Effects of hypertrophic cardiomyopathy (HCM) causing mutations on sequestration of human β-cardiac myosin via intra-molecular interactions

肥厚型心肌病 (HCM) 通过分子内相互作用引起突变对人 β-心肌肌球蛋白隔离的影响

• 批准号: 9469314
• 负责人: Dan Song
• 金额: $ 5.83万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9469314
• 关键词: ATP phosphohydrolase; Actins; Adopted; Adult; Affect; Affinity; Back; Binding; Biochemical; Biomechanics; Cardiac; Cardiac Myosins; Cardiovascular Diseases; Clinical; Cryoelectron Microscopy; Data; Development; Disease; Drug Targeting; Dyes; Electrostatics; Equilibrium; Genes; Goals; H-Meromyosin; Head; Heart Contractilities; Heritability; Homology Modeling; Human; Human Activities; Hypertrophic Cardiomyopathy; Kinetics; Label; Lead; Measures; Missense Mutation; Molecular; Molecular Conformation; Monitor; Motor; Motor output; Mutation; Myocardium; Myosin ATPase; Output; Pathogenesis; Patients; Proteins; Recombinants; Regulation; Research; Sarcomeres; Site; Sodium Chloride; Striated Muscles; Structure; Surface; Tail; Testing; Thick Filament; Ventricular; Work; base; biophysical analysis; cyanine dye 5; disease-causing mutation; early onset; heart function; human model; mutant; novel strategies; prevent; single-molecule FRET; small molecule; small molecule inhibitor; sudden cardiac death; targeted treatment; young adult

Project Summary/Abstract Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disease that is the leading cause of sudden cardiac death in young adults. More than half of all HCM patients are identified to carry missense mutations in genes encoding saromeric proteins, predominantly human β-cardiac myosin, the thick filament motor that powers ventricular contraction. Current treatment for HCM is limited to symptomatic relief. It is pressing to understand how HCM-causing mutations in human β-cardiac myosin alter the biomechanical function of the motor protein at the molecular level, which is a necessary prerequisite for the development of targeted therapies. Recent biochemical and biophysical studies using recombinant human β-cardiac myosin suggest that early-onset HCM-causing mutations significantly increase the power output of the motor by increasing velocity, intrinsic force, and ATPase activity, consistent with clinical observations that HCM-causing mutations lead to hyper-contractility of the heart muscle. However, similar studies of mutations that give rise to severe disease in adulthood have shown only subtle effects on these parameters. An overlooked parameter in the biomechanical function of the myosin motor protein is the number of myosin heads functionally available for interaction with actin (Na). CryoEM studies of striated muscle myosins suggest that myosin heads (S1) may fold back and interact with their proximal tail region (proxS2) and with each other. The only functional data to support this idea are from the Spudich lab demonstrating that recombinant human β-cardiac myosin S1 can bind to proxS2 in a salt-dependent manner. We hypothesize that this intra-molecular interaction possibly sequester myosin heads and prevent them from interacting with actin, thus regulating Na and imparting fine-tuned control of cardiac contractility. HCM-causing mutations located on the interacting surfaces will weaken this interaction and lead to an increase in Na, thus freeing myosin heads to interact with actin and causing hyper-contractility. To test this hypothesis, I propose to (1) Measure changes in the binding affinities between S1 myosin head and proxS2 myosin tail induced by HCM-causing mutations using Microscale Thermophoresis, (2) Directly visualize conformational change of human β-cardiac myosin between an open and a sequestered state using a novel approach based on single-molecule fluorescence resonance energy transfer, and (3) Determine the effects of HCM-causing mutations on sequestration-dependent changes in the actin-activated ATPase activity of human β-cardiac myosin. Our results will provide a more comprehensive understanding on how HCM-causing mutations affect the function of human β-cardiac myosin to generate power by determining whether these mutations alter the ability of myosin to adopt a sequestered conformation. Ultimately, this research will have a significant impact on the development of small molecule drugs targeted on specific changes in the structure and functions of the cardiac myosin induced by the disease-causing mutations.

项目摘要/摘要 肥厚性心肌病（HCM）是一种可遗传的心血管疾病，是突然的主要原因 年轻人心脏死亡。在所有HCM患者中，超过一半被确定为携带错义突变 编码讽刺蛋白的基因，主要是人β-心肌球蛋白，厚细丝运动 动力心室收缩。 HCM的当前治疗仅限于症状缓解。这是 了解人β-心肌球蛋白中引起HCM的突变如何改变生物力学功能 分子水平的运动蛋白的蛋白质，这是发展的必要先决条件 靶向疗法。最近使用重组人β-心肌球蛋白的生化和生物物理研究 表明早发性HCM引起的突变会大大增加电动机的功率输出 速度增加，内在力和ATPase活性，与临床观察结果一致 突变导致心肌超额收缩。但是，对引起的突变的类似研究 成年后的严重疾病对这些参数仅显示出微妙的影响。一个被忽略的参数 肌球蛋白运动蛋白的生物力学功能是可用于功能的肌球蛋白头的数量 与肌动蛋白（NA）的相互作用。纹状体肌球蛋白的冷冻研究表明肌球蛋白头（S1）可能 向后折叠并与它们的近端尾部区域（Proxs2）相互作用。唯一的功能数据 支持这个想法来自Spudich实验室，表明重组人β-甲肌球蛋白S1可以 以盐依赖性方式与Proxs2结合。我们假设这种分子内相互作用可能 隔离肌球蛋白头并防止它们与肌动蛋白相互作用，从而控制Na并赋予 对心脏收缩的微调控制。位于相互作用表面上的HCM引起的突变 将削弱这种相互作用并导致NA的增加，从而释放肌球蛋白头与 肌动蛋白并引起超收缩。为了检验这一假设，我建议（1）衡量的变化 使用HCM引起的突变引起的S1肌球蛋白头和Proxs2肌球蛋白尾之间的结合亲和力 显微镜热疗法，（2）直接可视化人β-心肌球蛋白的构象变化 使用基于单分子荧光共振的新方法开放和隔离状态 能量转移，（3）确定引起HCM的突变对隔离依赖性变化的影响 在肌动蛋白激活的人β-心肌球蛋白的ATPase活性中。我们的结果将提供更多 对HCM引起的突变如何影响人β-心肌球蛋白的功能的全面了解 通过确定这些突变是否会改变肌球蛋白采用隔离的能力来产生力量 构象。最终，这项研究将对小分子的发展产生重大影响 药物针对的是由诱导的心脏肌球蛋白结构和功能的特定变化 引起疾病的突变。