Molecular Mechanics of Mutant Cardiac Myosin

描述（由申请人提供）：家族性肥厚型心肌病（FHC）引起的心脏肥大、肌原纤维紊乱和猝死是由肌节蛋白的常染色体显性突变引起的。肌球蛋白是一种由两条重链组成的六聚体蛋白，是与肌动蛋白相互作用以驱动心肌收缩的肌节分子马达。每条重链结合两条轻链，一条必需（ELC），一条调节（RLC）。轻链结合（颈/杠杆）结构域放大源自肌球蛋白活性位点的ATP依赖性构象变化，以产生力和运动。考虑到肌球蛋白轻链结合（颈/杠杆）结构域在力产生中的重要性，在RLC中鉴定出几个FHC突变并不奇怪。该建议的目的是为RLC突变患者的FHC提供分子基础。由于肌球蛋白分子生物化学与力产生构象变化有关，因此预期肌球蛋白生物化学循环中的几个步骤是菌株依赖性的。因此，我们将研究外力通过肌球蛋白颈区传递到肌球蛋白活性部位。由于临床表现取决于特定的突变，这些研究是开发治疗方案的必要前提。 我们将测试的假设，即突变的肌球蛋白RLC降低的肌球蛋白颈域的能力，作为一个应变传感器，这改变了交付的力的活性位点，导致改变应变依赖性动力学和功率输出。选择用于研究的突变位于可磷酸化丝氨酸和RLC分子的EF-手附近（A13 T、N47 K、R58 Q和D166 V）。这些区域在历史上已被证明是重要的肌球蛋白功能，因此，我们的实验不仅提供了FHC的分子基础，但也将解决RLC功能的基本方面和肌球蛋白运动生成的分子基础。我们的方法将利用体外运动试验来评估RLC突变对功率输出（目标1）以及在系综（多分子）水平（目标2）的应变依赖性肌球蛋白动力学的影响。将在单个肌球蛋白分子水平上进一步研究整体应变依赖性的任何改变，以确定受突变影响的特定潜在应变依赖性肌动球蛋白动力学转变（目的3）。此外，与我们的初步数据一致，RLC磷酸化已被提出通过有助于增强收缩性能和效率来抑制肥大。因此，我们将确定RLC的磷酸化是否挽救RLC-FHC表型（目的4）。我们的方法测量分离的收缩蛋白的机械性质。因此，我们将确定FHC突变对肌动球蛋白的直接影响。RLC突变如何影响肌球蛋白的固有功能的知识将允许更高的功能单位，如心肌纤维，或心脏本身的改变程度与原发性收缩缺陷相关。 公共卫生相关性：家族性肥厚型心肌病（FHC）是一种遗传性心脏病，由允许心脏收缩的分子机制突变引起。本研究将研究心脏蛋白之一（肌球蛋白调节轻链）中的FHC突变如何改变心脏在分子水平上产生力和功率的能力。

DESCRIPTION (provided by applicant): The cardiac hypertrophy, myofibrillar disarray and sudden death caused by familial hypertrophic cardiomyopathy (FHC) results from autosomal dominant mutations in sarcomeric proteins. Myosin, the sarcomeric molecular motor that interacts with actin to power cardiac muscle contraction, is a hexameric protein consisting of two heavy chains. Each heavy chain binds two light chains, one essential (ELC) and one regulatory (RLC). The light chain binding (neck/lever) domain amplifies ATP dependent conformational changes originating in the myosin active site to generate force and motion. Given the importance of the light chain binding (neck/lever) domain of myosin in force production, it is not surprising that several FHC mutations have been identified in the RLC. The goal of this proposal is to provide a molecular basis for FHC in patients with mutations in the RLC. Since myosin molecule biochemistry is linked to force producing conformational changes, it is expected that several steps in the myosin biochemical cycle are strain dependent. Therefore, we will study the transmission of external forces to the myosin active site via the myosin neck region, . Since the clinical presentation depends on the specific mutation, these studies are a necessary precursor to development of therapeutic protocols. We will test the hypothesis that mutations in the myosin RLC decrease the ability of the myosin neck domain to act as a strain sensor, which alters the delivery of force to the active site, leading to altered strain dependent kinetics and power output. The mutations chosen for study are localized near the phosphorylatable serine and the EF-hand of the RLC molecule (A13T, N47K, R58Q and D166V). These regions have historically been shown to be important for myosin function; thus our experiments will not only provide a molecular basis for FHC but will also address fundamental aspects of RLC function and the molecular basis of myosin motion generation. Our approach will utilize in vitro motility assays to assess the effects of RLC mutations on power output (Aim 1) as well as strain dependent myosin kinetics at the ensemble (multiple molecule) level (Aim 2). Any alterations in ensemble strain dependence will be further pursued at the single myosin molecule level to determine the specific underlying strain dependent actomyosin kinetic transitions affected by the mutations (Aim 3). Furthermore, consistent with our preliminary data, RLC phosphorylation has been proposed to inhibit hypertrophy by contributing to enhanced contractile performance and efficiency. Therefore, we will determine if phosphorylation of the RLC rescues the RLC-FHC phenotypes (Aim 4). Our approach measures the mechanical properties of isolated contractile proteins. Therefore, we will determine the direct effects of the FHC mutations on actomyosin. Knowledge of how the RLC mutations affect myosin's inherent function will allow the degree of alteration of higher functional units, such as the cardiac muscle fiber, or the heart itself to be correlated with a primary contractile defect. PUBLIC HEALTH RELEVANCE: Familial hypertrophic cardiomyopathy (FHC) is a genetic heart disease that is caused by mutations in the molecular machinery that allows the heart to contract. This study will examine how FHC mutations in one of the proteins of the heart (the myosin regulatory light chain), alters the ability of the heart to generate force and power at the molecular level.