Post translational modifications tune cardiac myosin

翻译后修饰调节心肌肌球蛋白

• 批准号: 10291447
• 负责人: YURI NESMELOV
• 金额: $ 44万
• 依托单位: UNIVERSITY OF NORTH CAROLINA CHARLOTTE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10291447
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Acetylation; Actins; Active Sites; Actomyosin; Affect; Affinity; Amino Acids; Beds; Binding; Biological Assay; Blood Volume; Cardiac; Cardiac Muscle Contraction; Cardiac Myosins; Cartoons; Communication; Coupled; Disease; Electrostatics; Exercise; Filament; Goals; H-Meromyosin; Head; Heart; Human; Immobilization; In Vitro; Kinetics; Link; Lysine; Mediating; Microfilaments; Modification; Molecular; Molecular Motors; Motor; Mus; Muscle; Muscle Contraction; Muscle Fibers; Mutation; Myoblasts; Myocardium; Myosin ATPase; Myosin Heavy Chains; Nucleotides; Organism; Periodicity; Phosphorylation; Population; Post-Translational Modification Site; Post-Translational Protein Processing; Production; Protein Isoforms; Regulation; Research; Role; Rotation; Sarcomeres; Slide; Spatial Distribution; Structure; Surface; System; Tail; Testing; Thick Filament; Time; Tyrosine Phosphorylation; Work; actin 2; arm; base; biophysical techniques; blood pump; cell motility; dimer; experimental study; mimetics; mutant; new therapeutic target; skeletal; stroke recovery; success; theories; young adult

ABSTRACT We hypothesize that the post-translational modification (PTM) of the heavy chain of human cardiac myosin head (S1) regulates myosin allosteric transition between two distinct states, active, ready to interact with actin and the sequestered super-relaxed state when myosin S1 bound to the proximal S2 domain of myosin tail and sterically constrained from the interaction with actin. Rapid and reversible switch between myosin structural states could be a regulatory mechanism of muscle activation when more active heads become available in a sarcomere for force production. The proposal is based on (a) our recent success in the expression and purification of human cardiac myosin using the C2C12 murine myoblasts expression system, and (b) on the successful application of a toolset of complementary biophysical methods to study myosin and actomyosin kinetics, myosin motility, and myosin S1-S2 interactions. We propose to study three selected PTM-mimetic mutants of the human cardiac myosin. We will examine the effect of mutations on the kinetics of the actomyosin cycle, the binding affinity of myosin S1 and proximal S2, the population of myosin in the sequestered SRX state, and unloaded and loaded in vitro motility of the PTM-mimetic myosin constructs. Two aims of the proposal are complementary. The changed kinetics of the actomyosin cycle will be detected in the transient kinetics experiments and as the changed velocity of actin filament in the unloaded in vitro motility assay. The affected stability of the SRX state will be assessed directly in the single turnover ATP assay and will be detected as changed ensemble force of myosin bed in the loaded in vitro motility assay. As the result, we will examine our hypothesis that PTM destabilizes the SRX state of myosin and will characterize the effect of PTM on human cardiac myosin kinetics and motility.

摘要 我们假设人源性大肠杆菌重链的翻译后修饰（PTM） 心脏肌球蛋白头（S1）调节肌球蛋白在两种不同状态之间的变构转换， 活跃，准备与肌动蛋白相互作用，当肌球蛋白S1 与肌球蛋白尾的近端S2结构域结合，并在空间上受到相互作用的限制 actin的。肌球蛋白结构状态之间的快速和可逆转换可能是一种调节机制。 当肌节中有更多的活动头时， 力生产。这项建议是基于（a）我们最近在表达方面的成功， 使用C2 C12鼠成肌细胞表达系统纯化人心肌肌球蛋白，和 (b)关于成功应用一套补充生物物理方法研究 肌球蛋白和肌动球蛋白动力学、肌球蛋白运动性和肌球蛋白S1-S2相互作用。我们建议 研究三个选定的PTM模拟突变体的人心肌肌球蛋白。我们会研究 突变对肌动球蛋白循环动力学、肌球蛋白S1的结合亲和力和 近端S2，处于隔离SRX状态的肌球蛋白群体，以及未加载和加载 PTM模拟肌球蛋白构建体的体外运动性。该提案的两个目的是 互补的。将在瞬态中检测到肌动球蛋白循环的变化动力学。 动力学实验和体外无负荷条件下肌动蛋白丝的变化速度 运动性测定SRX状态的受影响稳定性将直接在单个 转换ATP测定，并将被检测为肌球蛋白床的整体力的变化， 加载的体外运动性测定。因此，我们将检验我们的假设，即PTM 使肌球蛋白的SRX状态不稳定，并将表征PTM对人心脏的作用。 肌球蛋白动力学和运动性。