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: 3726542
• 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/3726542
• 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.

该程序的一般目标包括信号的表征 调节阿尔茨海默氏淀粉样蛋白生物学的转导途径 打击乐蛋白（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提议详细研究 使用APP和APPR 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 磷酸化和分析它们对应用程序转录的影响。 这 将使用生化和分子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）和膜可渗透的第二使者和直接酶 激活剂将应用于细胞和从那些细胞中提取的RNA 将分析细胞以进行APP表达。 但是，这种方法 方便，受相对无法访问的可用性的限制 可渗透剂刺激。 为了引起刺激的活动 孤立的途径，单个酶的cDNA将转染到 细胞和对APP mRNA表达的影响将测量。 在相关 实验，同音蛋白和同音蛋白激酶或磷酸酶 （项目1确定）将被共转染并对应用程序影响 将表达与在转染的细胞中观察到的表达 要么一个人。 为了研究应用，将用蛋白激酶转化苍蝇， 包括蛋白激酶C和钙/钙调蛋白依赖性蛋白激酶 ii。 除了表型异常的形态检查外， 这些苍蝇将被研究以表达Appl。 初级文化， 从这些苍蝇中得出的也将被研究：这个项目将研究 在项目1时，APPL表达将研究应用磷酸化。 由于其预测的结构是整合跨膜蛋白的结构，因此 应用程序的正常功能（当前未知）可能包括转导 跨质膜的信号。 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 由应用程序域组成的嵌合分子连接到一个 特征信号转导蛋白的互补结构域。 将准备几类嵌合体，将应用程序加入到 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）。 在这三个中，存在 有关生化机制的详细数据 能力转导的能力。 将对嵌合体的应用程序进行测定 成功替换EGFR的相应域CD4的域 或n-cam。 成功带有应用程序域的成功嵌合体将形成 应用程序内源性“配体”的测定基础。 定义蛋白质磷酸化在表达中的作用的实验 阿尔茨海默氏病（AD）相关蛋白可能会产生与该数据有关的数据 病理生物学和AD的可能疗法。 嵌合分析可能表明 应用程序的生理功能，这些功能也可能成立 病因和临床意义。