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学的一个重要方面，该过程的破坏可导致许多人类疾病（包括肌营养不良症）的病理生理学。我们最近发现，MG 53，一种肌肉特异性TRIM家族蛋白，是膜修复机制的重要组成部分。虽然我们公布的数据定义了MG 53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MG 53可以区分完整和受损的膜，因此膜界定的信号可能参与MG 53与受损部位的束缚。在初步研究中，我们发现PTRF是MG 53介导的膜修复反应成核的强制性因子，因为缺乏PTRF内源性表达的细胞显示有缺陷的膜重新密封。虽然PTRF的RNAi沉默导致肌纤维中有缺陷的膜修复，但PTRF的过表达可以挽救dysferlin-/-肌肉中的这种缺陷，但不能挽救mg 53-/-肌肉中的这种缺陷，这表明PTRF在膜修复中的功能作用可能需要MG 53的存在。虽然许多研究已经探索了PTRF在调节质膜小窝结构中的功能，但我们的数据提出了PTRF作为MG 53的锚定分子用于启动细胞膜修复反应的新生物学功能。由于PTRF中的突变已经在具有脂肪营养不良和肌营养不良的人类病症中被鉴定，因此通常涉及受损的膜完整性或重新密封能力、靶向MG 53和PTRF之间的功能相互作用或恢复患病状态中被破坏的MG 53-PTRF相互作用的病症可能代表用于治疗或预防涉及受损的膜修复的退行性疾病的有吸引力的途径。该项目的长期目标是了解肌肉生理学和疾病中膜修复的细胞和分子机制。具体而言，我们将重点测试的假设，即“PTRF作为对接蛋白MG 53介导的细胞膜修复，并恢复膜完整性的肌营养不良症可以通过增强MG 53/PTRF功能的界面膜损伤”。我们提出的研究将集中于确定MG 53和PTRF之间的功能相互作用的分子机制，以启动骨骼肌中的细胞膜修复反应（目的1）;探索MG 53和PTRF在肌肉生理学和疾病中的生理作用，并测试PTRF-MG 53功能的增强是否可以改善肌营养不良症中的膜完整性（目的2）。通过MG 53和PTRF的定制表达以及生物化学标记物的使用、活细胞成像、离体和体内动物模型研究，所设计的实验将为靶向MG 53/PTRF介导的细胞膜修复治疗肌营养不良症提供关键的原理验证数据。

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.