Troponin and myosin in regulation of muscle contraction and heart disease

肌钙蛋白和肌球蛋白调节肌肉收缩和心脏病

基本信息

批准号：
8197235
负责人：
Zenon Grabarek
金额：
$ 50.24万
依托单位：
BOSTON BIOMEDICAL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-15 至 2013-03-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8197235
关键词：
ATP phosphohydrolase Actins Actomyosin Adenosinetriphosphatase Affect Affinity Binding C-terminal Cardiac Cessation of life Complement Complex Contractile Proteins Data Disease Disulfides Divalent Cations Drug Design Equilibrium Familial Hypertrophic Cardiomyopathy Fiber Fluorescence Resonance Energy Transfer Goals Head Heart Diseases Heart Hypertrophy Kinetics Label Lead Measurement Modeling Molecular Muscle Contraction Muscle Fibers Muscle function Mutation Myocardium Myosin ATPase Play Process Protein Isoforms Proteins Quality of life Regulation Relative (related person)Role Signal Transduction Skeletal Muscle Solutions Stretching Structure System Techniques Testing Thin Filament Titrations Tropomyosin Troponin Troponin C Troponin I actin-S1 base crosslink genetic regulatory protein mutant novel premature protein protein interaction reconstitution reconstruction research study response tool

项目摘要

A number of familial hypertrophic cardiomyopathy (FHC) causing mutations have been identified in the regulatory proteins, tropomyosin (Tm) and troponin (Tn). Most of these mutations cause an increase in the Ca2+-sensitivity of muscle contraction, i.e. the onset of force occurs at lower Ca2+ concentrations. Neither the molecular mechanisms underlying the increased Ca2+-sensitivity nor its relation to the hypertrophy of the heart are well understood. Stretch activation is another cardiac phenomenon whose molecular mechanism is not understood. The long-range goal is to understand the molecular basis of FHC and stretch activation. The main hypothesis that we will test is that both of these activations involve strongly bound cross bridges (myosin heads). We plan to: 1. Determine the contribution of the myosin head-induced vs. Ca2+-induced changes in the interactions of troponin I (TnI) with actin-Tm and with troponin C (TnC) in thin filaments reconstituted with skeletal and cardiac muscle isoforms of the regulatory proteins. The main techniques will be solution ATPase and FRET measurements. 2. Determine effects of FHC mutations on occupancy of the 3 thin filament regulatory states using equilibrium titrations and transient kinetics. Fluorescent labels on selected proteins will be used to obtain equilibrium constants and rates. 3. Determine effects of selected FHC mutations in TnI and Tm on ATPase in terms of myosin vs. Ca2+ activation. FRET measurements will be used to obtain structural information. 4. Test the hypothesis that the C-terminal domain of TnC is involved in the myosin head induced activation of the thin filament. Mutants of TnC having increased affinity for Mg2+ will be used to assess the role of divalent cation in the C-domain of TnC on thin filament function. A novel mutant of TnC which reconstitutes into the thin filament and binds Ca2+ but does not activate ATPase that was developed in this lab will be used. These experiments will lead to a better understanding of the regulatory mechanism in cardiac and skeletal muscle. In particular, a better understanding of the relative contribution of the Ca2+/troponin-dependent and the myosin S1/actin-dependent activation of the thin filament will be obtained. By identifying the protein-protein interactions that are altered in the disease state it will be possible to suggest potential targets for drug design for FHC.

许多家族性肥厚型心肌病（FHC）引起的突变已被证实在调节蛋白原肌球蛋白 (Tm) 和肌钙蛋白 (Tn) 中鉴定。其中大部分突变导致肌肉收缩的 Ca2+ 敏感性增加，即力量的开始发生在较低的 Ca2+ 浓度下。其背后的分子机制都不是 Ca2+ 敏感性增加及其与心脏肥大的关系已得到充分了解。牵张激活是另一种心脏现象，其分子机制尚不明确。明白了。长期目标是了解 FHC 和拉伸的分子基础激活。我们要测试的主要假设是这两种激活都强烈涉及结合的十字桥（肌球蛋白头）。我们计划： 1. 确定肌球蛋白头引起的与 Ca2+ 引起的变化的贡献细丝中肌钙蛋白 I (TnI) 与肌动蛋白-Tm 和肌钙蛋白 C (TnC) 的相互作用用调节蛋白的骨骼和心肌亚型重建。主要技术将是溶液 ATP 酶和 FRET 测量。 2. 确定 FHC 突变对 3 种细丝调节状态占用的影响使用平衡滴定和瞬态动力学。选定蛋白质上的荧光标记将用于获得平衡常数和速率。 3. 确定 TnI 和 Tm 中选定的 FHC 突变对 ATPase 的影响肌球蛋白与 Ca2+ 激活。 FRET 测量将用于获取结构信息。 4. 检验 TnC C 端结构域参与肌球蛋白头的假设诱导细丝激活。对 Mg2+ 亲和力增加的 TnC 突变体将是用于评估 TnC C 结构域中二价阳离子对细丝功能的作用。 TnC 的一种新型突变体，可重组为细丝并结合 Ca2+，但不将使用本实验室开发的不激活 ATP 酶。这些实验将导致更好地了解心肌和骨骼肌的调节机制。在特别是，更好地了解 Ca2+/肌钙蛋白依赖性和将获得细丝的肌球蛋白 S1/肌动蛋白依赖性激活。通过识别在疾病状态下改变的蛋白质-蛋白质相互作用可能表明 FHC 药物设计的潜在目标。