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.

抽象的 我们假设人类重链的翻译后修饰（PTM） 心肌肌球蛋白头 (S1) 调节两种不同状态之间的肌球蛋白变构转变， 活跃，准备与肌动蛋白相互作用，并且当肌球蛋白 S1 时处于隔离的超放松状态 与肌球蛋白尾部的近端 S2 结构域结合，并且在空间上受到相互作用的限制 与肌动蛋白。肌球蛋白结构状态之间的快速且可逆的转换可能是一种调节 当肌节中更活跃的头部变得可用时，肌肉激活的机制 力生产。该提案基于 (a) 我们最近在表达和 使用 C2C12 鼠成肌细胞表达系统纯化人心肌肌球蛋白，以及 (b) 成功应用补充生物物理方法工具集进行研究 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 研究三种选定的人类心肌肌球蛋白 PTM 模拟突变体。我们将检查 突变对肌动球蛋白循环动力学、肌球蛋白 S1 和肌球蛋白 S1 的结合亲和力的影响 近端 S2，处于隔离 SRX 状态的肌球蛋白群体，以及卸载和加载 PTM 模拟肌球蛋白构建体的体外运动性。该提案的两个目标是 补充。肌动球蛋白循环的动力学变化将在瞬态中检测到 动力学实验以及体外卸载时肌动蛋白丝速度的变化 运动测定。 SRX 状态受影响的稳定性将直接在单一评估中进行评估 周转 ATP 测定，并将被检测为肌球蛋白床中肌球蛋白床整体力的变化 加载体外运动测定。因此，我们将检验我们的假设：PTM 破坏肌球蛋白 SRX 状态的稳定性，并表征 PTM 对人类心脏的影响 肌球蛋白动力学和运动性。