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

项目摘要/摘要 肥厚性心肌病(HCM)是一种遗传性心血管疾病，是猝发的主要原因 年轻人的心源性死亡。超过一半的肥厚型心肌炎患者被确认携带错义突变 编码肌球蛋白的基因，主要是人类的β-心肌肌球蛋白，这种粗丝马达 增强了心室的收缩。目前对肥厚型心肌炎的治疗仅限于症状缓解。它迫在眉睫地 了解hCM引起的人β-心肌肌球蛋白突变如何改变生物力学功能 在分子水平上的马达蛋白，这是发展的必要前提 有针对性的治疗。重组人β-心肌肌球蛋白的生化和生物物理研究进展 提示早发性HCM引起的突变通过以下方式显著增加马达的功率输出 速度、内力和ATPase活性增加，与临床观察一致 基因突变会导致心肌过度收缩。然而，类似的研究导致了 成年期的严重疾病对这些参数只显示出微妙的影响。中被忽略的参数 肌球蛋白马达蛋白的生物力学功能是肌球蛋白头部的功能 与肌动蛋白(Na)的相互作用。对横纹肌肌球蛋白的冷冻EM研究表明，肌球蛋白头部(S1)可能 向后折叠，并与它们的近端尾部区域(ProxS2)相互作用。的唯一功能数据 支持这一想法的是斯普迪奇实验室证明的重组人β-心肌肌球蛋白S1可以 以依赖于盐的方式与proxS2结合。我们假设这种分子内相互作用可能 隔离肌球蛋白头部并阻止它们与肌动蛋白相互作用，从而调节钠和传递 对心脏收缩能力的微调控制。位于相互作用表面的引起HCM的突变 会削弱这种相互作用并导致钠的增加，从而释放肌球蛋白头部以与之相互作用 肌动蛋白和引起过度收缩。为了检验这一假设，我建议(1)测量 HCM突变诱导S1肌球蛋白头部与ProxS2肌球蛋白尾部结合亲和力的研究 微尺度热电泳法，(2)直接可视化人β-心肌肌球蛋白在 基于单分子荧光共振新方法的开关态和隔离态 能量转移，以及(3)确定HCM引起的突变对隔离依赖的改变的影响 肌动蛋白激活的人β-心肌肌球蛋白的ATPase活性。我们的结果将为我们提供更多 全面了解hCM基因突变对人β-心肌肌球蛋白功能的影响 通过确定这些突变是否改变肌球蛋白采用隔离的能力来产生能量 构象。最终，这项研究将对小分子的发展产生重大影响 靶向靶向改变心肌肌球蛋白结构和功能的药物 致病突变。

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.