REGULATION OF THE ACTIN FILAMENT POINTED END DYNAMICS IN HEALTH AND DISEASE

健康和疾病中肌动蛋白丝尖头动力学的调节

• 批准号: 10296898
• 负责人: Vitold Galkin
• 金额: $ 60.89万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10296898
• 关键词: 3-Dimensional; Actin-Binding Protein; Actins; Affect; Animals; Architecture; Arizona; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Biophysics; C-terminal; Cardiac; Cardiac Myocytes; Cellular biology; Clinical Research; Complement; Complex; Cryoelectron Microscopy; Crystallography; Data; Development; Developmental Biology; Diagnostic; Disease; Electron Microscopy; Family; Fluorescence Resonance Energy Transfer; Functional disorder; Goals; Health; In Vitro; Interdisciplinary Study; Knockout Mice; Laboratories; Learning; Length; Link; Maintenance; Methods; Microfilaments; Minus End of the Actin Filament; Modeling; Molecular; Molecular Biology; Molecular Conformation; Monitor; Muscle; Muscle Fibers; Muscle Weakness; Mutate; Mutation; Myocardium; Myofibrils; Myopathy; Myosin ATPase; Nuclear Magnetic Resonance; Organ; Physiology; Protein Fragment; Proteins; Public Health; Regulation; Research; Resolution; Role; Sarcomeres; Side; Site; Skeletal Muscle; Slide; Striated Muscles; Structural Models; Structure; Telomere Length Maintenance; Testing; Thick Filament; Thin Filament; Time; Tropomyosin; Troponin; Universities; Virginia; Washington; biophysical properties; cardiac muscle disease; combat; cryogenics; experimental study; improved; in vivo; medical schools; muscular structure; new therapeutic target; novel; prevent; protein structure; structural biology; tropomodulin; vector

In striated muscles, actin thin filament architecture is critical for efficient contractile activity, and alterations in thin filament integrity are linked to severe and often lethal skeletal and cardiac muscle diseases. Our long-term goal is to identify the components and molecular mechanisms regulating actin architecture in striated muscle during normal development and disease. Our short-term goal is to evaluate how actin-binding proteins of the tropomodulin family (e.g. leiomodin or Lmod and tropomoduin or Tmod) affect the formation and then the structure of the thin filament. We will test our recently proposed molecular mechanism for the Lmod/Tmod-dependent regulation of the pointed end of the thin filaments. We will also study the structural and functional consequences of Lmod binding to sides of the already formed thin filaments. Finally, we will establish mechanisms of regulation of Lmod functions. We propose three aims to identify underlying molecular mechanisms of the full spectrum of Lmod and Tmod functions: (1) to decipher the role of Lmod in the maintenance of proper thin filament lengths via pointed end regulation; (2) to establish the role of Lmod’s actin-binding sites in thin filament activation; (3) to test the hypothesis that Lmod functions are regulated by Ca2+. By employing high resolution cryogenic electron microscopy (cryo-EM) in conjunction with 3-dimensional nuclear magnetic resonance and Förster resonance energy transfer), we will recreate the full picture of Ca2+- dependent Lmod interactions with the thin filament and reveal the biological significance of these interactions. The robustness of structural models will be evaluated by monitoring of development and contractility of cardiac and skeletal muscles in knockout mice in vivo via the identification and utilization of mutations specifically affecting newly discovered Lmod’s and Tmod’s functionalities. To achieve these goals, we established a powerful multidisciplinary collaboration between the Kostyukova laboratory at the Washington State University (expert in protein structure, biochemical and biophysical properties of actin-binding proteins), the Gregorio laboratory at the University of Arizona (expert in the molecular, cellular and developmental biology of myofibril assembly) and the Galkin laboratory at the Eastern Virginia Medical School (expert in high resolution cryo-EM of actin complexes). Our data will provide a comprehensive identification of critical components of the regulatory mechanisms underlying thin filament assembly and maintenance in health and disease.

在横纹肌中，肌动蛋白细丝结构对于有效的收缩活动至关重要， 细丝完整性的改变与严重的且通常致命的骨骼和心脏疾病有关。 肌肉疾病。我们的长期目标是确定其成分和分子机制 在正常发育和疾病期间调节横纹肌中的肌动蛋白结构。我们 短期目标是评估原调节蛋白家族的肌动蛋白结合蛋白（例如leiomodin）如何 或Lmod和tropomoduin或Tmod）影响细丝的形成，然后影响细丝的结构。 我们将测试我们最近提出的Lmod/Tmod依赖的分子机制， 调节细细丝的尖端。我们还将研究其结构和功能 Lmod结合到已经形成的细丝的侧面的结果。最后我们将 建立Lmod功能调节机制。我们提出了三个目标，以确定 Lmod和Tmod功能的全谱的潜在分子机制：（1）至 解读Lmod在通过尖端维持适当的细丝长度中的作用 调节;（2）确定Lmod的肌动蛋白结合位点在细丝激活中的作用;（3） 验证Lmod功能受Ca 2+调节的假设。通过采用高分辨率 低温电子显微镜（cryo-EM）结合三维核磁共振 共振和Förster共振能量转移），我们将重现Ca 2+的全貌- 依赖的Lmod与细丝的相互作用，并揭示这些的生物学意义。 交互.结构模型的稳健性将通过监测发展情况进行评估。 和心肌和骨骼肌的收缩性， 利用特异性影响新发现的Lmod和Tmod功能的突变。 为了实现这些目标，我们建立了强大的多学科合作， 华盛顿州立大学的科斯秋科娃实验室（蛋白质结构、生物化学 和生物物理特性的肌动蛋白结合蛋白），格雷戈里奥实验室在大学 Arizona（肌原纤维组装的分子、细胞和发育生物学专家）， 东弗吉尼亚医学院的Galkin实验室（高分辨率冷冻电镜专家， 肌动蛋白复合物）。我们的数据将提供一个全面的识别关键组成部分， 细丝组装和维持健康的调节机制， 疾病