ADHESION OF MYELIN PO PROTEIN

髓磷脂PO蛋白的粘附

• 批准号: 2717298
• 负责人: Marie T. Filbin
• 金额: $ 27.52万
• 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/2717298
• 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：（改编自申请人的摘要）：轴突是绝缘的， 是由多层膜，髓鞘，有效地发挥作用。 周围神经系统髓鞘由P0紧密保持，P0是一种蛋白质， 含有单个免疫球蛋白（IG）结构域。 P0将髓磷脂结合在一起， 细胞外（通过嗜同性相互作用）和细胞质 （通过与脂质的相互作用产生的脓毒症）膜小叶。 此外，委员会认为， 通过测定转染细胞中P0的粘附， P0的细胞外和细胞质结构域之间的相互作用 被证明;细胞质结构域必须是完整的，最初， 与细胞骨架相互作用，用于细胞外 序列发生。 类似地，P0在细胞外 或者P0的胞质结构域可以对 粘附，意味着膜内的顺式相互作用。 任何损失 P0的这些相互作用可能导致分子功能的丧失。 最近，P0的突变和缺失已经与肿瘤的发生有关。 周围神经病变 Charcot-Marie-Tooth（CMT），1B. 本申请的长期目标是表征 P0作为所有Ig成员的原型的相互作用，并确定如何 P0突变导致CMT 1B。 在这一建议中， 将通过识别和表征来研究相互作用 P0相关蛋白，可能与细胞骨架有关。 还有， 使用转染/粘附策略，如何翻译后修饰 影响粘附和细胞骨架相互作用 将得到解决。 P0聚集的能力，一个指示， 相互作用，将由交联表面P0确定。 此外，本发明还提供了一种方法， 单克隆抗体的可溶性，细胞外结构域的P0，在其 天然形式，将提高和用于区分突变，破坏 与那些直接影响结合的构象不同。 最后， 信息，与CMT 1B相关的P0突变如何影响其 将在转染的细胞和转基因小鼠中解决功能。 各种CMT 1B突变的P0蛋白将通过以下方式在细胞中表达： 转染 表面表达、聚集、抗体结合和粘附 将被评估。 然后突变的蛋白质将与野生型共表达， 类型P0，以确定它们是否产生显性负效应。 到 作为对这些研究的补充，转基因小鼠最初表达标记的P0 将从形态学和生化学两方面进行检查。 最终，老鼠 将产生共表达野生型和突变体P0，并检查其是否存在。 髓鞘形成障碍 这样，P0的各种突变如何导致 CMT 1B中的疾病表型可以直接解决。