Molecular Mechanics of Mutant Cardiac Myosin

突变心肌肌球蛋白的分子力学

• 批准号: 7888730
• 负责人: JEFFREY R MOORE
• 金额: $ 41.95万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7888730
• 关键词: Actins; Active Sites; Actomyosin; Address; Affect; Attenuated; Binding; Biochemical; Biochemistry; Biological Assay; Cardiac; Cardiac Muscle Contraction; Cardiac Myosins; Clinical; Communication; Contractile Proteins; Contracts; Data; Defect; Dependence; Disease; EF Hand Motifs; Exhibits; Familial Hypertrophic Cardiomyopathy; Feedback; Generations; Genetic; Goals; Heart; Heart Diseases; Heart Hypertrophy; Hypertrophy; In Vitro; Induced Mutation; Isometric Exercise; Kinetics; Knowledge; Laboratories; Light; Link; Measurement; Measures; Mechanics; Molecular; Molecular Motors; Motion; Muscle Fibers; Mutation; Myocardium; Myosin ATPase; Myosin Regulatory Light Chains; Neck; Nucleotides; Output; Pathway interactions; Patients; Performance; Phenotype; Phosphorylation; Production; Property; Proteins; Protocols documentation; Serine; Sudden Death; System; Techniques; Testing; Therapeutic Agents; Work; arm; base; cell motility; molecular mechanics; mutant; novel; optical traps; public health relevance; research study; sensor; single molecule; sudden cardiac death; therapeutic development; transmission process; young adult

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.

描述（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。肌球蛋白是一种与肌动蛋白相互作用以驱动心肌收缩的肌节分子马达，是一种由两条重链组成的六聚体蛋白质。每条重链结合两条轻链，一条必需轻链 (ELC) 和一条调节轻链 (RLC)。轻链结合（颈/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。鉴于肌球蛋白的轻链结合（颈/杆）结构域在力产生中的重要性，在 RLC 中发现多个 FHC 突变也就不足为奇了。该提案的目标是为 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产生构象变化的力有关，因此预计肌球蛋白生化循环中的几个步骤是应变依赖性的。因此，我们将研究外力通过肌球蛋白颈部区域传递到肌球蛋白活性位点。由于临床表现取决于特定的突变，这些研究是制定治疗方案的必要前提。 我们将测试这样的假设：肌球蛋白 RLC 的突变会降低肌球蛋白颈域作为应变传感器的能力，从而改变向活性位点传递力的能力，从而导致应变依赖性动力学和功率输出的改变。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（A13T、N47K、R58Q 和 D166V）。历史证明，这些区域对于肌球蛋白功能很重要。因此，我们的实验不仅将为 FHC 提供分子基础，还将解决 RLC 功能的基本方面和肌球蛋白运动产生的分子基础。我们的方法将利用体外运动测定来评估 RLC 突变对功率输出的影响（目标 1）以及整体（多分子）水平上的应变依赖性肌球蛋白动力学（目标 2）。整体菌株依赖性的任何改变都将在单个肌球蛋白分子水平上进一步追踪，以确定受突变影响的特定潜在菌株依赖性肌动球蛋白动力学转变（目标 3）。此外，与我们的初步数据一致，RLC 磷酸化被认为可以通过增强收缩性能和效率来抑制肥大。因此，我们将确定 RLC 磷酸化是否可以挽救 RLC-FHC 表型（目标 4）。我们的方法测量分离的收缩蛋白的机械特性。因此，我们将确定 FHC 突变对肌动球蛋白的直接影响。了解 RLC 突变如何影响肌球蛋白的固有功能将使高级功能单位（例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。 公众健康相关性：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。这项研究将研究心脏一种蛋白质（肌球蛋白调节轻链）中的 FHC 突变如何在分子水平上改变心脏产生力量和功率的能力。