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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要肥厚性心肌病 (HCM) 是一种相对常见的疾病，影响超过五百分之一的人也是年轻人和运动员猝死的主要原因。 HCM 是一项未得到满足的医疗需求没有 FDA 批准的治疗方法。约 40% 的 HCM 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，对于心肌功能正常；它位于肌节的中央，调节之间的相互作用肌球蛋白跨桥和肌动蛋白细丝负责力量的发展。我们之前有过证明 MyBP-C 磷酸化的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，而磷酸化减少导致肌动蛋白肌球蛋白减少接近和减速收缩。然而，尚不清楚 MyBP-C 磷酸化如何改变其与肌动蛋白和/或肌球蛋白相互作用的结构动力学，以调节正常情况下的力量发展心肌或突变如何改变最终导致 HCM 发病机制的功能。我们有开发了创新的生物物理工具，首次能够评估：（1） 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 端 MyBP-C 的 HCM 突变改变了 MyBP-C 的功能上重要的结构特性，与肌动蛋白和肌球蛋白的相互作用。目标 1 将评估磷酸化、HCM 突变和在 MyBP-C 结构动力学上与肌动蛋白或肌球蛋白结合。将采用光谱方法来检测由于磷酸化、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）模拟将作为补充方法应用。目标2 将确定 MyBP-C 磷酸化和 HCM 突变体如何影响关键的邻近性和动态心肌蛋白。我们将在带皮（去膜）心脏纤维中利用定点探针技术确定磷酸化/突变体如何影响原位蛋白质结构/相互作用以调节收缩性。拟议的研究实时捕获结构动力学并解决真实心肌空间中的相互作用使用新颖的高分辨率方法。这些目标是逐步发展的，为研究收缩周期中正常和突变的 MyBP-C。该范例涉及监控距离蛋白质上的点之间以及生理条件下这些距离的顺序（或无序），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。时间分辨荧光数据组件、细/粗丝动力学、力学和模拟将用于将突变体分离到可识别的箱中，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。