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）：细丝相关的肌动蛋白结合蛋白控制基于肌动肌球蛋白的肌肉收缩和细胞骨架形成。为了阐明肌肉薄细丝起作用所需的机制，确定涉及调节蛋白的结构相互作用至关重要。作为实现我们的目标的一种手段，可以理解心脏，骨骼和平滑肌控制系统的生理，我们将检查肌肉薄丝在基本结构水平上的结构，并表征调节肌肉活性的细丝连接蛋白的不断变化的相互作用。我们将使用最先进的电子显微镜和电子断层扫描以及图像分析和3D重建来建立肌动蛋白结合蛋白在薄丝肌动蛋白上的大分子结构。使用这些技术：（1）我们旨在通过分析肌肌蛋白和肌蛋白在薄丝上的相互作用，通过分析肌蛋白和肌蛋白在薄丝上的相互作用，该结构基础对心脏和骨骼肌的活性进行了调节，而肌蛋白和肌蛋白的相互作用由Ca2+与肌动蛋白和肌蛋白蛋白杂交桥的结合在薄丝上控制。为了实现这一目标，（a）我们将通过产生单个颗粒和电子断层造影重建来测试我们新提出的肌钙蛋白 - 肌球蛋白定位的原子模型； （b）我们将检验以下假设：肌钙蛋白I锁定在肌动蛋白上的移动结构域在抑制性肌肉的抑制性“阻断”状态特征中限制了肌动蛋白； （c）我们将既检验了肌球蛋白假设F-肌动蛋白螺旋的轮廓为相对僵硬的盘绕螺旋线圈，又要检验到肌球蛋白是柔性的替代观点。 （2）我们将检验以下假设：突变心脏肌钙蛋白和不同的肌动蛋白变体通过导致肌动蛋白位置的不平衡来改变薄丝细丝的调节状态，从而扰乱肌肉。 （3）我们将评估薄丝连接的Caldesmon和calponin在定义肌动蛋白在血管和内脏肌肉中的位置的调节作用。 （4）我们将确定与肌动蛋白结合的雾蛋白的结构，以完成我们的细丝图。在每项研究中，拟合到F-肌动蛋白原子分辨率图的重建将划定结合蛋白与肌动蛋白与肌动蛋白的分子接触（“杂交晶体学”）。摘要摘要：对肌钙蛋白 - 肌球蛋白调节的细丝的研究，特别注意源自心肌的正常和突变蛋白，将导致阐明控制心脏收缩的分子调节机制，这对于追踪心血管疾病过程至关重要。对平滑肌丝的研究将有助于理解平滑肌收缩的微调，从而揭示了血管张力和肺气道耐药性的关键控制，例如高血压和哮喘。肌动蛋白丝和相关蛋白是主要参与各种细胞系统，强调了拟议工作的广泛意义。我们的目标是阐明调节心血管和骨骼肌活动的控制机制。我们将检查通过调节蛋白精心策划的分子水平的结构变化，并控制肌肉缩短和产生肌肉。了解有关心肌和血管中收缩和放松的基本分子生理学是破译心血管疾病过程，控制血压并确定药物发育的新目标的关键。