Improving cell membrane repair to treat muscular dystrophy

项目摘要 杜氏肌营养不良症（DMD）是一种致命的退行性肌肉疾病，由X- 连锁肌营养不良蛋白基因。DMD是最常见的肌营养不良症，发病率为1：5，000 活产男婴。肌营养不良蛋白突变导致蛋白质表达的丧失和受损的肌膜 膜完整性机械应力损伤通常会导致肌纤维死亡， 增强肌肉的再生能力。反复的损伤严重地减少了骨骼肌 在生命的第二到第四个十年中导致过早死亡的力量。目前的治疗方案可以 延长生存期和改善生活质量;然而，它们不是治愈性的。因此，仍然需要 靶向疾病病理学基础的分子机制的新疗法。以前的研究 确定了三重基序蛋白72/Mitsugumin 53（TRIM 72/MG 53）是正常细胞生长所必需的。 通过与磷脂酰丝氨酸（PS）结合，修复骨骼肌和心肌的膜。MG 53的损失 功能与肌营养不良症的发展和对 心脏损伤MG 53是一种富含肌肉的TRIM家族E3泛素连接酶蛋白，我们实验室在2004年发现了MG 53。 以前报道过的可以通过过表达或外源性应用 重组MG 53（rhMG 53）用于改善多种肌营养不良模型中的疾病病理。这 一个奖学金项目的目的是询问rhMG 53蛋白结构域在这种观察到的膜效应中的作用 修复.我的初步数据表明，E3泛素连接酶的活性不是增强的细胞凋亡所必需的。 膜修复表型这一观察结果支持了经典的TRIM E3泛素 MG 53的连接酶活性不是治疗性膜修复能力所必需的，因此治疗效果 可以通过可以结合PS的MG 53蛋白的紧凑版本来实现。在这份奖学金申请中， 我建议用三个具体而独立的目标来检验这一假设。目的1筛选rhMG 53突变体 蛋白构建体，以鉴定膜修复所必需的rhMG 53蛋白结构域。目标2定义 磷脂酰丝氨酸结合rhMG 53的机制作用，通过识别蛋白质结构域结合伴侣。 目的3：评价经鉴定的rhMG 53突变蛋白构建体在外源性 递送至离体的完整肌纤维。这些研究将提供一个很好的培训经验， 阐明了以前未表征的MG 53蛋白相互作用，这将使得能够开发新的 DMD的蛋白质疗法。这些实验性的努力将与额外的技能发展相结合 活动，以产生一个综合的培训经验，让我朝着职业生涯的持续发展， 神经肌肉疾病领域的独立研究者

Project Summary Duchenne Muscular Dystrophy (DMD) is a fatal degenerative muscle disease caused by mutations in the X- linked dystrophin gene. DMD is the most prevalent form of muscular dystrophy with an incidence of 1:5,000 live male births. Dystrophin mutations lead to the loss of protein expression and compromised sarcolemmal membrane integrity. Injury from mechanical stress often leads to muscle fiber death that eventually overwhelms the regenerative capacity of the muscle. Repeated injury severely reduces skeletal muscle strength contributing to early death by the second to fourth decade of life. Current treatment options can prolong survival and improve quality of life; however, they are not curative. Therefore, there remains a need for novel therapies that target the molecular mechanisms underlying disease pathology. Previous studies determined that the tripartite motif protein 72/mitsugumin 53 (TRIM72/MG53) is essential for proper cell membrane repair in skeletal and cardiac muscle through its binding of phosphatidylserine (PS). Loss of MG53 function has been associated with the development of muscular dystrophy and increased susceptibility to cardiac injury. MG53 is a muscle-enriched TRIM family E3 ubiquitin ligase protein that our laboratory previously reported can increase membrane repair by overexpression or by exogenous application of recombinant MG53 (rhMG53) to ameliorate disease pathology in multiple models of muscular dystrophy. This fellowship project aims to interrogate the role of rhMG53 protein domains in this observed effect on membrane repair. My preliminary data suggest that the E3 ubiquitin ligase activity is not required for the enhanced membrane repair phenotype. This observation supports the hypothesis that the canonical TRIM E3 ubiquitin ligase activity of MG53 is not required for therapeutic membrane repair capacity, therefore therapeutic effects may be achieved by compact versions of the MG53 protein that can bind PS. In this fellowship application I propose to test this hypothesis using three specific and independent aims. Aim 1 screens rhMG53 mutant protein constructs to identify rhMG53 protein domains that are essential for membrane repair. Aim 2 defines the mechanistic role of phosphatidylserine binding to rhMG53 by identifying protein domain binding partners. Aim 3 evaluates the therapeutic potential of identified rhMG53 mutant protein constructs when exogenously delivered to intact muscle fibers ex vivo. These studies will provide an excellent training experience by elucidating previously uncharacterized MG53 protein interactions that will enable the development of novel protein therapeutics for DMD. These experimental efforts will be coupled with additional skill development activities to produce an integrated training experience to allow my continued development toward a career as an independent investigator in the neuromuscular disease field.