Thin Filaments and Muscle Regulation

细丝和肌肉调节

• 批准号: 7998182
• 负责人: WILLIAM J LEHMAN
• 金额: $ 40.63万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-30 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7998182
• 关键词: Actin-Binding Protein; Actins; Actomyosin; Airway Resistance; Architecture; Asthma; Attention; Binding; Binding Proteins; Blood Vessels; Cardiac; Cardiovascular Diseases; Cardiovascular system; Characteristics; Coupled; Crystallography; Electron Microscopy; Electrons; F-Actin; Filament; Goals; Hybrids; Hypertension; Image Analysis; Lead; Link; Lung; Maps; Microfilaments; Modeling; Molecular; Molecular Structure; Muscle; Muscle Contraction; Myocardium; Myosin ATPase; Names; Physiology; Positioning Attribute; Principal Investigator; Process; Production; Proteins; Regulation; Relaxation; Research; Resolution; Role; Skeletal Muscle; Smooth Muscle; Structure; System; Techniques; Testing; Thin Filament; Tropomyosin; Troponin; Troponin I; Variant; Visceral; Work; base; blood pressure regulation; caldesmon; calponin; drug development; electron tomography; flexibility; genetic regulatory protein; link protein; mutant; nebulin; novel; particle; programs; reconstruction

DESCRIPTION (provided by applicant): Thin filament-associated actin-binding proteins control both actomyosin-based muscle contraction and cytoskeletal formation. To elucidate the mechanisms required for muscle thin filaments to function, it is crucial to determine the structural interactions of the regulatory proteins involved. As a means of achieving our objective to understand the physiology of cardiac, skeletal and smooth muscle control systems, we will examine the architecture of muscle thin filaments at a fundamental structural level and characterize the changing interactions of thin filament-linked proteins that regulate muscle activity. We will use state-of- the-art electron microscopy and electron tomography coupled with image analysis and 3D reconstruction to establish the macromolecular structure of actin-binding proteins on thin filament actin. Using these techniques: (1) We aim to determine the structural basis of troponin-tropomyosin regulation of cardiac and skeletal muscle activity by analyzing interactions of tropomyosin and troponin on thin filaments, which are governed by Ca2+binding to troponin and myosin-crossbridge binding on actin. To accomplish this goal, (A) we will test our newly proposed atomic model for troponin-tropomyosin localization on thin filaments by generating single particle and electron tomographic reconstructions; (B) we will test the hypothesis that mobile domains of troponin-I latch onto actin to constrain tropomyosin in the inhibitory "blocking" state characteristic of relaxed muscle; (C) we will test both the hypothesis that tropomyosin assumes the contours of the F-actin helix as a relatively stiff coiled coiled-coil and the alternative view that tropomyosin is flexible. (2) We will test the hypothesis that mutant cardiac troponin and different tropomyosin variants perturb muscle regulation by causing an imbalance in tropomyosin's position that alters the regulatory state of thin filaments. (3) We will assess the regulatory role of thin filament-linked caldesmon and calponin in defining tropomyosin position in vascular and visceral muscle. (4) We will determine the structure of nebulin bound to actin to complete our map of thin filaments. In each study, reconstructions fitted to the atomic resolution maps of F-actin will demarcate molecular contacts of binding proteins with actin at near atomic resolution ("hybrid crystallography"). Lay summary: Studies on troponin-tropomyosin regulated filaments, with particular attention devoted to normal and mutant proteins derived from cardiac muscle, will lead to an elucidation of the molecular regulatory mechanisms governing cardiac contraction, which is essential for tracing cardiovascular disease processes. Studies on smooth muscle filaments will aid in understanding the fine-tuning of smooth muscle contraction thus revealing key controls for vascular tone and pulmonary airway resistance, determinants in, e.g., hypertension and asthma. Actin filaments and associated proteins are major participates in diverse cellular systems, underscoring the broad significance of the proposed work. Our goal is to elucidate the control mechanisms that regulate cardiovascular and skeletal muscle activity. We will examine structural changes at a molecular level that are orchestrated by regulatory proteins and which control muscle shortening and force production. Understanding the underlying molecular physiology governing contraction and relaxation in heart muscle and blood vessels is key to deciphering cardiovascular disease processes, controlling blood pressure and identifying novel targets for drug development.

描述（由申请人提供）：薄的与肌动蛋白相关的肌动蛋白结合蛋白控制基于肌动球蛋白的肌肉收缩和细胞骨架形成。为了阐明肌肉细丝发挥功能所需的机制，确定所涉及的调节蛋白的结构相互作用至关重要。作为实现我们的目标，以了解心脏，骨骼和平滑肌控制系统的生理学的一种手段，我们将检查在基本结构水平上的肌肉细丝的架构，并表征调节肌肉活动的薄的顺应性连接蛋白质的变化的相互作用。我们将使用最先进的电子显微镜和电子断层扫描加上图像分析和三维重建，以建立肌动蛋白结合蛋白的大分子结构的细丝肌动蛋白。使用这些技术：（1）通过分析肌纤维上原肌球蛋白和肌钙蛋白的相互作用，确定肌钙蛋白-原肌球蛋白调节心肌和骨骼肌活动的结构基础，这种相互作用是由Ca ~（2+）与肌钙蛋白的结合和肌球蛋白-桥与肌动蛋白的结合控制的。为了实现这一目标，（A）我们将通过产生单粒子和电子断层重建来测试我们新提出的肌钙蛋白-原肌球蛋白在细丝上定位的原子模型;（B）我们将测试肌钙蛋白-I的移动的结构域锁定在肌动蛋白上以将原肌球蛋白限制在松弛肌肉的抑制性“阻断”状态特征的假设;（C）我们将检验原肌球蛋白假定F-肌动蛋白螺旋的轮廓是相对坚硬的卷曲螺旋的假设和原肌球蛋白是柔性的替代观点。(2)我们将测试的假设，即突变的心肌肌钙蛋白和不同的原肌球蛋白变体扰乱肌肉调节引起的原肌球蛋白的位置，改变细丝的监管状态的不平衡。(3)我们将评估薄的顺应性连接的钙调蛋白和钙调蛋白在定义血管和内脏肌原肌球蛋白的位置的调节作用。(4)我们将确定与肌动蛋白结合的星云蛋白的结构，以完成我们的细丝图。在每一项研究中，适合于F-肌动蛋白的原子分辨率图的重建将在近原子分辨率下划分结合蛋白与肌动蛋白的分子接触（“混合晶体学”）。敷设总结：研究肌钙蛋白原肌球蛋白调节丝，特别是关注来自心肌的正常和突变蛋白，将导致阐明的分子调控机制，心脏收缩，这是必不可少的跟踪心血管疾病的过程。对平滑肌细丝的研究将有助于理解平滑肌收缩的微调，从而揭示血管张力和肺气道阻力的关键控制，例如，高血压和哮喘。肌动蛋白丝和相关蛋白是多种细胞系统的主要参与者，强调了所提出的工作的广泛意义。我们的目标是阐明调节心血管和骨骼肌活动的控制机制。我们将在分子水平上研究结构变化，这些变化是由调节蛋白协调的，并控制肌肉缩短和力量产生。了解控制心肌和血管收缩和舒张的潜在分子生理学是破译心血管疾病过程、控制血压和确定药物开发新靶点的关键。