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: 3808978
• 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/3808978
• 关键词: Alzheimer's disease; 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确定分子 神经元萎缩和死亡的潜在机制， Alzheimer病神经元内和神经元外损害的发展 疾病（AD）。我们最近发现了一群 视网膜神经节细胞神经元中转运磷蛋白， 包括主要微管相关蛋白（MAPS）和 纤维和膜细胞骨架，包括已知的蛋白质， 与AD有关。主要目标将是在体内使用神经元特异性 方法来识别这些蛋白质，并确定如何磷酸化 可能控制其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 与Triton不溶性蛋白结合的磷蛋白 细胞骨架和与微管共组装的能力。他们的 将使用良好的特征， 已知的脑MAPs和细胞骨架蛋白的抗体， SDS-PAGE和2-D碘肽图谱。数量和相对位置 在体内，放射性标记蛋白质上的磷酸化位点将被 通过2-D磷酸肽图谱确定并与 在体外被四种主要蛋白质中的每一种磷酸化的相同多肽 激酶，以鉴定能够介导 特定位点的体内磷酸化及其与 功能将研究蛋白质上的位点特异性磷酸盐周转 在轴浆运输过程中， 具有特定移动和固定细胞骨架元件的多肽。 磷酸化状态、周转率和 将建立与特定细胞骨架细胞器的结合。 这些信息将用于对 AD脑内淀粉样蛋白积聚机制。在单独的实验中， 阿尔茨海默病的淀粉样前体蛋白将被研究， 关于其在体外对纯化的人脑蛋白的易感性， 它的蛋白水解切割模式和它的周转， 磷酸化的神经细胞转染的cDNA编码 淀粉样蛋白通过关注已知或疑似 对反倾销的重要性以及对 这些研究有望阐明 神经元内和神经元外病变之间的关系 阿尔茨海默病，并将提供直接相关的信息， 淀粉样蛋白和tau蛋白在AD脑中积累的机制。