PROTEOMIC MAPPING OF MYELIN AND ITS MEMBRANE SUBDOMAINS

描述(申请人提供)：髓鞘是一种动态的、功能活跃的膜，与轴突进行双向通信，并接收和处理来自环境的信号。我们认为，了解髓鞘的活性生物学功能的关键是从功能上鉴定和表征完整的髓鞘蛋白谱系，即髓鞘蛋白质组。我们对髓鞘进行了广泛的蛋白质组学分析，开发了髓鞘蛋白质的2D-PAGE图谱，迄今已鉴定出125种此类蛋白质，并通过比较蛋白质组学的具体应用说明了这种方法的力量和用途。这种蛋白质组学图谱和相关的概念方法为研究髓鞘膜的不同生物学作用提供了一个重大进展，并为其他神经系统的类似研究提供了一个范例。该项目的长期目标是确定和表征髓鞘膜的分子组成；开发这些分子在髓鞘膜中的排列模型，特别侧重于糖鞘磷脂-胆固醇微域的作用；指定与髓鞘生物发生、维持和活动至关重要的蛋白质的功能相关；并将这些概念与更好地理解多发性硬化症和其他脱髓鞘疾病中的脱/再髓鞘作用有关。髓鞘蛋白鉴定：在目标I中，我们进一步发展了髓鞘蛋白质组，应用新的方法来鉴定未分解的蛋白质。这项研究为整个髓鞘生物界提供了一个无价的髓鞘蛋白数据库，为许多项目奠定了基础。髓鞘蛋白功能：在AIM II中，我们使用直接凝胶内电泳法(DGE)和同位素编码亲和标签(ICAT)质谱学方法研究了OL分化和生理介导的蛋白质表达变化，并使用抗体阻断、RNAi技术和转基因小鼠突变体对蛋白质表达和功能进行了调控。髓鞘蛋白病：在AIM III中，我们研究了在正常的OL生理学和髓鞘生物学疾病中，细胞信号通路被环境因素激活的机制。重点是模拟自然配体和神经免疫学疾病条件的抗体扰动的蛋白质组学分析，包括髓鞘少突胶质细胞糖蛋白(MOG)、半乳糖磷脂和糖鞘磷脂-胆固醇微域在启动信号转导中的作用。

DESCRIPTION (provided by applicant): Myelin is a dynamic, functionally active membrane in bi-directional communication with the axon and receiving and processing signals from the environment. We propose that a key to understanding the active biological functions of myelin is to identify and characterize functionally the complete repertoire of myelin proteins, i.e., the Myelin Proteome. We have undertaken an extensive proteomic analysis of myelin, developed a 2D-PAGE map of myelin proteins, identified to date 125 of these proteins, and illustrated the power and utility of this approach through specific applications of comparative proteomics. This proteomic map and related conceptual approaches offers a major advance for investigations into the diverse biological roles of the myelin membrane and suggest a paradigm for similar studies in other neural systems. The long range goals of this project are to identify and characterize the molecular components of the myelin membrane; develop a model for the arrangement of these molecules in the myelin membrane, with a particular focus on the role of glycosphingolipid-cholesterol microdomains; assign functional correlates to proteins central to myelin biogenesis, maintenance and activity; and relate these concepts to a better understanding of de/remyelination in multiple sclerosis and other demyelinating diseases. Myelin Protein Identity: In Aim I we further develop the Myelin Proteome, applying new approaches for identification of unresolved proteins. This study is providing an invaluable myelin protein database for the myelin biology community at large, forming the basis for numerous projects. Myelin Protein Function: In Aim II we investigate OL differentiation- and physiology-mediated changes in protein expression using Direct In-Gel Electrophoresis (DIGE) and Isotope-Coded Affinity Tag (ICAT) mass spectrometric methodologies, and manipulation of protein expression and function using antibody blocking, RNAi technology and genetically modified mouse mutants. Myelin Protein Disease: In Aim III, we study mechanisms by which cell signaling pathways become activated by environmental factors in normal OL physiology and in myelin biology disease. This focuses on proteomic analyses of antibody perturbations that mimic natural ligands and neuroimmunological disease conditions, including the roles of Myelin Oligodendrocyte Glycoprotein (MOG), galactosphingolipids, and glycosphingolipid-cholesterol microdomains in the initiation of signal transaction.