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） 心脏肌球蛋白头（S1）调节两个不同状态之间的肌球蛋白变构过渡， 活跃，准备与肌动蛋白相互作用和肌动蛋白S1时隔离的超浮肿状态 与肌球蛋白尾的近端S2结构域结合，并在相互作用上限制在空间上 与肌动蛋白。肌球蛋白结构状态之间的快速和可逆切换可能是调节性的 肌肉激活的机制当更活跃的头在肌膜中可用时 力量产生。该提议基于（a）我们最近在表达方面的成功和 使用C2C12鼠肌细胞表达系统纯化人心脏肌球蛋白，并 （b）成功地应用互补生物物理方法的工具集研究 肌球蛋白和肌动蛋白动力学，肌球蛋白运动和肌球蛋白S1-S2相互作用。我们建议 研究人类心脏肌球蛋白的三个选择的PTM模拟突变体。我们将检查 突变对肌球蛋白周期动力学的影响，肌球蛋白S1和 近端S2，静脉内肌球蛋白的种群，并卸载和加载 PTM模拟肌球蛋白构建体的体外运动。该提案的两个目标是 补充。在瞬态中将检测到肌动蛋白周期的动力学变化 动力学实验和随着肌动蛋白丝的变化在体外卸载的速度 运动性测定。 SRX状态的影响稳定性将直接在单个中进行评估 营业额ATP测定法，将被检测为在肌球蛋白床中更改的合奏力 加载体外运动测定法。结果，我们将研究PTM的假设 破坏肌球蛋白的SRX状态，并将表征PTM对人心脏的影响 肌球蛋白动力学和运动。