Troponin and myosin in regulation of muscle contraction and heart disease

描述（由申请人提供）：在调节蛋白原肌球蛋白（Tm）和肌钙蛋白（Tn）中发现了许多家族性肥厚性心肌病（FHC）引起的突变。大多数这些突变引起Ca2+对肌肉收缩的敏感性增加，即在较低的Ca2+浓度下发生力的发作。Ca2+敏感性增加的分子机制及其与心脏肥大的关系都没有得到很好的理解。拉伸激活是另一种分子机制尚不清楚的心脏现象。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的与Ca2+诱导的肌钙蛋白I （TnI）与肌动蛋白- tm和肌钙蛋白C （TnC）在由调节蛋白的骨骼肌和心肌同种型重构的细丝中的相互作用变化的贡献。主要技术将是溶液atp酶和FRET测量。2. 利用平衡滴定和瞬态动力学确定FHC突变对3种细丝调控状态占用的影响。选定蛋白质上的荧光标记将用于获得平衡常数和速率。3. 确定选定的FHC突变在TnI和Tm对肌球蛋白和Ca2+激活方面的atp酶的影响。FRET测量将用于获得结构信息。4. 验证TnC的c端结构域参与肌凝蛋白头诱导的细丝活化的假设。对Mg2+亲和力增加的TnC突变体将用于评估TnC c结构域二价阳离子对细丝功能的作用。将使用在本实验室开发的一种新的TnC突变体，该突变体重组成细丝并结合Ca2+，但不激活atp酶。这些实验将有助于更好地理解心脏和骨骼肌的调控机制。特别是，将更好地了解Ca2+/肌钙蛋白依赖和肌球蛋白S1/肌动蛋白依赖的细丝激活的相对贡献。通过鉴定在疾病状态下发生改变的蛋白-蛋白相互作用，将有可能为FHC的药物设计提出潜在的靶点。肌肉收缩关系是一个复杂的过程，涉及多种蛋白质之间的相互作用。对家族性肥厚性心肌病（FHC）的研究表明，心肌对激活信号的反应即使发生轻微变化，也可能导致严重的疾病、生活质量下降和过早死亡。提出的研究将有助于更好地了解心脏和骨骼肌的调节机制。通过确定蛋白质-蛋白质相互作用在疾病状态下发生改变的方面，将有可能为FHC的药物设计提出潜在的靶点

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