MG53-Mediated Membrane Repair in Muscle Physiology and Disease

描述（由申请人提供）：质膜急性损伤的修复是正常细胞生理的一个重要方面，这一过程的破坏可导致许多人类疾病的病理生理，包括肌肉萎缩症。我们最近发现MG53，一种肌肉特异性trimm家族蛋白，是膜修复机制的重要组成部分。虽然我们发表的数据定义了MG53在细胞膜修复中的传感器功能，但成核过程的分子机制仍有待确定。由于MG53可以区分完整和受损的膜，因此膜分隔的信号可能参与了MG53与受损部位的捆绑。在初步研究中，我们发现PTRF是mg53介导的膜修复反应成核的必要因子，因为缺乏内源性PTRF表达的细胞会出现膜重封缺陷。RNAi沉默PTRF会导致肌纤维的膜修复缺陷，而过表达PTRF可以修复dysferlin-/-肌中的这种缺陷，而在mg53-/-肌中则不能，这表明PTRF在膜修复中的功能作用可能需要mg53的存在。虽然许多研究已经探索了PTRF在调节质膜小泡结构中的功能，但我们的数据显示了PTRF作为MG53启动细胞膜修复反应的锚定分子的新生物学功能。由于PTRF突变已在脂肪营养不良和肌肉营养不良的人类疾病中被发现，通常涉及膜完整性受损或重封能力受损的疾病，以MG53和PTRF之间的功能相互作用为目标，或在患病状态下恢复被破坏的MG53-PTRF相互作用，可能是治疗或预防涉及膜修复受损的退行性疾病的有吸引力的途径。本项目的长期目标是了解肌肉生理和疾病中膜修复的细胞和分子机制。具体而言，我们将重点验证“PTRF作为MG53介导的细胞膜修复的对接蛋白，通过增强MG53/PTRF在膜损伤界面的功能，可以实现肌萎缩症中膜完整性的恢复”的假设。我们提出的研究将侧重于确定MG53和PTRF之间功能相互作用的分子机制，以启动骨骼肌的细胞膜修复反应（Aim 1）；探索MG53和PTRF在肌肉生理和疾病中的生理作用，并测试增强PTRF-MG53功能是否可以改善肌营养不良患者的膜完整性（Aim 2）。通过MG53和PTRF的特异性表达，利用生化标志物、活细胞成像、离体和体内动物模型研究，设计的实验将为靶向MG53/PTRF介导的细胞膜修复治疗肌肉萎缩症提供关键的原理证明数据。

DESCRIPTION (provided by applicant): Repair of acute damage to the plasma membrane is an important aspect of normal cellular physiology and disruption of this process can result in pathophysiology in a number of human diseases including muscular dystrophy. We recently discovered that MG53, a muscle-specific TRIM-family protein, is an essential component of the membrane repair machinery. While our published data define the sensor function for MG53 in cell membrane repair, the molecular mechanisms underlying the nucleation process remain to be defined. Since MG53 can discriminate between intact and injured membrane, a membrane-delimited signal would likely be involved in tethering of MG53 to the injured site. In pilot studie, we found that PTRF is an obligatory factor for MG53-mediated nucleation of the membrane repair response, for cells lacking endogenous expression of PTRF show defective membrane resealing. While RNAi- silencing of PTRF leads to defective membrane repair in muscle fibers, overexpression of PTRF can rescue this defect in dysferlin-/- muscle but not in mg53-/- muscle, suggesting that the functional role of PTRF in membrane repair likely requires the presence of MG53. While many studies have explored the function of PTRF in regulating caveolae structure of the plasma membrane, our data present a new biological function for PTRF as an anchoring molecule for MG53 for initiation of the cell membrane repair response. Since mutations in PTRF have been identified in human disorders with lipodystrophy and muscular dystrophy, conditions that often involve compromised membrane integrity or resealing capacity, targeting the functional interaction between MG53 and PTRF, or restoration of the disrupted MG53-PTRF interaction in the diseased states, may represent an attractive avenue for treatment or prevention of degenerative diseases involving compromised membrane repair. The long-term goal of this project is to understand the cellular and molecular mechanism for membrane repair in muscle physiology and diseases. Specifically, we will focus on testing the hypothesis that "PTRF acts as a docking protein for MG53-mediated cell membrane repair, and restoration of membrane integrity in muscular dystrophy can be achieved through enhancement of MG53/PTRF function at the interface of membrane injury". Our proposed studies will focus on defining the molecular mechanism underlying the functional interaction between MG53 and PTRF for initiation of the cell membrane repair response in skeletal muscle (Aim 1); and exploring the physiological role of MG53 and PTRF in muscle physiology and diseases and test if enhancement of PTRF-MG53 function can improve membrane integrity in muscular dystrophy (Aim 2). Through tailored-expression of MG53 and PTRF and the use of biochemical markers, live cell imaging, ex vivo and in vivo animal model studies, the designed experiments will provide key proof-of-principle data for targeting MG53/PTRF-mediated cell membrane repair in treatment of muscular dystrophy.