Improving cell membrane repair to treat muscular dystrophy

项目摘要 Duchenne肌营养不良症(DMD)是一种致命的退行性肌肉疾病，由X-基因突变引起。 连锁的抗肌营养不良基因。DMD是最常见的肌营养不良症，发病率为1：5000 活的男婴。肌营养不良蛋白突变导致蛋白表达丧失和肌膜受损 膜的完整性。机械应激造成的损伤通常会导致肌肉纤维死亡，最终 压倒肌肉的再生能力。反复损伤严重减少骨骼肌 在生命的第二到第四个十年，力量会导致早逝。当前的治疗选项可以 延长生存时间和提高生活质量；然而，它们并不能治愈。因此，仍然需要 针对疾病病理基础的分子机制的新疗法。以前的研究 确定三体基序蛋白72/Mitsugumin 53(TRIM72/MG53)是正常细胞所必需的 骨骼肌和心肌的膜修复通过其与磷脂酰丝氨酸(PS)的结合。MG53的损失 功能与肌营养不良的发展和增加的易感性 心脏损伤。MG53是本实验室发现的一种富含肌肉的TRIM家族E3泛素连接酶蛋白 以前报道的可以通过过度表达或外源应用 重组MG53(RhMG53)改善多种肌营养不良模型的疾病病理。这 联谊会项目的目的是询问rh MG53蛋白结构域在这种观察到的膜效应中的作用。 修理。我的初步数据表明，E3泛素连接酶活性不是增强的 膜修复表型。这一观察结果支持了经典修剪E3泛素的假设 MG53的连接酶活性不是治疗膜修复能力所必需的，因此治疗效果 可能是通过可以结合PS的MG53蛋白的紧凑型版本实现的。在这份奖学金申请中，我 建议使用三个具体和独立的目标来检验这一假设。AIM 1筛选重组人MG53突变体 蛋白质构建以确定对膜修复至关重要的rhMG53蛋白结构域。目标2定义 磷脂酰丝氨酸通过识别蛋白结构域结合伙伴与重组人MG53结合的机制。 目的3评估已鉴定的重组人MG53突变蛋白构建物在外源时的治疗潜力 体外输送到完整的肌肉纤维中。这些研究将通过以下方式提供出色的培训体验 阐明以前未表征的MG53蛋白相互作用将使新的 蛋白质疗法治疗DMD。这些试验性的努力将伴随着额外的技能发展。 提供综合培训体验的活动，使我的职业生涯得以继续发展 神经肌肉疾病领域的独立研究员。

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.