Deciphering how a human mutation in leiomodin-3 leads to muscle disease

解读人类 leiomodin-3 突变如何导致肌肉疾病

• 批准号: 10228711
• 负责人: Lauren Elisa Schultz
• 金额: $ 3.85万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-21 至 2022-08-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10228711
• 关键词: Actin-Binding Protein; Actins; Adenoviruses; Affect; Amino Acids; Animals; Binding; Binding Proteins; Biochemical; Biological Assay; Cardiac Myocytes; Cardiovascular system; Cell physiology; Circular Dichroism; Dependovirus; Development; Disease; Disease Progression; Disease model; Family; Family member; Filament; Fluorescence Recovery After Photobleaching; Goals; Heart Abnormalities; Human; Immunofluorescence Immunologic; Immunofluorescence Microscopy; Impairment; Individual; Knockout Mice; Lead; Length; Leucine-Rich Repeat; Link; Measures; Mechanics; Microfilaments; Microscopy; Minus End of the Actin Filament; Missense Mutation; Molecular; Mus; Muscle Contraction; Muscle Development; Muscle Fibers; Muscle function; Mutate; Mutation; Myocardium; Myofibrils; Myopathy; Myosin ATPase; Nemaline Myopathies; Nuclear Magnetic Resonance; Patients; Play; Protein Isoforms; Proteins; Pyrenes; Regulation; Research; Research Personnel; Role; Sarcomeres; Sedimentation process; Skeletal Muscle; Striated Muscles; Structure; Techniques; Testing; Thick Filament; Thin Filament; Tropomyosin; cell type; clinically relevant; congenital myopathy; experimental study; heart function; in vivo; insight; mouse model; multidisciplinary; novel; polymerization; prevent; programs; single molecule; skeletal; skeletal muscle weakness; therapeutic target; tool; tropomodulin

PROJECT SUMMARY Striated muscle cell contraction is dependent on the proper overlap of myosin (thick) filaments and actin (thin) filaments. Leiomodin (Lmod) and tropomodulin (Tmod) are proteins that bind to the pointed end of thin filaments in order to fine-tune their lengths. Tmod1 and Lmod2 are the major isoforms in cardiac muscle. Lmod3 is the major skeletal isoform, however it is also expressed in cardiac muscle. Mutations in Tmod and Lmod have been shown to result in dysregulated thin filament lengths and lead to the development of myopathies. The goal of this proposal is to identify molecular mechanisms for how Tmod and Lmod proteins regulate thin filament assembly. We plan to create a novel model of disease by studying a mutation that has been identified in Lmod3 in patients with nemaline myopathy (a skeletal muscle disorder). We hypothesize that introducing this mutation in Tmod and Lmod will result in altered thin filament lengths and perturbed actin assembly, leading to disease development. We have obtained a Lmod3 knockout mouse model, which will serve as an important tool for this study. We propose the following aims: Aim 1 is to examine the role of Lmod3 in striated muscle by utilizing a Lmod3 knockout (KO) mouse line. Immunofluorescence deconvolution microscopy will be used to assess overall sarcomere structure and changes in thin filament lengths in these KO mice. Contractile force of individual skeletal and cardiac myocytes will be measured. We will attempt to prevent skeletal and cardiac defects in these mice by introducing Lmod3 via adeno-associated virus. Aim 2 will determine the effect of a nemaline myopathy-linked mutation on thin filament lengths and actin dynamics. Mutated Lmod and Tmod proteins will be expressed in both skeletal and cardiac myocytes via adenovirus. Thin filaments will be visualized and measured using immunofluorescence microscopy, while fluorescence recovery after photobleaching will test mutated Lmod and Tmod's ability to assemble to the pointed ends. Aim 3 is to determine how a nemaline myopathy-linked mutation affects structure and function of Lmod/Tmod. Circular dichroism will be used to investigate the ability of mutated Lmod and Tmod to fold properly, and nuclear magnetic resonance will be used to determine how structural alterations could potentially affect mutated Tmod and Lmod's binding interfaces with other proteins, such as actin and tropomyosin. We will assess functional changes in mutated Lmod and Tmod by performing pyrene-actin polymerization assays and co-sedimentation assays. The long-term goal of this multidisciplinary project, that spans from single molecule to whole animal studies, is to determine how perturbation of actin-thin filament lengths leads to muscle disease. This is significant because actin is the most abundant protein in most cell types and is involved in numerous essential cellular processes. The results obtained in this project will allow us to decipher the connection between thin filament lengths and muscle function, and in vivo mechanistic information on how a single mutation in Lmod3 leads to human myopathy.

项目概要 横纹肌细胞收缩取决于肌球蛋白（厚）丝和肌动蛋白的适当重叠 （细）细丝。 Leiomodin (Lmod) 和原调节蛋白 (Tmod) 是结合到薄层尖端的蛋白质 细丝以微调其长度。 Tmod1 和 Lmod2 是心肌中的主要亚型。 Lmod3 是主要的骨骼亚型，但它也在心肌中表达。 Tmod 和 Lmod 的突变 已被证明会导致细丝长度失调并导致肌病的发展。目标 该提案的目的是确定 Tmod 和 Lmod 蛋白如何调节细丝的分子机制 集会。我们计划通过研究 Lmod3 中发现的突变来创建一种新的疾病模型 线状肌病（一种骨骼肌疾病）患者。我们假设引入这种突变 Tmod 和 Lmod 会导致细丝长度改变和肌动蛋白组装扰乱，从而导致疾病 发展。我们获得了Lmod3基因敲除小鼠模型，该模型将作为此研究的重要工具 学习。我们提出以下目标： 目标 1 是通过利用 Lmod3 敲除 (KO) 小鼠系。免疫荧光反卷积显微镜将用于评估整体 这些 KO 小鼠的肌节结构和细丝长度的变化。个体骨骼的收缩力 并将测量心肌细胞。我们将尝试预防这些小鼠的骨骼和心脏缺陷 通过腺相关病毒引入 Lmod3。目标 2 将确定线状肌病相关的影响 细丝长度和肌动蛋白动力学的突变。突变的 Lmod 和 Tmod 蛋白将在 通过腺病毒感染骨骼肌细胞和心肌细胞。细丝将使用可视化和测量 免疫荧光显微镜，而光漂白后的荧光恢复将测试突变的 Lmod 和 Tmod 能够组装到尖端。目标 3 是确定线状肌病相关突变如何 影响 Lmod/Tmod 的结构和功能。圆二色性将用于研究突变的能力 Lmod 和 Tmod 正确折叠，核磁共振将用于确定结构如何 改变可能会影响突变的 Tmod 和 Lmod 与其他蛋白质（例如肌动蛋白）的结合界面 和原肌球蛋白。我们将通过芘-肌动蛋白评估突变 Lmod 和 Tmod 的功能变化 聚合测定和共沉降测定。这个多学科项目的长期目标是 从单分子到整个动物研究，旨在确定肌动蛋白细丝的扰动如何 长度会导致肌肉疾病。这很重要，因为肌动蛋白是大多数细胞类型中最丰富的蛋白质 并参与许多重要的细胞过程。该项目取得的成果将使我们能够 破译细丝长度和肌肉功能之间的联系以及体内机械信息 Lmod3 的单一突变如何导致人类肌病。