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

• 批准号: 7994840
• 负责人: Zenon Grabarek
• 金额: $ 50.75万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-15 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7994840
• 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; Health; 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

DESCRIPTION (provided by applicant): 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 f FHC. PUBLIC HEALTH RELEVANCE Lation of muscle contraction is a complex process involving interactions among multiple proteins. As studies on familial hypertrophic cardiomyopathy (FHC) have shown even a slight change in the response of the heart muscle to the activating signal may lead to a severe disease, a diminished quality of life and premature death. The proposed studies will lead to a better understanding of the regulatory mechanism in cardiac and skeletal muscles. By identifying the aspects of the protein-protein interactions that are altered in the disease state it will be possible to suggest potential targets for drug design for FHC

描述（由申请人提供）：已在调节蛋白原肌球蛋白（Tm）和肌钙蛋白（Tn）中鉴定出许多引起家族性肥厚型心肌病（FHC）突变的突变。大多数这些突变会导致肌肉收缩的 Ca2+ 敏感性增加，即力量的开始发生在较低的 Ca2+ 浓度下。 Ca2+敏感性增加的分子机制及其与心脏肥大的关系尚不清楚。牵张激活是另一种心脏现象，其分子机制尚不清楚。长期目标是了解 FHC 和拉伸激活的分子基础。我们将测试的主要假设是这两种激活都涉及强结合的交叉桥（肌球蛋白头）。我们计划： 1. 确定肌球蛋白头诱导与 Ca2+ 诱导的变化对骨骼肌和心肌重建细丝中肌钙蛋白 I (TnI) 与肌动蛋白-Tm 和肌钙蛋白 C (TnC) 相互作用的影响调节蛋白的亚型。主要技术是溶液 ATP 酶和 FRET 测量。 2. 使用平衡滴定和瞬态动力学确定 FHC 突变对 3 种细丝调节状态占据的影响。选定蛋白质上的荧光标记将用于获得平衡常数和速率。 3. 根据肌球蛋白与 Ca2+ 激活的情况，确定 TnI 和 Tm 中选定的 FHC 突变对 ATP 酶的影响。 FRET 测量将用于获取结构信息。 4. 检验以下假设：TnC 的 C 末端结构域参与肌球蛋白头诱导的细丝激活。对 Mg2+ 亲和力增加的 TnC 突变体将用于评估 TnC C 结构域中二价阳离子对细丝功能的作用。将使用本实验室开发的一种新型 TnC 突变体，该突变体可重组为细丝并结合 Ca2+，但不会激活 ATP 酶。这些实验将有助于更好地了解心肌和骨骼肌的调节机制。特别是，将更好地了解细丝的 Ca2+/肌钙蛋白依赖性和肌球蛋白 S1/肌动蛋白依赖性激活的相对贡献。通过识别疾病状态下改变的蛋白质-蛋白质相互作用，将有可能为 FHC 药物设计提出潜在目标。公共卫生相关性 肌肉收缩是一个复杂的过程，涉及多种蛋白质之间的相互作用。对家族性肥厚型心肌病 (FHC) 的研究表明，即使心肌对激活信号的反应发生轻微变化，也可能导致严重疾病、生活质量下降和过早死亡。拟议的研究将有助于更好地了解心肌和骨骼肌的调节机制。通过识别疾病状态下改变的蛋白质-蛋白质相互作用的各个方面，将有可能为 FHC 药物设计提出潜在目标