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.

概括 该提案重点是nebulin的结构和功能。 用骨骼肌表达。巨大的树星大小使阐明其功能的挑战，但 重要性得到了导致杀线虫肌病（NEM2）的许多雾化蛋白突变的支持，最重要的是 常见的非营养性先天性肌病。云母的蛋白质结构由大量 肌动蛋白结合的简单重复序列，其中大多数被组织成超级重复（SRS）。方法 治疗NEM2非常缺乏和深入了解肌肉中的许多角色 结构和功能至关重要。我们将基于我们的重大进展，全面研究云蛋白 近年来其他人做出了。为了帮助实现我们的目标，我们利用鼠标模型，其中一些 模仿严重和其他温和的NEM2，是我们基础科学和翻译研究的强大工具。我们 将使用多学科研究云蛋白的功能从单分子到完整的肌肉水平 涉及转录组学，蛋白质组学，超分辨率成像，低角度X射线衍射和的方法 生物力学。 AIM 1专注于细丝长度调节。我们最近的工作支持在慢速肌肉中 Nebulin与Leiomodin-2（LMOD2）合作，Nebulin调节近端细丝的长度 段和LMOD2调节无云母蛋白的远端段的长度。在这里我们将进行批判性测试 这种双重长度调节模型并通过确定是否是否具有转化潜力 上调LMOD2是针对严重基于Nebulin的Nemaline肌病的有效治疗方法。 AIM 2研究 中心位置的肾上腺素SR的弱肌动蛋白结合的功能显着性比肌动蛋白更弱地结合肌动蛋白 那些附近分子的末端。考虑到一个突变，这可能在功能上很重要 增加中央SR的结合亲和力会导致全世界患者的骨骼肌肌病。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使用机械测定和完整肌肉的X射线衍射。 AIM 3研究nebulin的C末端， 它的布局在Z磁盘中，以及Nebulin差异表达的Z重复的功能。许多NEM2患者 具有截断的突变，导致大多数C端（位于Z磁盘内）的损失，但很少 研究研究了该分子的这一区域。 Nebulin的C末端包含Z-DISK重复序列 或者剪接（在具有宽Z磁盘的肌肉中以高水平表示）。我们将建立布局 Z-磁带中的树星，Z重复的位置，识别蛋白质结合伴侣，并研究 删除Z-DISK结构和功能上差异表达的Nebulin Z-重复。利用我们的 >创新的云蛋白研究的15年历史记录，并利用我们的经验丰富的科学家团队和 这项提议有才华的学员，设定了雄心勃勃的目标，这些目标有望加速对 云蛋白的生物学，其在疾病中的作用，并确定可能的新型治疗途径。