Molecular Mechanics of Mutant Cardiac Myosin

突变心肌肌球蛋白的分子力学

• 批准号: 8236854
• 负责人: JEFFREY R MOORE
• 金额: $ 40.26万
• 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/8236854
• 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; 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（由申请人提供）：由家族性肥厚性心肌病（FHC）引起的心脏肥大，肌原纤维混乱和猝死，这是由于肉类蛋白中常染色体显性突变的结果。肌球蛋白是一种与肌动蛋白相互作用的肌肉分子运动，是一种由两个重链组成的六聚体蛋白。每个重型链结合了两个光链，一个必需（ELC）和一个调节（RLC）。轻链结合（颈部/杆）结构域放大了依赖ATP的构象变化，该构象变化起源于肌球蛋白活性位点以产生力和运动。鉴于肌球蛋白在力产生中的轻链结合（颈/杆）结构域的重要性，在RLC中已经确定了几个FHC突变也就不足为奇了。该提案的目的是为RLC突变患者提供FHC的分子基础。由于肌球蛋白分子生物化学与产生构象变化有关，因此预计肌球蛋白生化循环的几个步骤取决于菌株。因此，我们将通过肌球蛋白颈部区域研究外力向肌球蛋白活性部位的传播。由于临床表现取决于特定突变，因此这些研究是治疗方案开发的必要先驱。 我们将测试以下假设：肌球蛋白RLC中的突变降低了肌球蛋白颈域充当应变传感器的能力，从而改变力向活性位点传递，从而改变应变依赖性动力学和功率输出。选择的研究突变位于可磷酸磷酸丝氨酸和RLC分子的EF手（A13T，N47K，R58Q和D166V）附近。从历史上看，这些区域对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，而且还将解决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法评估RLC突变对功率输出（AIM 1）以及依赖性肌球蛋白动力学的影响（多分子）水平（AIM 2）。集合应变依赖性的任何改变将在单个肌球蛋白分子水平上进一步追求，以确定受突变影响的特定基础菌株依赖性菌株动力学转变（AIM 3）。此外，与我们的初步数据一致，已提出RLC磷酸化来抑制肥大，从而提高收缩性能和效率。因此，我们将确定RLC的磷酸化是否拯救了RLC-FHC表型（AIM 4）。我们的方法测量了分离的收缩蛋白的机械性能。因此，我们将确定FHC突变对肌动蛋白的直接影响。了解RLC突变如何影响肌球蛋白的固有功能将允许更高功能单元的改变程度，例如心肌纤维，或心脏本身与主要收缩缺陷相关。 公共卫生相关性：家族性肥厚性心肌病（FHC）是一种遗传性心脏病，是由分子机械中突变引起的，可以使心脏收缩。这项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。