Structural Dynamics of Cardiac Myosin-Binding Protein C Regulation

心肌肌球蛋白结合蛋白 C 调节的结构动力学

• 批准号: 10320335
• 负责人: Brett A Colson
• 金额: $ 38.38万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320335
• 关键词: Accounting; Actins; Address; Affect; Artificial skin; Binding; Biological Assay; Cardiac; Cardiac Muscle Contraction; Cardiac Myosins; Cardiomyopathies; Computer Simulation; Data; Development; Disease; Equilibrium; Evaluation; FDA approved; Fiber; Filament; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Frequencies; Genes; Health; Heart; Heart Diseases; Human; Hypertrophic Cardiomyopathy; In Situ; In Vitro; Individual; Inherited; Kinetics; Knowledge; Label; Lead; Measurement; Measures; Mechanics; Medical; Microfilaments; Molecular; Molecular Conformation; Monitor; Muscle; Muscle function; Mutation; Myocardial; Myocardium; Myosin ATPase; N-terminal; Pathogenesis; Pathogenicity; Pathologic; Performance; Phenotype; Phosphorylation; Physiological; Positioning Attribute; Property; Proteins; Publishing; Recombinants; Regulation; Reporter; Reporting; Research; Resolution; Role; Sarcomeres; Site; Skin; Stress; Structure; Sudden Death; Technology; Testing; Therapeutic; Thick Filament; Thin Filament; Thinness; Time; Work; base; biophysical tools; cardiac muscle disease; data exchange; design; effective therapy; heart cell; improved; in silico; in vivo; innovation; molecular dynamics; mutant; myosin-binding protein C; novel; protein function; protein structure; simulation; tool

PROJECT SUMMARY Hypertrophic cardiomyopathy (HCM) is a relatively common disease affecting more than 1 in 500 individuals and the leading cause of sudden death in young individuals and athletes. HCM is an unmet medical need with no FDA-approved treatments. ~40% of all HCM cases are associated with mutations in the gene encoding cardiac myosin-binding protein C (MyBP-C). MyBP-C is a thick filament-associated protein that is critical for normal myocardial performance; it is centrally positioned in the sarcomere to regulate interactions between myosin cross-bridges and actin thin filaments that are responsible for force development. We have previously demonstrated that increased phosphorylation of MyBP-C enhances actin-myosin interactions leading to accelerated contraction kinetics in myocardium, whereas reduced phosphorylation led to reduced actin-myosin proximity and decelerated contraction. However, it is not understood how MyBP-C phosphorylation alters the structural dynamics of its interactions with actin and/or myosin to modulate force development in normal myocardium or how mutations alter functions that ultimately contribute to HCM pathogenesis. We have developed innovative biophysical tools that, for the first time, enable evaluation of: (1) the structural dynamics of MyBP-C, (2) how it interacts with actin and/or myosin in muscle, and (3) how these interactions are affected by phosphorylation and known pathologic mutations. We will test the central hypothesis that phosphorylation and HCM mutations of N-terminal MyBP-C alter functionally significant structural properties of MyBP-C and interactions with actin and myosin. Aim 1 will evaluate the effects of phosphorylation, HCM mutations, and binding to actin or myosin on MyBP-C structural dynamics. Spectroscopic approaches will be employed to detect conformational changes (structure) within MyBP-C due to phosphorylation, HCM mutation, and actin/myosin binding (function). Molecular dynamics (MD) simulations will be applied as a complementary approach. Aim 2 will determine how MyBP-C phosphorylation and HCM mutants affect proximities and dynamics of key myocardial proteins. We will utilize site-directed probe technologies in skinned (demembranated) cardiac fibers to determine how phosphorylation/mutants affect protein structure/interactions in situ to regulate contractility. The proposed studies capture structural dynamics in real time and resolve interactions in real myocardial space using novel high-resolution approaches. These aims are a stepwise progression developing a new paradigm for studying normal and mutant MyBP-C during the contractile cycle. This paradigm involves monitoring distances between points on proteins and the order (or disorder) of those distances under physiological conditions, in interacting proteins and functioning myocardium. Not all HCM mutants impact the same functions of MyBP-C. Time-resolved fluorescence data components, thin/thick filament dynamics, mechanics, and simulations will be used to separate mutants into identifiable bins, setting the stage for identifying mechanistic-based therapies to specifically treat different classes of mutations.

项目总结 肥厚型心肌病(HCM)是一种相对常见的疾病，每500人中就有1人患病 也是年轻人和运动员猝死的主要原因。HCM是一种未得到满足的医疗需求 没有FDA批准的治疗方法。约40%的肥厚型心肌炎病例与编码基因突变有关 心肌肌球蛋白结合蛋白C(MyBP-C)。MyBP-C是一种粗丝相关蛋白，对 正常的心肌功能；它位于肌节的中央，调节 肌球蛋白跨桥和肌动蛋白细丝负责力量的发展。我们之前已经 证明MyBP-C的磷酸化增加增强了肌动蛋白-肌球蛋白的相互作用，从而导致 心肌收缩动力学加速，而磷酸化减少则导致肌动蛋白-肌球蛋白减少 接近和减速收缩。然而，目前还不清楚MyBP-C磷酸化是如何改变 其与肌动蛋白和/或肌球蛋白相互作用调控力量发育的结构动力学 或突变如何改变最终导致肥厚型心肌病发病的功能。我们有 开发了创新的生物物理工具，首次能够评估：(1)结构动力学 MyBP-C，(2)它如何与肌肉中的肌动蛋白和/或肌球蛋白相互作用，以及(3)这些相互作用如何受到 磷酸化和已知的病理突变。我们将检验这一中心假设，即磷酸化和 N-末端MyBP-C的HCM突变改变了MyBP-C和 肌动蛋白和肌球蛋白的相互作用。目标1将评估磷酸化、HCM突变和 肌动蛋白或肌球蛋白与MyBP-C结构动力学的结合。将使用光谱方法来检测 磷酸化、HCM突变和肌动蛋白/肌球蛋白引起的MyBP-C的构象变化(结构) 绑定(函数)。分子动力学(MD)模拟将作为补充方法应用。目标2 将决定MyBP-C磷酸化和HCM突变如何影响KEY的亲和力和动力学 心肌蛋白质。我们将在去皮(去膜)的心肌纤维中使用定点探测技术 确定磷酸化/突变体如何在原位影响蛋白质结构/相互作用以调节收缩能力。 建议的研究实时捕捉结构动力学，并解决真实心肌空间中的相互作用 使用新的高分辨率方法。这些目标是一个循序渐进的过程，为 研究收缩周期中正常和突变的MyBP-C。这一范例涉及到监控距离 在生理条件下，蛋白质上的点和这些距离的顺序(或无序)之间， 相互作用的蛋白质和功能心肌。并不是所有的HCM突变体都影响MyBP-C的相同功能。 时间分辨荧光数据组件、细/粗细丝动力学、力学和模拟 用于将突变体分离到可识别的垃圾箱中，为识别基于机械的疗法奠定了基础 专门治疗不同类别的突变。