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

基本信息

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
项目类别：
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

项目摘要

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至关重要，包括肌肉收缩、细胞分裂和细胞内运输。肌球蛋白在其各个阶段的原子水平结构。酶循环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化和结构数据表明，肌球蛋白通过耦合核苷酸结合区域中的小构象变化来产生力。轻链结合区（杠杆臂）的大幅摆动，而肌球蛋白与肌动蛋白强烈结合。人类β心肌肌球蛋白的突变与多种形式的心肌病有关，但尚不清楚这些突变如何导致不同的疾病病理。我们认为突变通过破坏肌动蛋白激活 ATP 水解产物（磷酸盐和 ADP）释放并触发杠杆臂摆动所需的子域协调来改变力产生的保守结构机制。将研究突变如何影响肌动蛋白结合、核苷酸结合和杠杆臂区域的特定构象变化，将战略性地放置新型外在荧光探针，以利用荧光共振能量转移（FRET）来测量这三个关键区域的构象变化。此外，瞬态动力学实验将用于将构象变化与肌动球蛋白 ATP 酶循环中的特定生化步骤相关联，将通过瞬态时间解析来检查关键生化转变期间结构状态整体的变化。 FRET。我们还将研究突变如何改变肌球蛋白的酶和力生成特性，这将使我们能够开发出突变如何损害运动结构和功能的详细模型，我们将确定心脏肌球蛋白激活剂药物如何改变构象动力学。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性的治疗药物，并确定与心力衰竭相关的结构缺陷。心肌病的肌球蛋白突变。