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: 10687109
• 负责人: Vitold Galkin
• 金额: $ 57.06万
• 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/10687109
• 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; Complement; Complex; Cryoelectron Microscopy; Crystallography; Data; Development; Developmental Biology; Diagnostic; Disease; 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; experimental study; improved; in vivo; medical schools; monomer; 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有效的收缩活动至关重要，并且 细丝完整性的改变与严重且往往致命的骨骼和心脏有关 肌肉疾病。我们的长期目标是确定其成分和分子机制 在正常发育和疾病过程中调节横纹肌的肌动蛋白结构。我们的 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 或Lmod和troomoduin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 对细丝尖端的调节。我们还将研究结构和功能 Lmod结合到已经形成的细丝的侧面的后果。最后，我们会 建立LMOD功能调节机制。我们提出了三个目标来确定 Lmod和Tmod功能全谱的潜在分子机制：(1) 破译Lmod在通过尖端保持适当细丝长度中的作用 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功能受钙离子调控的假设。通过采用高分辨率 低温电子显微镜(CRYO-EM)结合三维核磁 共振和Förster共振能量转移)，我们将重现钙离子的全貌- 依赖的Lmod与细丝的相互作用，并揭示这些作用的生物学意义 互动。结构模型的稳健性将通过对发展的监测来评估 以及体内基因敲除小鼠心肌和骨骼肌的收缩性能 利用专门影响新发现的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关系， 克斯特尤科娃华盛顿州立大学实验室(蛋白质结构、生化专家 和肌动蛋白结合蛋白的生物物理特性)，加州大学格雷戈里奥实验室 亚利桑那州(肌原纤维组装的分子、细胞和发育生物学专家)和 东弗吉尼亚医学院的加尔金实验室(高分辨率低温电磁技术专家 肌动蛋白复合体)。我们的数据将全面识别关键组件 细丝组装和维护的调节机制在健康和 疾病。