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: 10673594
• 负责人: Henk L. GRANZIER
• 金额: $ 52.67万
• 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/10673594
• 关键词: Acceleration; Actins; Actomyosin; Address; Affinity; Alternative Splicing; Basic Science; Binding; Binding Proteins; Biological Assay; Biology; Biomechanics; Collaborations; Disease; Distal; Environment; Fiber; Generations; Goals; Kinetics; Length; Location; Mechanics; Microfilaments; Missense Mutation; Modeling; Muscle; Muscle Proteins; Muscle function; Mutation; Myopathy; Nemaline Myopathies; Patients; Play; Productivity; Proteins; Proteomics; Publishing; RNA Splicing; Regulation; Research; Role; Scientist; Severity of illness; Skeletal Muscle; Striated Muscles; Structure; Talents; Testing; Thick Filament; Thin Filament; Thinness; Width; Work; X ray diffraction analysis; 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; superresolution imaging; tool; transcriptomics; translational goal; translational potential; translational study; ultra high resolution

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 该提案重点关注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， 表达于骨骼肌。星云蛋白的巨大尺寸使得阐明其功能具有挑战性，但它 引起线状肌病 (NEM2) 的许多星云蛋白突变也证明了其重要性，这是最常见的 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）。接近 治疗NEM2非常缺乏并深入了解nebulin在肌肉中的许多作用 结构和功能至关重要。我们将在我们取得的重大进展的基础上全面研究星云蛋白 和其他人近年来所做的。为了帮助实现我们的目标，我们利用小鼠模型，其中一些 模仿严重和其他较温和的 NEM2，作为我们基础科学和转化研究的强大工具。我们 将利用多学科研究从单分子到完整肌肉水平的星云蛋白功能 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 生物力学。目标 1 侧重于细丝长度调节。我们最近的工作支持在慢肌中， nebulin 与 leiomodin-2 (Lmod2) 合作，nebulin 调节近端细丝的长度 节段和 Lmod2 调节无云蛋白的远端节段的长度。在这里我们将严格测试 这种双长度调控模型并通过确定是否具有转化潜力来研究其是否具有转化潜力 上调 Lmod2 是治疗严重星云蛋白线状肌病的有效方法。目标 2 研究 位于中心的星云蛋白 SR 与肌动蛋白的结合较弱，其弱肌动蛋白结合的功能意义 靠近分子末端的那些。考虑到突变 增加中枢 SR 的结合亲和力会导致全世界患者出现骨骼肌肌病。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 对完整肌肉使用机械测定和 X 射线衍射进行 SR。目标 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 具有截短突变，导致大部分 C 末端（位于 Z 盘内）丢失，但很少有 研究调查了分子的这个区域。 Nebulin 的 C 末端包含 Z 盘重复序列 选择性剪接（在具有宽 Z 盘的肌肉中高水平表达）。我们将确定布局 Z 盘中的星云蛋白（Z 重复的位置），识别蛋白质结合伙伴，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。充分利用我们的 > 15 年的创新星云研究记录，并利用我们经验丰富的科学家团队和 该提案设定了雄心勃勃的目标，预计将加速对才华横溢的学员的理解 星云蛋白的生物学、其在疾病中的作用，并确定可能的新治疗途径。

项目成果

Lifting the nebula: novel insights into skeletal muscle contractility.

提升星云：对骨骼肌收缩力的新见解。

• 发表时间: 2010-10
• 作者: Ottenheijm, Coen A C;Granzier, Henk
• 通讯作者: Granzier, Henk

Nebulin and titin modulate cross-bridge cycling and length-dependent calcium sensitivity.

星云蛋白和肌联蛋白调节跨桥循环和长度依赖性钙敏感性。

• 发表时间: 2019
• 作者: Mijailovich, Srboljub M;Stojanovic, Boban;Nedic, Djordje;Svicevic, Marina;Geeves, Michael A;Irving, Thomas C;Granzier, Henk L
• 通讯作者: Granzier, Henk L

In vivo characterization of skeletal muscle function in nebulin-deficient mice.

星云蛋白缺陷小鼠骨骼肌功能的体内表征。

• 发表时间: 2020
• 影响因子: 3.4
• 作者: Gineste, Charlotte;Ogier, Augustin C;Varlet, Isabelle;Hourani, Zaynab;Bernard, Monique;Granzier, Henk;Bendahan, David;Gondin, Julien
• 通讯作者: Gondin, Julien

Removal of MuRF1 Increases Muscle Mass in Nemaline Myopathy Models, but Does Not Provide Functional Benefits.

去除 MuRF1 会增加线状肌病模型中的肌肉质量，但不会提供功能益处。

• 发表时间: 2022-07-23
• 影响因子: 5.6
• 作者: Lindqvist, Johan;Kolb, Justin;de Winter, Josine;Tonino, Paola;Hourani, Zaynab;Labeit, Siegfried;Ottenheijm, Coen;Granzier, Henk
• 通讯作者: Granzier, Henk

Nebulin alters cross-bridge cycling kinetics and increases thin filament activation: a novel mechanism for increasing tension and reducing tension cost.

Nebulin 改变跨桥循环动力学并增加细丝激活：一种增加张力和降低张力成本的新机制。

• 发表时间: 2009-11-06
• 作者: Chandra, Murali;Mamidi, Ranganath;Ford, Steven;Hidalgo, Carlos;Witt, Christian;Ottenheijm, Coen;Labeit, Siegfried;Granzier, Henk
• 通讯作者: Granzier, Henk