The Role of Nebulin's C-terminus in Sarcomeric Structure and Function

 描述(申请人提供)：星云蛋白是已知最大的蛋白质之一，是一种丝状蛋白质，几乎横跨肌肉收缩单位肌节的一半长度。这种蛋白的突变可能会导致一种名为线虫肌病的肌肉疾病，患者无法维持正常的肌肉质量和收缩功能，导致行动和呼吸困难。对基因敲除小鼠模型和大量患者队列的研究发现，在Z盘内相互作用的星云蛋白的C-末端结构域，即单个肌瘤之间的连接连接，可能对健康肌瘤中星云蛋白的正常功能以及当这些区域丢失时线状肌病的发病起到重要作用。该项目的目标是确定星云蛋白这一区域的功能，以及它如何有助于形成健康的肌节。这项提议将检验这样一种假设，即失去星云蛋白的C末端足以导致肌肉结构的变化，随后失去力量，使肌肉无法发展和经历肥大。我们将研究一种新的小鼠模型，该模型缺少星云蛋白的C-末端结构域。目的1是确定在这种缺乏星云蛋白C末端的模型中，肌肉结构和功能发生的变化。小鼠的体重将在整个发育过程中被记录下来，骨骼肌重量将在野生型小鼠和纯合子小鼠之间进行比较，以进行星云蛋白截断。结构变化将通过透射电子显微镜和免疫荧光观察，而功能变化将通过使用整体肌肉力学进行量化。初步研究显示，截短星云蛋白的小鼠总体体重不足，某些骨骼肌的重量也发生了变化。目的2是研究蛋白质与星云蛋白C末端相互作用的变化。肌动蛋白结合蛋白CAPZ之前被报道与星云蛋白和肌动蛋白细丝长度的已知调节因子相互作用，将被分析导致细丝长度中断的适当定位的丢失。最近发现的诱导肌肉肥大的分支肌动蛋白组装蛋白神经元Wiskott-Aldrich综合征蛋白(N-WASP)与星云蛋白C末端之间的相互作用也将通过诱导生长因子-1(IGF-1)激活这一机制来研究。这一目标的结果将更好地阐明星云蛋白的蛋白质功能，在以前的基因敲除模型中看到的肌小节变化，以及为什么星云蛋白改变的肌肉不能肥大。目的3是评估交替的肌肉肥大途径的贡献。虽然IGF-1治疗可能表明肌肉无法经历肥大，但这一目标将表明截短星云蛋白的肌肉是否能够 通过超负荷肥大和治疗性运动进行生长，这两种途径都可以绕过星云蛋白C末端调控相互作用的丧失。这项研究的发现将有助于阐明雾化蛋白在肌肉肌节和生长调节中的作用。

 DESCRIPTION (provided by applicant): Nebulin, one of the largest known proteins, is a filamentous protein that spans almost half the length of the sarcomere, the contractile units of the muscle. Mutations in this protein can lead to a muscular disorder called nemaline myopathy, wherein patients cannot maintain normal muscle mass and contractile function, leading to difficulties in mobility and respiration. Studies in knockout mouse models coupled with large patient cohorts have led to the observation that the C-terminal domains of nebulin interacting within the Z-discs, the connective junctions between individual sarcomeres, may contribute significantly to both nebulin's normal function in healthy sarcomeres, as well as the onset of nemaline myopathy when these domains are lost. The goal of this project is to define the function of this region of nebulin and how it contributes to the formation of healthy sarcomeres. This proposal will test the hypothesis that loss of nebulin's C-terminus is sufficient for structurl changes in muscle, subsequent loss of force, and an inability for muscle to develop and undergo hypertrophy. We will study a novel mouse model that we have made which lacks the C-terminal domains of nebulin. Aim 1 is to determine the changes that occur in muscle structure and function in this model that lacks the C- terminus of nebulin. Mouse weights will be recorded throughout development and skeletal muscle weights will be compared between wild-type mice and mice homozygous for the nebulin truncation. Structural changes will be observed through transmission electron microscopy and immunofluorescence while functional changes will be quantified through the use of whole muscle mechanics. Preliminary studies have revealed an overall weight deficit in mice with truncated nebulin as well as changes in the weights of certain skeletal muscles. Aim 2 is to investigate changes protein-protein interactions with nebulin's C-terminus. The actin-binding protein CapZ, previously reported to interact with nebulin and a known regulator of actin thin filament length, will be analyzed for loss of proper localization leading to disruption of thin filament lengths. A proposed interaction between nebulin's C-terminus and the branched actin assembly protein neuronal Wiskott-Aldrich Syndrome protein (N-WASP), recently found to induce muscle hypertrophy will also be studied through the activation of this mechanism with induced growth factor-1 (IGF-1). The results of this aim will better clarify nebulin's protein function, the sarcomeric changes seen in prior knockout models, and why muscles with altered nebulin cannot undergo hypertrophy. Aim 3 is to assess the contributions of alternate muscle hypertrophy pathways. While the IGF-1 treatment may indicate an inability to undergo hypertrophy, this aim will show if muscle with truncated nebulin is able to undergo growth through both overload hypertrophy and therapeutic exercise, both of which act through pathways that could circumvent the loss of regulatory interactions in nebulin's C-terminus. The findings of this study will help elucidate nebulin's function in muscle sarcomeres and growth regulation.