Structure and Mechanism of Cardiomyopathy Myosin Mutants

心肌病肌球蛋白突变体的结构和机制

• 批准号: 8903525
• 负责人: Eva Forgacs
• 金额: $ 38.73万
• 依托单位: EASTERN VIRGINIA MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8903525
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Actomyosin; Address; Affect; Automobile Driving; Benign; Beryllium; Binding; Biochemical; Biological Assay; Cardiac; Cardiac Myosins; Cardiomyopathies; Cells; Chemicals; Clinical Management; Clinical Treatment; Coupling; Development; Dilated Cardiomyopathy; Disease; Dissociation; Docking; Family suidae; Gap Junctions; Generations; Genes; Goals; H-Meromyosin; Health; Heart; Heart Diseases; Heterogeneity; Human; Hydrolysis; Hypertrophic Cardiomyopathy; In Vitro; Infection; Inherited; Kinetics; Lead; Link; Measurement; Measures; Mechanics; Methods; Missense Mutation; Molecular; Motion; Motor; Movement; Mutation; Myocardial Contraction; Myopathy; Myosin ATPase; Myosin Heavy Chains; Output; Pathway interactions; Patients; Pharmaceutical Preparations; Phase II Clinical Trials; Phenotype; Population; Power stroke; Production; Property; Proteins; Recovery; Specimen; Spectrum Analysis; Striated Muscles; Structure; Symptoms; System; Systolic heart failure; Tissues; Variant; Work; adenoviral-mediated; arm; base; biophysical properties; cell motility; design; disease characteristic; driving force; inorganic phosphate; insight; multidisciplinary; mutant; prevent; single molecule; small molecule; stroke recovery; sudden cardiac death

DESCRIPTION (provided by applicant): Mutations in the β-cardiac myosin heavy chain are responsible for a large number of inherited Hypertrophic (HCM) and Dilated Cardiomyopathies (DCM). The objective of the proposal is the characterization of biophysical and biochemical properties of the mutant human cardiac myosin as an essential first step in identifying the changes in the structure and mechanism resulting in inherited cardiomyopathies. The difficulties in studying human cardiac myosin were due to the instability and heterogeneity of the protein obtained from patient tissues and the lack of an adequate expression system to produce high quality human β-cardiac myosin in quantities that are sufficient for structural and kinetic analysis. We developed a mammalian expression system based on adenoviral infection of C2C12 cells enabling us to produce mg quantities of the human β -cardiac motor domain and heavy meromyosin required for kinetic and structural studies. The crystal structure of the wild type β-cardiac motor domain reveals a cluster of HCM and DCM mutations in a region linking the SH-1 helix, the converter domain, the wedge loop, and the relay helix/relay loop (Coupling Region) by a cluster of hydrophobic residues (Hydrophobic Nexus). The concerted movement of these elements is critical for mechanochemical coupling in the myosin motor. Docking and structural studies of the motor domain with the small molecule activator, omecamtiv mecarbil, indicate that it binds adjacent to the Hydrophobic Nexus and may act by influencing the mechanochemical coupling mechanism. Eight HCM/DCM mutants representing the benign and severe phenotypes residing in the Coupling Region were selected to study the changes in the structure and mechanism of the mutant motor by steady-state and rapid kinetics (Aim 1.) in vitro motility assays and single molecule force measurements (Aim 2.) and crystallographic structure (Aim 3). The effect of omecamtiv mecarbil on the coupling mechanism of mutant cardiac myosins may provide insights into the clinical management of the disease specifically for DCM patients. Understanding the molecular mechanism of cardiomyopathies will provide the basis for the development of new assays for targeted therapies.

描述（由申请方提供）：β-心肌肌球蛋白重链突变导致大量遗传性肥厚型（HCM）和扩张型心肌病（DCM）。该提案的目的是表征突变型人类心肌肌球蛋白的生物物理和生物化学特性，作为确定导致遗传性心肌病的结构和机制变化的重要第一步。研究人心肌肌球蛋白的困难是由于从患者组织中获得的蛋白质的不稳定性和异质性以及缺乏足够的表达系统来产生足以进行结构和动力学分析的高质量人β-心肌肌球蛋白。我们开发了一种基于腺病毒感染C2 C12细胞的哺乳动物表达系统，使我们能够产生动力学和结构研究所需的mg量的人β -心脏运动结构域和重肌球蛋白。野生型β-心脏运动结构域的晶体结构揭示了在通过疏水残基簇（疏水连接）连接SH-1螺旋、转换器结构域、楔形环和中继螺旋/中继环（偶联区）的区域中的HCM和DCM突变簇。这些元素的协同运动对于肌球蛋白马达中的机械化学偶联是至关重要的。马达结构域与小分子激活剂omecamtiv mecarbil的对接和结构研究表明，它与疏水性连接附近结合，并可能通过影响机械化学偶联机制发挥作用。选择了8个HCM/DCM突变体，代表了位于偶联区的良性和严重表型，通过稳态和快速动力学研究突变马达的结构和机制的变化（目的1）。体外运动性测定和单分子力测量（Aim 2.）和晶体结构（目标3）。Omecamtiv Mecarbil对突变心肌肌球蛋白偶联机制的影响可能为DCM患者的临床管理提供见解。了解心肌病的分子机制将为开发新的靶向治疗方法提供基础。