Troponin and myosin in regulation of muscle contraction and heart disease

一些引起突变的家族性肥厚型心肌病(FHC)已经被 在调节蛋白中发现原肌球蛋白(TM)和肌钙蛋白(TN)。其中大多数都是 突变导致肌肉收缩对钙的敏感性增加，即力量的开始 在较低的钙离子浓度下发生。无论是潜在的分子机制， 钙敏感性的增加及其与心脏肥厚的关系是众所周知的。 伸展激活是另一种心脏现象，其分子机制不是 明白了。长期目标是了解FHC和Stretch的分子基础 激活。我们将测试的主要假设是，这两种激活都强烈地涉及 捆绑的交叉桥(肌球蛋白头部)。我们计划： 1.确定肌球蛋白头部诱导的与钙离子诱导的改变之间的关系 肌钙蛋白I(TnI)与肌动蛋白-TM和肌钙蛋白C(TNC)在细丝中的相互作用 由骨骼肌和心肌调节蛋白的异构体重组。主 技术将是溶液ATPase和FRET测量。 2.确定FHC突变对3种细丝调节状态占用的影响 使用平衡滴定和瞬时动力学。选定蛋白质上的荧光标记将是 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VS钙激活。将使用FRET测量来获得结构信息。 4.验证TNC的C-末端结构域参与肌球蛋白头部的假设 诱导细丝的激活。TNC对镁离子亲和力增加的突变体将是 用于评估TNC C结构域中的二价阳离子对细丝功能的作用。 一种新的TNC突变体，它重组成细丝并与钙结合，但 将使用在本实验中开发的不激活ATPase。这些实验将导致 更好地了解心肌和骨骼肌的调节机制。在……里面 特别是，更好地理解钙/肌钙蛋白依赖的和 肌球蛋白S1/肌动蛋白依赖的细丝激活将被获得。通过识别 在疾病状态下改变的蛋白质-蛋白质相互作用将可能表明 FHC药物设计的潜在靶点。

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.