Roles of Nebulin in Structure and Function of Striated Muscle

摘要 这项建议关注的是星云蛋白的结构和功能，星云蛋白是一种异常大的肌节蛋白，它是 在骨骼肌中表达。星云的巨大尺寸使人们很难阐明它的功能，但它的 许多导致线状肌病(NEM2)的星云蛋白突变支持了这一重要性，其中 常见的非营养不良先天性肌病。星云蛋白的蛋白质结构由大量的 与肌动蛋白结合的简单重复序列，其中大部分被组织成超重复序列(SR)。实现以下目标的方法 治疗NEM2严重缺乏，并对雾化蛋白在肌肉中的许多作用有了深入的了解 结构和功能是必不可少的。我们将在我们取得的重大进展的基础上，全面研究星云 还有一些人在最近几年取得了进展。为了帮助实现我们的目标，我们使用了鼠标模型，其中一些 模仿严重的NEM2和其他较温和的NEM2，作为我们基础科学和翻译研究的强大工具。我们 将利用多学科研究，从单分子到完整肌肉水平研究星云蛋白的功能 涉及转录组学、蛋白质组学、超分辨率成像、低角X射线衍射以及 生物力学。目标1侧重于细丝长度的调节。我们最近的研究在慢肌中支持了这一点， 星云蛋白与Leiomodin-2(Lmod2)协同作用，调节近端细丝的长度 片段和Lmod2调节不含云雾蛋白的末端片段的长度。在这里，我们将严格测试 这种双重长度调节模型，并研究它是否具有翻译潜力，通过确定是否 上调Lmod2是治疗以雾化蛋白为基础的线状肌病的有效方法。Aim 2研究了 与肌动蛋白结合较弱的中心定位星云蛋白SRs与肌动蛋白弱结合的功能意义 那些靠近分子末端的分子。这可能在功能上很重要，考虑到 增加中枢SR的结合亲和力会导致全世界患者的骨骼肌病。我们 我将研究中心位置弱结合的SRS转化为强结合的小鼠模型 用机械分析和X射线衍射法对完整的肌肉进行SRS。目的3研究星云蛋白的C末端， 它在Z盘中的布局，以及星云蛋白差异表达的Z-重复序列的功能。许多NEM2患者 有导致大部分C末端(位于Z盘内)丢失的截断突变，但很少 研究人员已经对该分子的这一区域进行了研究。星云蛋白的C-末端包含Z-盘重复序列 选择性剪接(在有宽Z盘的肌肉中高水平表达)。我们将建立 星云蛋白在Z-盘中的位置，Z-重复序列，确定蛋白质结合伙伴，并研究其影响 删除Z盘结构和功能上差异表达的星云蛋白Z-重复序列。利用我们的 &gt；15年创新星云研究的跟踪记录，并利用我们经验丰富的科学家和 这项建议设定了雄心勃勃的目标，预计将加快对 云雾蛋白的生物学，其在疾病中的作用，并确定可能的新的治疗途径。

Summary This proposal focuses on the structure and function of nebulin, an unusually large sarcomeric protein that is expressed in skeletal muscle. The giant size of nebulin has made it challenging to elucidate its functions but its importance is supported by the many nebulin mutations that cause nemaline myopathy (NEM2), the most common non-dystrophic congenital myopathy. The protein structure of nebulin consists of a large number of simple repeats that are actin-binding, most of which are organized into super-repeats (SRs). Approaches to treat NEM2 are sorely lacking and gaining an in-depth understanding of the many roles of nebulin in muscle structure and function is essential. We will comprehensively study nebulin, building on major advances that we and others have made in recent years. To help achieve our goals we utilize mouse models, some of which mimic severe and others milder NEM2, as powerful tools for our basic science and translational studies. We will investigate nebulin's functions from the single-molecule to the intact muscle levels, using multidisciplinary approaches that involve transcriptomics, proteomics, super-resolution imaging, low-angle X-ray diffraction, and biomechanics. Aim 1 focuses on thin filament length regulation. Our recent work supports that in slow muscle, nebulin collaborates with leiomodin-2 (Lmod2), with nebulin regulating the length of a proximal thin filament segment and Lmod2 regulating the length of a distal segment that is nebulin-free. Here we will critically test this dual length regulation model and study whether it has translational potential, by determining whether upregulating Lmod2 is an effective treatment for severe nebulin-based nemaline myopathy. Aim 2 studies the functional significance of weak actin-binding of centrally-located nebulin SRs that bind actin more weakly than those near the ends of the molecule. This is likely functionally important, considering that a mutation that increases the binding affinity of a central SR causes a skeletal muscle myopathy in patients world-wide. We will study mouse models in which centrally located weak-binding SRs have been converted into strong-binding SRs using mechanical assays and X-ray diffraction on intact muscle. Aim 3 studies the C-terminus of nebulin, its layout in the Z-disk, and the functions of nebulin's differentially expressed Z-repeats. Many NEM2 patients have truncating mutations that result in the loss of most of the C-terminus (located within the Z disk), yet few studies have investigated this region of the molecule. Nebulin's C-terminus contains Z-disk repeats that are alternatively spliced (expressed at high levels in muscles with wide Z-disks). We will establish the layout of nebulin in the Z-disk, the location of the Z-repeats, identify protein binding partners, and study the effects of deleting differentially expressed nebulin Z-repeats on the Z-disk structure and function. Capitalizing on our >15-year track record of innovative nebulin research, and utilizing our team of experienced scientists and talented trainees, this proposal sets ambitious goals that are expected to accelerate the understanding of the biology of nebulin, its role in disease, and to identify possible novel therapeutic avenues.