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

 描述（由申请人提供）：星云蛋白是已知最大的蛋白质之一，是一种丝状蛋白质，跨越几乎一半的肌节长度，肌节是肌肉的收缩单位。这种蛋白质的突变会导致一种称为线状体肌病的肌肉疾病，患者无法维持正常的肌肉质量和收缩功能，导致活动和呼吸困难。在敲除小鼠模型中的研究加上大的患者队列已经导致观察到，在Z盘内相互作用的星云蛋白的C-末端结构域，个体肌节之间的连接点，可能显著有助于健康肌节中星云蛋白的正常功能，以及当这些结构域丢失时线状体肌病的发作。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健康肌节的形成。这项提议将检验这样的假设，即星云蛋白C末端的缺失足以导致肌肉的结构变化，随后的力量损失，以及肌肉无法发育和肥大。我们将研究一种新的小鼠模型，我们已经没有了星云蛋白的C-末端结构域。目的1是确定在缺乏星云蛋白C末端的模型中肌肉结构和功能发生的变化。在整个发育过程中记录小鼠体重，并比较野生型小鼠和星云蛋白截短纯合子小鼠之间的骨骼肌重量。将通过透射电子显微镜和免疫荧光观察结构变化，而功能变化将通过使用整个肌肉力学进行量化。初步研究显示，具有截短的星云蛋白的小鼠的总体体重不足以及某些骨骼肌重量的变化。目的二是研究nebulin蛋白质C端蛋白质相互作用的变化。肌动蛋白结合蛋白CapZ，以前报道与星云蛋白和肌动蛋白细丝长度的已知调节剂相互作用，将进行分析的损失，导致破坏细丝长度的适当定位。一个建议之间的相互作用星云蛋白的C-末端和分支肌动蛋白组装蛋白神经元Wiskott-Aldrich综合征蛋白（N-WASP），最近发现诱导肌肉肥大也将通过激活这种机制与诱导生长因子-1（IGF-1）进行研究。这一目标的结果将更好地阐明nebulin的蛋白质功能，在先前的敲除模型中看到的肌节变化，以及为什么具有改变的nebulin的肌肉不能经历肥大。目的3是评估替代肌肉肥大途径的贡献。虽然IGF-1治疗可能表明无法进行肥大，但这一目标将显示具有截短的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，这两者都通过可以避免在星云蛋白C-末端失去调节相互作用的途径起作用。本研究的发现将有助于阐明星云蛋白在肌肉肌节和生长调节中的功能。

 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.