ADHESION OF MYELIN PO PROTEIN

髓磷脂PO蛋白的粘附

• 批准号: 2839322
• 负责人: Marie T. Filbin
• 金额: $ 24.22万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-19 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2839322
• 关键词: CHO cells; adhesions; cytoskeleton; gene mutation; genetically modified animals; intermolecular interaction; laboratory mouse; molecular cloning; nerve /myelin protein; posttranslational modifications; protein structure function; transfection

DESCRIPTION: (Adapted from Applicant's Abstract): Axons are insulated and are allowed to function efficiently by the multi-lamellar membrane, myelin. Peripheral nervous system myelin is held compact by P0, a protein that contain a single immunoglobulin (Ig)-domain. P0 holds myelin together at both the extracellular (via homophilic interactions) and at the cytoplasmic (putatively via interactions with lipids) membrane leaflets. Furthermore, by measuring the adhesion of P0 in transfected cells, a dynamic relationship between the interaction of the extracellular and cytoplasmic domain of P0 was demonstrated; the cytoplasmic domain must be intact, initially, interacting with the cytoskeleton, for adhesion of the extracellular sequences to take place. Similarly, P0 mutated in either the extracellular or the cytoplasmic domain of P0 can exert a dominant negative effect on adhesion, implying a cis interaction, within the membrane. Loss of any of these interactions of P0 could result in a loss of function of the molecule. Recently, mutations and deletions of P0 have been associated with the peripheral neuropathy. Charcot-Marie-Tooth (CMT),1B. The longterm objectives of this application are to characterize the interactions of P0 as a prototype for all Ig-members and to determine how mutations in P0 bring about CMT 1B. In this proposal, the cytoskeletal interactions will be investigated by identifying and characterizing P0-associated proteins, which may be a link to the cytoskleleton. Also, using a transfection/adhesion strategy, how post-translational modifications of P0 s cytoplasmic domain affect adhesion and cytoskeletal interactions will be addressed. The ability of P0 to cluster, an indication cis interactions, will be determined by cross-linking surface P0. In addition, monoclonal antibodies to a soluble, extracellular domain of P0, in its native form, will be raised and used to distinguish mutations that disrupt conformation from those that affect binding directly. Finally, with this information, how the mutations in P0 associated with CMT1B, affect its function will be addressed both in transfected cells and in transgenic mice. The various CMT1B-mutated P0 protein will be expressed in cells by transfection. Surface expression, clustering, antibody binding and adhesion will be assessed. Then the mutated proteins will be co-expressed with wild type P0 to determine if they exert a dominant negative effect. To complement these studies, transgenic mice, initially, expressing a tagged P0 will be examined both morphologically and biochemically. Eventually, mice co-expressing wildtype and mutant P0 will be produced and examined for a dysmyelination. In this way, how the various mutations in P0 bring about the disease phenotype in CMT1B can be addressed directly.

描述：(改编自申请者摘要)：轴突是绝缘的， 被多层膜髓磷脂有效地发挥作用。 外周神经系统髓磷脂由P0保持致密，P0是一种 含有单一的免疫球蛋白(Ig)结构域。P0将髓鞘结合在一起 胞外(通过亲和性相互作用)和细胞质 (可能是通过与脂质的相互作用)膜小叶。此外， 通过测量P0在转基因细胞中的粘附性，建立了一个动态关系 P0胞外区和胞内区的相互作用 细胞质区域必须是完整的，最初， 与细胞骨架相互作用，促进细胞外的黏附 要发生的序列。同样，P0在细胞外的两种情况下都发生突变 或者，P0的细胞质结构域可以对 粘附性，暗示顺式相互作用，在膜内。任何一项损失 P0的这些相互作用可能导致分子功能的丧失。 最近，P0的突变和缺失被认为与 周围神经病。Charcot-Marie-Tooth(CMT)，1B. 这个应用程序的长期目标是描述 P0的相互作用作为所有Ig-成员的原型并确定如何 P0基因突变导致CMT-1B。在这项提案中，细胞骨架 交互作用将通过识别和表征 P0相关蛋白，这可能与细胞骨架有关。另外， 使用转染法/粘附法，翻译后修饰 P0 S胞浆结构域影响黏附和细胞骨架相互作用 将会得到解决。P0的集群能力，这是一个指示顺式 相互作用，将由交联面P0确定。此外, 抗P0可溶胞外区的单抗在其 原生形式，将被提升并用于区分破坏的突变 构象不同于那些直接影响结合的构象。最后，有了这个 信息，与CMT1B相关的P0突变如何影响其 功能将在转基因细胞和转基因小鼠中得到解决。 CMT1B突变的各种P0蛋白将通过 转染法。表面表达、聚集、抗体结合和黏附 将会被评估。然后，突变的蛋白质将与野生的 键入P0以确定它们是否产生显性负面影响。至 补充这些研究，转基因小鼠，最初，表达标记的P0 将从形态和生化两方面进行检查。最终，老鼠 将产生共表达野生型和突变体P0并对其进行检测 髓鞘障碍。通过这种方式，P0的各种突变是如何导致 CMT1B中的疾病表型可以直接解决。