicted structure as an integral transmembrane protein, the normal function of APP (currently unknown) may include the transduction of signals across the plasma membrane. In order to address this possibility, we propose in Project 4 to prepare, express and analyze chimeric molecules which are composed of a domain of APP joined to a complementary domain of a characterized signal transducing protein. Several classes of chimeras will be prepared, joining APP to the complementary domain of the epidermal growth factor receptor, CD4 receptor or neural cell adhesion molecule (N-CAM). For all three, there exist detailed data concerning the biochemical mechanisms responsible for their ability to transduce. Chimeras will be assayed for the ability of an APP domain to successfully substitute for the corresponding domain of EGFR, CD4 or N-CAM. Successful chimeras bearing the APP ectodomain will form the basis of assays for endogenous "ligands" for APP. Experiments defining the role of protein phosphorylation in the expression of Alzheimer's disease (AD)-related proteins may yield data relevant to the pathobiology and possible therapy of AD. Chimeric analysis may suggest physiological functions for APP, and these functions may also hold etiologic and clinical implications.

该计划的总体目标包括信号的表征 调节阿尔茨海默淀粉样蛋白生物学的转导途径 打击蛋白 (APP)、其果蝇同源物、APPL 和 APP 启动子结合同源蛋白。 其他项目的重点是 这些磷酸化的调节和生物学后果 基材。 项目4建议详细研究监管 APP 和 APPL mRNA 转录的蛋白质磷酸化，使用 几种方法。 第一种方法是使用激活蛋白质的特定治疗方法 磷酸化并分析其对 APP 转录的影响。 这 将使用生化和分子技术在完整的细胞中进行 生物技术。 第一信使（神经递质、神经肽、 生长因子），以及膜渗透性第二信使和直接酶 激活剂将应用于细胞并从中提取RNA 将检测细胞的 APP 表达。 这种做法虽然 方便，但受到相对难以到达的可用性的限制 渗透剂的刺激。 为了引起刺激活动 分离的途径，单个酶的 cDNA 将被转染到 细胞并测量对 APP mRNA 表达的影响。 在相关的 实验，同源蛋白和同源蛋白激酶或磷酸酶 （由项目1确定）将被共转染并对APP产生影响 表达将与转染细胞中观察到的表达进行比较 要么独自一人。 为了研究APPL，苍蝇将被蛋白激酶改造， 包括蛋白激酶 C 和钙/钙调蛋白依赖性蛋白激酶 二. 除了表型异常的形态学检查外， 将研究这些果蝇的 APPL 表达。 主要文化， 来自这些苍蝇的，也将被研究：该项目将研究 APPL 表达，而项目 1 将研究 APPL 磷酸化。 由于其预测结构为完整的跨膜蛋白， APP的正常功能（目前未知）可能包括转导 穿过质膜的信号。 为了解决这个问题 可能性，我们在项目4中建议准备、表达和分析 嵌合分子由 APP 结构域连接到 特征信号转导蛋白的互补结构域。 将准备几类嵌合体，加入APP 表皮生长因子受体、CD4 受体的互补结构域 或神经细胞粘附分子（N-CAM）。 对于这三者，都存在 有关其生化机制的详细数据 转换能力。 将测试嵌合体的 APP 能力 成功替代EGFR、CD4相应结构域 或 N-CAM。 成功携带 APP 胞外域的嵌合体将形成 APP 内源“配体”测定的基础。 定义蛋白质磷酸化在表达中的作用的实验 阿尔茨海默病（AD）相关蛋白的研究可能会产生与 AD 的病理生物学和可能的治疗方法。 嵌合分析可能表明 APP的生理功能，这些功能也可能具有 病因学和临床意义。