Mechanisms governing myosin turnover and exchange in vivo.

体内控制肌球蛋白周转和交换的机制。

• 批准号: 10182478
• 负责人: Michael Joseph Previs
• 金额: $ 47.69万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10182478
• 关键词: Address; Adult; Affect; Amino Acids; Animal Model; Animals; Biochemical; Biological Assay; Biophysical Process; Biophysics; Cardiac; Cardiac Myosins; Cytosol; Data; Electron Microscopy; Equilibrium; Exhibits; Filament; Fluorescence Recovery After Photobleaching; Half-Life; Head; Heart; Human; Individual; Isotope Labeling; Knowledge; Label; Macromolecular Complexes; Mass Spectrum Analysis; Modeling; Molecular; Molecular Conformation; Mus; Muscle; Myocardium; Myosin ATPase; Nature; Organ; Pathology; Pharmaceutical Preparations; Polymers; Property; Proteins; Resolution; Sarcomeres; Stable Isotope Labeling; Stochastic Processes; Striated Muscles; Structure; Testing; Thick Filament; Ursidae Family; Viral; Viral Proteins; confocal imaging; design; in vivo; inhibitor/antagonist; innovation; insight; microscopic imaging; monomer; mouse model; muscle form; muscular structure; muscular system; novel; novel therapeutics; prevent; protein expression; reduced muscle mass; single molecule; small molecule

ABSTRACT Striated muscle myosin is highly organized into thick filaments that bear the molecular forces generated by the myosin heads. While thick filament structure and stability are essential for contractility, the mechanisms that allow fully developed muscles to replace myosin molecules while maintaining contractile fidelity are unclear. Critical questions include; what are the temporal dynamics of myosin synthesis and degradation (i.e. turnover) and how are molecules selected for degradation? Do striated myosin molecules exist in a dynamic equilibrium with thick filaments to allow for their exchange out of and into thick filaments? If thick filament structure is dynamic, what are the molecular mechanisms governing this equilibrium? Most importantly, is this mechanism tunable to modify striated muscle structure and/or function? We will address these questions in an adult mouse model in three aims. Our overall hypothesis is that myosin turnover is a stochastic process which involves the exchange of individual myosin molecules between a cytosolic pool of monomers and thick filaments, by a mechanism governed by the folding of the monomers within the cytosol. Aim 1 will define the turnover rate of cardiac myosin in our model and determine whether myosin degradation occurs via a stochastic (i.e. random) mechanism by using a combination of isotope labeling strategies and mass spectrometry. Aim 2 will test the hypothesis that the organization of striated muscle myosin is highly dynamic to allow for the rapid exchange of individual molecules between thick filaments and a cytosolic pool of monomers by virally labeling myosin with a fluorescent tag in vivo and examining the mobility of the myosin within hearts using multiphoton fluorescence recovery after photobleaching. Aim 3 will test the hypotheses that the structural conformation (i.e. folded vs. extended) of individual myosin molecules in the cytosol regulates the exchange of myosin molecules between pools. Aim 3 will take advantage of a drug that folds myosin and reduces cardiac mass. We will test our overall hypothesis that tuning myosin folding, affects the effective concentration of myosin with the cytosol, and regulates its availability for degradation. The proposed studies will be the first to examine myosin turnover and macromolecular exchange in a striated muscle system in any intact animal model. The results will provide conceptual innovation that fully developed muscle is designed in such a way to allow for structural rearrangement of myosin on a minute-to-minute timescale. The mechanistic findings have the potential to add to the current paradigm regarding thick filament structure and explain how striated muscle is maintained from the single molecule to whole organ level. The new knowledge gained may allow us to take advantage of this mechanism for tuning striated muscle structure and/or function in whole animals.

抽象的 横纹肌肌球蛋白高度组织成粗丝，承受由肌球蛋白产生的分子力。 肌球蛋白头。虽然粗丝结构和稳定性对于收缩性至关重要，但其机制 是否允许完全发育的肌肉替换肌球蛋白分子，同时保持收缩保真度尚不清楚。 关键问题包括：肌球蛋白合成和降解（即周转）的时间动态是什么 以及如何选择分子进行降解？横纹肌球蛋白分子是否处于动态平衡状态 与粗丝以允许它们交换出和进入粗丝？如果粗丝结构是 动态的，控制这种平衡的分子机制是什么？最重要的是这个机制 可调节以改变横纹肌结构和/或功能？我们将在成年小鼠身上解决这些问题 三个目标的模型。我们的总体假设是肌球蛋白周转是一个随机过程，涉及 单个肌球蛋白分子在单体池和粗丝之间进行交换，通过 机制由细胞质内单体的折叠控制。目标 1 将定义周转率 我们模型中的心肌肌球蛋白，并确定肌球蛋白降解是否通过随机（即随机）发生 通过使用同位素标记策略和质谱分析相结合的机制。目标 2 将测试 假设横纹肌肌球蛋白的组织是高度动态的，以允许快速交换 通过用病毒标记肌球蛋白，在粗丝和单体胞质池之间形成单个分子 体内荧光标签并使用多光子荧光检查心脏内肌球蛋白的流动性 光漂白后恢复。目标 3 将测试结构构象（即折叠与折叠）的假设。 细胞质中各个肌球蛋白分子的扩展）调节肌球蛋白分子之间的交换 水池。目标 3 将利用一种可折叠肌球蛋白并减少心脏质量的药物。我们将测试我们的整体 假设调整肌球蛋白折叠，影响肌球蛋白与细胞质的有效浓度，以及 调节其降解的可用性。拟议的研究将是第一个检查肌球蛋白周转率和 任何完整动物模型中横纹肌系统中的大分子交换。结果将提供 概念创新，充分发展的肌肉被设计成允许结构 肌球蛋白在每分钟的时间尺度上重新排列。机制研究结果有可能增加 目前有关粗丝结构的范例，并解释横纹肌是如何维持的 从单个分子到整个器官水平。获得的新知识可以让我们利用这一点 调整整个动物横纹肌结构和/或功能的机制。