Impact of cardiomyopathy mutations on cardiac myosin structure and function

心肌病突变对心肌肌球蛋白结构和功能的影响

• 批准号: 9220678
• 负责人: CHRISTOPHER M YENGO
• 金额: $ 46.78万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-15 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9220678
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Actins; Actomyosin; Actomyosin Adenosinetriphosphatase; Binding; Biochemical; Biological Process; Cardiac Myocytes; Cardiac Myosins; Cardiomyopathies; Cell division; Classification; Communication; Congenital cardiomyopathy; Coupled; Coupling; Data; Defect; Depressed mood; Development; Disease; Dissociation; Drug Targeting; Fluorescence; Fluorescence Resonance Energy Transfer; Foundations; Functional disorder; Generations; Goals; Heart; Heart failure; Human; Impairment; In Vitro; Intracellular Transport; Kinetics; Label; Lead; Light; Link; Measures; Mediating; Microfilaments; Modeling; Molecular; Molecular Conformation; Molecular Motors; Motion; Motor; Motor Activity; Movement; Muscle Contraction; Mutation; Myocardium; Myosin ATPase; Myosin Type V; Nucleotides; Pathogenesis; Pathology; Pathway interactions; Patients; Periodicity; Pharmaceutical Preparations; Phenotype; Point Mutation; Power stroke; Property; Proteins; Research; Site; Structural defect; Structure; Sudden Death; System; Therapeutic; Thermodynamics; Time; Ventricular; arm; base; beta-Myosin; cell motility; experimental study; heart function; inorganic phosphate; insight; motor impairment; mutant; novel; novel therapeutics; programs; public health relevance; small molecule

 DESCRIPTION (provided by applicant): The ability of myosin to generate force and motion through its interaction with actin filaments is essential to many biological processes including muscle contraction, cell division, and intracellular transport. The atomic level structures of myosin in various stages of its enzymatic cycle have provided a framework of the molecular mechanism of force generation utilized by myosin. These structures as well as other biochemical and structural data suggest that myosin generates force by coupling small conformational changes in the nucleotide-binding region to a large swing of the light-chain binding region (lever arm) while myosin is strongly bound to actin. Mutations in human beta cardiac myosin are associated with several forms of cardiomyopathies, while it is unclear how the mutations lead to different disease pathologies. We propose the mutations alter the conserved structural mechanism of force generation by disrupting the subdomain coordination necessary for actin to activate the release of the products of ATP hydrolysis (phosphate and ADP) and trigger the force generating swing of the lever arm. We will investigate how the mutations impact specific conformational changes in the actin-binding, nucleotide-binding, and lever arm regions. Novel extrinsic fluorescence probes will be strategically placed to measure conformational changes in these three critical regions using fluorescence resonance energy transfer (FRET). In addition, transient kinetic experiments will be used to correlate the conformational changes with specific biochemical steps in the actomyosin ATPase cycle. The shift in the ensemble of structural states during key biochemical transitions will be examined by transient time resolved FRET. We will also investigate how the mutations alter the enzymatic and force generating properties of myosin, which will allow us to develop detailed models of how the mutations impair motor structure and function. We will determine how the cardiac myosin activator drug alters the conformational dynamics of human beta cardiac myosin and determine if it can rescue the altered motor structure-function in the cardiomyopathy mutants. Overall, our studies will be instrumental in developing therapeutic drugs that target myosin motor activity in heart failure and establishing the structural defects associated with cardiomyopathy mutations in myosin.

 描述（由申请人提供）：肌球蛋白通过与肌动蛋白丝相互作用产生力和运动的能力对于许多生物过程（包括肌肉收缩、细胞分裂和细胞内转运）是必不可少的。肌球蛋白在其酶循环的各个阶段的原子水平结构提供了肌球蛋白利用的力产生的分子机制的框架。这些结构以及其他生物化学和结构数据表明，肌球蛋白产生的力量耦合小的构象变化的核苷酸结合区的轻链结合区（杠杆臂）的大摆动，而肌球蛋白强烈结合肌动蛋白。人类β心肌肌球蛋白的突变与几种形式的心肌病有关，但目前还不清楚这些突变如何导致不同的疾病病理。我们提出的突变改变保守的结构机制的力产生破坏所需的肌动蛋白激活释放的ATP水解产物（磷酸盐和ADP）的子域协调，并触发力产生摆动的杠杆arm. We将调查突变如何影响特定的构象变化的肌动蛋白结合，核苷酸结合，杠杆臂区域。新型外源性荧光探针将被战略性地放置在这三个关键区域使用荧光共振能量转移（FRET）来测量构象变化。此外，瞬态动力学实验将被用来关联的构象变化与特定的生化步骤中的肌动球蛋白ATP酶循环。在关键的生化转变期间的结构状态的合奏的转变将通过瞬态时间分辨FRET检查。我们还将研究突变如何改变肌球蛋白的酶和力产生特性，这将使我们能够开发详细的模型，突变如何损害运动结构和功能。我们将确定心肌肌球蛋白激活剂药物如何改变人β心肌肌球蛋白的构象动力学，并确定它是否可以挽救心肌病突变体中改变的运动结构-功能。总体而言，我们的研究将有助于开发针对心力衰竭中肌球蛋白运动活动的治疗药物，并确定与肌球蛋白中心肌病突变相关的结构缺陷。