Roles of Nebulin in Structure and Function of Striated Muscle

星云蛋白在横纹肌结构和功能中的作用

• 批准号: 10362940
• 负责人: Henk L. GRANZIER
• 金额: $ 49.97万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10362940
• 关键词: Actins; Actomyosin; Address; Affinity; Alternative Splicing; Basic Science; Binding; Binding Proteins; Biological Assay; Biology; Biomechanics; Collaborations; Disease; Distal; Environment; Fiber; Generations; Goals; Image; Kinetics; Length; Location; Mechanics; Microfilaments; Missense Mutation; Modeling; Muscle; Muscle Proteins; Muscle function; Mutation; Myopathy; Nemaline Myopathies; Patients; Play; Proteins; Proteomics; Publishing; RNA Splicing; Regulation; Research; Resolution; Role; Scientist; Severity of illness; Signaling Molecule; Skeletal Muscle; Striated Muscles; Structure; Talents; Testing; Thick Filament; Thin Filament; Thinness; Width; Work; X ray diffraction analysis; base; congenital myopathy; differential expression; effective therapy; experience; filamin; innovation; insight; interdisciplinary approach; mouse model; muscular structure; nebulin; novel; novel therapeutic intervention; novel therapeutics; protein structure; single molecule; tool; transcriptomics; translational goal; translational potential; translational study

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.

总结 这项建议的重点是nebulin的结构和功能，nebulin是一种异常大的肌节蛋白， 在骨骼肌中表达。星云蛋白的巨大尺寸使得阐明其功能具有挑战性， 许多引起线状体肌病（NEM 2）的星云蛋白突变支持了这一重要性， 常见的非营养不良性先天性肌病。星云蛋白的蛋白质结构由大量的 肌动蛋白结合的简单重复序列，其中大部分被组织成超级重复序列（SR）。方法来 治疗NEM 2是严重缺乏和获得深入了解的许多作用，星云蛋白在肌肉 结构和功能至关重要。我们将全面研究星云蛋白，建立在我们所取得的重大进展的基础上， 和其他人在最近几年所做的。为了帮助实现我们的目标，我们利用小鼠模型，其中一些 模拟严重和其他温和的NEM 2，作为我们的基础科学和转化研究的强大工具。我们 将研究星云蛋白的功能，从单分子到完整的肌肉水平，使用多学科 包括转录组学、蛋白质组学、超分辨率成像、低角度X射线衍射和 生物力学目标1集中于细丝长度调节。我们最近的研究支持在慢肌中， 星云蛋白与leiomodin-2（Lmod 2）协同作用，星云蛋白调节近端细丝的长度 段和Lmod 2调节无星云蛋白的远端段的长度。在这里，我们将严格测试 这个双长度调节模型，并研究它是否具有翻译潜力，通过确定是否 上调Lmod 2是严重的基于星云蛋白的线状体肌病的有效治疗。目标2研究了 位于中心的星云蛋白SR的弱肌动蛋白结合的功能意义， 分子末端附近的分子。这可能在功能上很重要，考虑到突变， 增加了中枢SR的结合亲和力，导致了世界范围内的骨骼肌肌病患者。我们 将研究小鼠模型，其中位于中心的弱结合SR已转化为强结合SR， 在完整肌肉上使用机械测定和X射线衍射的SR。目的3研究星云蛋白的C-末端， 其布局在Z-磁盘，和功能的星云蛋白的差异表达的Z-重复。许多NEM 2患者 具有截短突变，导致大部分C-末端（位于Z盘内）丢失，但很少 研究已经调查了分子的这一区域。星云蛋白的C端含有Z盘重复序列， 选择性剪接（在具有宽Z盘的肌肉中以高水平表达）。我们将建立布局， 星云蛋白在Z-盘，Z-重复的位置，确定蛋白质结合伙伴，并研究影响 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 >15年的创新星云蛋白研究记录，并利用我们经验丰富的科学家团队， 有才华的学员，这一建议设定了雄心勃勃的目标，预计将加快对 星云蛋白的生物学，它在疾病中的作用，并确定可能的新的治疗途径。