PHOSPHORYLATION AND TURNOVER OF NEURONAL CYTOSKELETON-ASSOCIATED PROTEINS

神经元细胞骨架相关蛋白的磷酸化和周转

• 批准号: 3802332
• 负责人: RALPH A NIXON
• 金额: --
• 依托单位: MCLEAN HOSPITAL
• 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/3802332
• 关键词: Alzheimer's disease; SDS polyacrylamide gel electrophoresis; amyloid proteins; brain cell; cytoskeleton; gel electrophoresis; gene expression; immunochemistry; laboratory mouse; membrane proteins; microtubule associated protein; molecular pathology; nerve /myelin protein; neuronal transport; phosphoproteins; phosphorylation; posttranslational modifications; protein biosynthesis; protein kinase; protein metabolism; proteolysis; radiotracer; retinal ganglion; tau proteins; transfection

The overall objective of this research is to define the molecular mechanisms underlying the atrophy and death of neurons and the development of intraneuronal and extraneuronal lesion in Alzheimer's disease (AD). We have recently detected a population of slowly transported phosphoproteins in retinal ganglion cell neurons which comprise major microtubule-associated proteins (MAPS) and constituents of the fibrous and membrane cytoskeleton, including proteins with known relevance to AD. Major goals will be to use neuron specific in vivo approaches to identify these proteins and to define how phosphorylation may govern their turnover and interactions with cytoskeletal elements and the membrane. Our specific aims are to characterize the neuronal phosphoproteins with respect to associations with the Triton-insoluble cytoskeleton and ability to co-assemble with micro tubules. Their identities will be established immunochemically using well-character antibodies to known brain MAPs and cytoskeletal proteins and by 2-D SDS-PAGE and 2-D iodopeptide mapping. The number and relative locations of phosphorylated sites on the radiolabeled proteins in vivo will be determined by 2-D phosphopeptide mapping and compared with maps of the same polypeptide phosphorylated in vitro by each of four major protein kinases in order to identify the kinase(s) that are capable of mediating the vivo phosphorylation of specific sites and their relation to function. Site-specific phosphate turnover on proteins will be studied during axoplasmic transport in relation to changing associations of these polypeptides with specific moving and stationary cytoskeletal elements. Interrelationships between phosphorylation state, turnover rate and association with specific cytoskeletal organelles will be established. This information will be applied to additional investigations on the mechanism of amyloid accumulate AD brain. In separate experiments, the amyloid precursor protein of Alzheimer's disease will be studied with respect to its susceptibility to purified human brain proteins in vitro, its proteolytic cleavage patterns and its turnover and possible phosphorylation in cultured neural cells transfected with cDNA encoding the amyloid protein. By focusing on proteins of known or suspected importance in AD and on regulatory processes that govern cytoskeleton-membrane, these studies are expected to clarify the relationship between intraneuronal and extraneuronal lesions in Alzheimer's disease and will provide information directly relevant to the mechanism by which amyloid and tau proteins accumulate in AD brain.

这项研究的总体目标是定义分子 神经元萎缩和死亡的潜在机制和 阿尔茨海默病神经元内和神经元外损害的发展 疾病(AD)。我们最近检测到了一个缓慢的种群 在视网膜神经节细胞神经元中运输的磷蛋白 由主要的微管相关蛋白(MAP)和 纤维和膜细胞骨架，包括已知的蛋白质 与AD的相关性。主要目标将是在体内使用神经元特异性 鉴定这些蛋白质并定义如何磷酸化的方法 可能支配它们的周转和与细胞骨架元素的相互作用 膜。我们的具体目标是描述神经元的特征 磷酸蛋白与Triton不解性的关系 细胞骨架和与微管共同组装的能力。他们的 将使用Well-Character通过免疫化学方法确定身份 针对已知脑图和细胞骨架蛋白的抗体以及2-D 十二烷基硫酸钠-PAGE和二维碘肽作图。数量和相对位置 体内放射性标记蛋白质上的磷酸化位点的数量将是 由二维磷酸肽图确定，并与 四种主要蛋白质各自在体外磷酸化的相同多肽 为了确定能够中介的激酶(S) 体内特定位点的磷酸化及其与细胞因子的关系 功能。将研究蛋白质上特定部位的磷酸盐周转 在轴浆运输过程中与这些改变的联系有关 具有特定运动和固定细胞骨架元件的多肽。 磷酸化状态、周转率和细胞周期的相互关系 将建立与特定细胞骨架细胞器的联系。 这些信息将被应用于对 淀粉样蛋白蓄积AD脑的机制。在单独的实验中， 阿尔茨海默病的淀粉样前体蛋白将与 关于它对体外纯化的人脑蛋白的敏感性， 它的蛋白水解性裂解模式及其周转和可能 转染编码基因的神经细胞的磷酸化 淀粉样蛋白。通过关注已知或可疑的蛋白质 在AD和监管流程中的重要性 细胞骨架-膜，这些研究有望澄清 脑神经元内、外损害的关系 阿尔茨海默氏症，并将提供与 淀粉样蛋白和tau蛋白在AD脑内积聚的机制。