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: 10545008
• 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 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10545008
• 关键词: Acceleration; Accounting; Actins; Address; Affect; Artificial skin; Binding; Biological Assay; Cardiac; Cardiac Muscle Contraction; Cardiac Myosins; Cardiomyopathies; Computer Simulation; Data; Deceleration; 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; Protein Dynamics; 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; Visualization; Work; 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 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.

项目摘要 肥厚性心肌病（HCM）是一种相对常见的疾病，影响了500个以上的人 以及年轻人和运动员突然死亡的主要原因。 HCM是未满足的医疗需求 没有FDA批准的治疗方法。在所有HCM病例中，约有40％与编码基因中的突变有关 心脏肌球蛋白结合蛋白C（MYBP-C）。 MYBP-C是一种与细丝相关的蛋白质，对 正常心肌表现；它是在肌膜中集中放置的，以调节之间的相互作用 肌球蛋白跨桥和肌动蛋白细丝，负责发育。我们以前有 证明MYBP-C的磷酸化增加会增强肌动蛋白 - 肌球蛋白相互作用，导致 心肌的加速收缩动力学，而磷酸化降低导致肌动蛋白肌球蛋白减少 接近性和减速收缩。但是，尚不了解MyBP-C磷酸化如何改变 与肌动蛋白和/或肌球蛋白相互作用的结构动力学，以调节正常的力发育 心肌或突变如何改变最终导致HCM发病机理的功能。我们有 开发了创新的生物物理工具，该工具首次可以评估：（1） 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。我们将检验磷酸化和 N末端MYBP-C的HCM突变改变了MYBP-C和MYBP-C和 与肌动蛋白和肌球蛋白的相互作用。 AIM 1将评估磷酸化，HCM突变和 在MYBP-C结构动力学上与肌动蛋白或肌球蛋白结合。光谱方法将采用检测 由于磷酸化，HCM突变和肌动蛋白/肌球蛋白，MYBP-C内的构象变化（结构） 绑定（功能）。分子动力学（MD）模拟将作为互补方法应用。目标2 将确定MYBP-C磷酸化和HCM突变体如何影响钥匙的接近和动力学 心肌蛋白。我们将在皮肤（爆炸）心脏纤维中利用现场定向的探针技术 确定磷酸化/突变体如何影响蛋白质结构/相互作用以调节收缩力。 拟议的研究实时捕获结构动力学，并解决实际心肌空间中的相互作用 使用新型的高分辨率方法。这些目标是开发新范式的逐步发展 在收缩周期研究正常和突变的MYBP-C。此范式涉及监视距离 在生理条件下蛋白质点的点与这些距离的顺序（或混乱）之间 相互作用的蛋白质和功能性心肌。并非所有HCM突变体都会影响MYBP-C的相同功能。 时间分辨的荧光数据成分，薄/厚的细丝动力学，力学和模拟将是 用于将突变体分离成可识别的垃圾箱，为识别基于机械的疗法的阶段 特别处理不同类别的突变。