LGMD 2A Protein Calpain 3 and Its Binding to Titin

描述(申请人提供)：Calain 3(C3)是钙依赖蛋白水解酶家族中肌肉特有的成员，突变导致一种形式的肌营养不良症，称为肢体带状肌营养不良症2A型(LGMD2A型)。在这笔赠款的前一个资助期，我们在了解C3的生物学功能方面取得了良好的进展。我们发现，在体外和体内，C3对于合适的肌节结构都是重要的。根据我们的发现，我们假设在正常发育和肌肉适应过程中，C3与泛素/蛋白酶体系统一起取代和移除肌原纤维蛋白，并允许随后被新的蛋白取代。一旦肌节完全组装起来，它们就形成了一个致密致密的实体，以至于它们的亚单位受到保护，不受蛋白质降解作用的影响。C3‘S定位于肌节，由于其与肌动蛋白的相互作用，使其能够接触到肌原纤维蛋白，这些蛋白在损伤或重塑后被翻转。我们进一步表明，致病的LGMD2A突变不影响C3S的蛋白降解能力可以影响其结合Titin的能力，而Titin是C3的重要锚。最后，我们已经证明，在C3KO肌肉中存在普遍的蛋白质周转损失，导致蛋白质聚集和细胞应激反应。上述观察已阐明了C3的生物学功能，但仍有未解的问题。C3‘S正常活动丧失如何导致肌肉疾病？肌动蛋白失去锚定如何影响C3‘S的正常细胞作用？在我们的初步数据中，我们发现C3有两种主要的细胞底物，包括肌原纤维蛋白和线粒体蛋白。这一发现表明，除了在肌原纤维蛋白周转中的作用外，C3可能在线粒体蛋白周转中起重要作用。这一假设得到了我们初步数据的支持，这些数据显示C3基因敲除小鼠的线粒体形态和氧化应激异常。在这一资助阶段，我们将研究以下目标：目标1：研究calain 3-titin相互作用中断对calain 3活性和底物可及性的生化影响。目的2：研究钙蛋白酶3-titin相互作用中断对肌肉形态和功能的生物学影响。目的：研究Calain 3 KO肌肉线粒体缺陷和氧化应激的特点。

DESCRIPTION (provided by applicant): Calpain 3 (C3) is the muscle specific member of the calcium dependent protease family, mutations in which result in a form of muscular dystrophy called limb girdle muscular dystrophy type 2A (LGMD2A). In the previous funding period for this grant, we made good progress in understanding the biological function of C3. We showed that C3 is important for proper sarcomere structure, in vitro and in vivo. Based on our findings, we hypothesize that during normal development and muscle adaptation, C3, in conjunction with the ubiquitin/proteasome-system, displaces and removes myofibrillar proteins, and allows for the subsequent replacement by new proteins. Once sarcomeres are fully assembled they form such a dense compact entity that their subunits are protected from proteolytic action. C3's placement in the sarcomere, due to its interaction with titin, allows it accessibility to myofibrillar proteins that are to be turned over following damage or remodeling. We further showed that pathogenic LGMD2A mutations that do not affect C3s proteolytic capabilities can affect its ability to bind titin, and that titin serves as an important anchor for C3. Finally, we have shown that there is a generalized loss of protein turnover in C3KO muscles, leading to protein aggregates and a cellular stress response. The above observations have elucidated the biological function of C3 but have left unanswered questions. How does loss of C3's normal activity result in muscle disease? How does loss of anchorage to titin affect C3's normal cellular role? In our preliminary data, we show that C3 has cellular substrates that fall into two major categories that include myofibrillar proteins and mitochondrial proteins. This finding suggests that in addition to its role in myofibrillar protein turnover, C3 might be important in mitochondrial protein turnover. This hypothesis is supported by our preliminary data showing abnormal mitochondrial morphology and oxidative stress in C3 knock out mice. In this funding period, we will investigate the following aims: Aim 1: To characterize the biochemical consequences of disrupted calpain 3-titin interactions on calpain 3 activity and substrate accessibility. Aim 2: To characterize the biological consequences of disrupted calpain 3-titin interactions on muscle morphology and function. Aim 3: To characterize the mitochondrial defect and features of oxidative stress in calpain 3 KO muscles.