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

项目总结/摘要 肥厚型心肌病（HCM）是一种遗传性心血管疾病，是导致心脏骤停的主要原因。 年轻人的心源性死亡超过一半的HCM患者被鉴定为携带错义突变， 编码染色体蛋白的基因，主要是人类β-心肌肌球蛋白， 为心室收缩提供动力目前对HCM的治疗仅限于症状缓解。迫切需要 了解HCM引起的人类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，这是一个必要的先决条件的发展， 靶向治疗。重组人β-心肌肌球蛋白的生物化学和生物物理学研究进展 表明早发性HCM引起的突变显著增加了电机的功率输出， 增加速度，内力和ATP酶活性，与临床观察结果一致，HCM引起的 突变导致心肌过度收缩。然而，类似的突变研究，引起 成年期严重疾病对这些参数的影响很小。一个被忽略的参数， 肌球蛋白马达蛋白的生物力学功能是肌球蛋白头的数量， 与肌动蛋白（Na）的相互作用。横纹肌肌球蛋白的冷冻EM研究表明肌球蛋白头（S1）可能 折叠并与它们的近端尾区（CD 4S 2）相互作用。唯一的功能数据， Spudich实验室证实了重组人β-心肌肌球蛋白S1可以 以盐依赖性方式结合到BNS 2。我们假设这种分子间的相互作用可能 隔离肌球蛋白头，并防止它们与肌动蛋白相互作用，从而调节钠和赋予 对心脏收缩力的微调控制。位于相互作用表面的HCM引起突变 将减弱这种相互作用，导致Na增加，从而释放肌球蛋白头与 肌动蛋白并导致过度收缩。为了验证这一假设，我建议（1）测量 使用HCM引起的突变诱导的S1肌球蛋白头和S1 S2肌球蛋白尾之间的结合亲和力， （2）直接观察人β-心肌肌球蛋白在不同温度下的构象变化， 使用基于单分子荧光共振的新方法的开放和隔离状态 能量转移，和（3）确定HCM引起的突变对螯合依赖性变化的影响 人β-心肌肌球蛋白肌动蛋白激活的ATP酶活性。我们的结果将提供一个更 全面了解HCM引起的突变如何影响人β-心肌肌球蛋白的功能 通过确定这些突变是否改变了肌球蛋白采用隔离蛋白的能力来发电， 构象最终，这项研究将对小分子的发展产生重大影响。 靶向于由心肌纤维化引起的心肌肌球蛋白结构和功能的特异性变化的药物， 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.