Molecular mechanics of mutant cardiac myosin

突变型心肌肌球蛋白的分子力学

基本信息

批准号：
9231099
负责人：
JEFFREY R MOORE
金额：
$ 13.5万
依托单位：
UNIVERSITY OF MASSACHUSETTS LOWELL
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-04-01 至 2017-05-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9231099
关键词：
Molecular mechanics mutant cardiac myosin

项目摘要

Abstract The cardiac hypertrophy, myofibrillar disarray and sudden death caused by familial hypertrophic cardiomyopathy (FHC) results from autosomal dominant mutations in sarcomeric proteins. Myosin, the sarcomeric molecular motor that interacts with actin to power cardiac muscle contraction, is a hexameric protein consisting of two heavy chains. Each heavy chain binds two light chains, one essential (ELC) and one regulatory (RLC). The light chain binding (neck/lever) domain amplifies ATP dependent conformational changes originating in the myosin active site to generate force and motion. Given the importance of the light chain binding (neck/lever) domain of myosin in force production, it is not surprising that several FHC mutations have been identified in the RLC. The goal of this proposal is to provide a molecular basis for FHC in patients with mutations in the RLC. Since myosin molecule biochemistry is linked to force producing conformational changes, it is expected that several steps in the myosin biochemical cycle are strain dependent. Therefore, we will study the transmission of external forces to the myosin active site via the myosin neck region, . Since the clinical presentation depends on the specific mutation, these studies are a necessary precursor to development of therapeutic protocols. We will test the hypothesis that mutations in the myosin RLC decrease the ability of the myosin neck domain to act as a strain sensor, which alters the delivery of force to the active site, leading to altered strain dependent kinetics and power output. The mutations chosen for study are localized near the phosphorylatable serine and the EF-hand of the RLC molecule (A13T, N47K, R58Q and D166V). These regions have historically been shown to be important for myosin function; thus our experiments will not only provide a molecular basis for FHC but will also address fundamental aspects of RLC function and the molecular basis of myosin motion generation. Our approach will utilize in vitro motility assays to assess the effects of RLC mutations on power output (Aim 1) as well as strain dependent myosin kinetics at the ensemble (multiple molecule) level (Aim 2). Any alterations in ensemble strain dependence will be further pursued at the single myosin molecule level to determine the specific underlying strain dependent actomyosin kinetic transitions affected by the mutations (Aim 3). Furthermore, consistent with our preliminary data, RLC phosphorylation has been proposed to inhibit hypertrophy by contributing to enhanced contractile performance and efficiency. Therefore, we will determine if phosphorylation of the RLC rescues the RLC-FHC phenotypes (Aim 4). Our approach measures the mechanical properties of isolated contractile proteins. Therefore, we will determine the direct effects of the FHC mutations on actomyosin. Knowledge of how the RLC mutations affect myosin's inherent function will allow the degree of alteration of higher functional units, such as the cardiac muscle fiber, or the heart itself to be correlated with a primary contractile defect.

抽象的家族性肥大引起的心脏肥大，肌原纤维混乱和猝死心肌病（FHC）是由肉瘤蛋白中常染色体显性突变引起的。肌球蛋白，与肌动蛋白相互作用与心脏肌肉收缩相互作用的肉瘤分子运动是一种六聚体蛋白质由两个重链组成。每个重链都结合了两个轻链，一个必需（ELC）和一个监管（RLC）。轻链结合（颈/杠杆）结构域扩增ATP依赖构象源自肌球蛋白活性部位以产生力和运动的变化。鉴于光的重要性肌球蛋白的链结合（颈部/杠杆）域在力产生中，几个FHC突变也就不足为奇了在RLC中已确定。该提案的目的是为患者提供FHC的分子基础与RLC中的突变。由于肌球蛋白分子生物化学与产生构象有关变化，预计肌球蛋白生化周期的几个步骤取决于应变。因此，我们将通过肌球蛋白颈部区域研究外力向肌球蛋白活性部位的传播。自从临床表现取决于特定突变，这些研究是制定治疗方案。我们将检验以下假设：肌球蛋白RLC中的突变降低了肌球蛋白颈的能力域可充当应变传感器，从而改变力向活性位点传递，从而改变应变依赖动力学和功率输出。选择用于研究的突变位于磷酸化的附近丝氨酸和RLC分子的EF手（A13T，N47K，R58Q和D166V）。这些地区历史上有被证明对肌球蛋白功能很重要；因此，我们的实验不仅将提供分子基础对于FHC，但还将解决RLC功能的基本方面和肌球蛋白运动的分子基础一代。我们的方法将利用体外运动测定法来评估RLC突变对功率的影响输出（AIM 1）以及依赖性肌球蛋白动力学在集合（多分子）水平（AIM 2）。合奏应变依赖性的任何改变将在单个肌球蛋白分子水平上进一步追求确定受突变影响的特异性下菌株依赖性肌球蛋白动力学转变（AIM 3）。此外，与我们的初步数据一致，已提出RLC磷酸化来抑制通过提高收缩性能和效率来促进肥大。因此，我们将确定是否 RLC的磷酸化营救了RLC-FHC表型（AIM 4）。我们的方法测量机械孤立的收缩蛋白的特性。因此，我们将确定FHC突变的直接影响在肌动蛋白上。了解RLC突变如何影响肌球蛋白的固有功能将允许的程度改变较高功能单位的改变，例如心肌纤维，或与A的心脏本身相关的心脏本身原发性收缩缺陷。