PROTEOMIC MAPPING OF MYELIN AND ITS MEMBRANE SUBDOMAINS

描述（由申请人提供）：髓磷脂是一种动态的、功能活跃的膜，与轴突进行双向通信，接收和处理来自环境的信号。我们提出理解髓磷脂活性生物学功能的关键是鉴定和表征髓磷脂蛋白的完整曲目，即髓磷脂蛋白质组。我们对髓磷脂进行了广泛的蛋白质组学分析，开发了髓磷脂蛋白的2D-PAGE图谱，迄今为止鉴定了125种这些蛋白质，并通过比较蛋白质组学的特定应用说明了这种方法的力量和实用性。这一蛋白质组学图谱和相关的概念方法为研究髓磷脂膜的多种生物学作用提供了重大进展，并为其他神经系统的类似研究提供了范例。该项目的长期目标是鉴定和表征髓磷脂膜的分子成分；开发这些分子在髓鞘膜中的排列模型，特别关注鞘糖脂-胆固醇微域的作用；分配与髓磷脂生物发生、维持和活性相关的蛋白质的功能；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的脱髓鞘形成联系起来。髓磷脂蛋白鉴定：在Aim I中，我们进一步开发髓磷脂蛋白质组，应用新方法鉴定未解析蛋白。这项研究为髓磷脂生物学社区提供了一个宝贵的髓磷脂蛋白质数据库，形成了许多项目的基础。髓磷脂蛋白功能：在Aim II中，我们使用直接凝胶电泳（DIGE）和同位素编码亲和标签（ICAT）质谱方法研究了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来操纵蛋白质表达和功能。髓磷脂蛋白疾病：在Aim III中，我们研究了正常OL生理和髓磷脂生物学疾病中细胞信号通路被环境因素激活的机制。本研究的重点是模拟天然配体和神经免疫疾病状况的抗体扰动的蛋白质组学分析，包括髓鞘少突胶质糖蛋白（MOG）、半乳糖鞘脂和鞘脂-胆固醇微结构域在信号交易起始中的作用。

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.