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 条纹肌肉肌球蛋白高度组织成厚细丝，这些细丝具有由 肌球蛋白头。虽然较厚的细丝结构和稳定性对于收缩性至关重要 尚不清楚允许完全发育的肌肉替代肌球蛋白分子，同时保持收缩的保真度。 关键问题包括；肌球蛋白合成和降解的时间动力学是什么（即营业额） 如何选择分子降解？横纹肌球蛋白分子是否存在于动态平衡中 用厚的细丝允许它们从厚细丝中交换和交换？如果较厚的细丝结构是 动态，控制这种平衡的分子机制是什么？最重要的是这种机制 可调以修饰横纹肌的结构和/或功能？我们将在成年鼠标中解决这些问题 模型三个目标。我们的总体假设是，肌球蛋白更新是一个随机过程，涉及 通过A之间的单个胞质池之间的单个肌球蛋白分子交换 由细胞质内单体折叠的机制。 AIM 1将定义周转率 我们模型中的心脏肌球蛋白，并确定肌球蛋白降解是否通过随机发生（即随机）发生 通过使用同位素标记策略和质谱法的组合来机理。 AIM 2将测试 假设条纹肌肉肌球蛋白的组织具有高度动态性，可以快速交流 厚细丝和单体胞质池之间的单个分子通过用A的肌球蛋白标记 体内荧光标签，并使用多光子荧光检查心脏内肌球蛋白的迁移率 光漂白后恢复。 AIM 3将检验结构构象的假设（即折叠Vs. 细胞质中单个肌球蛋白分子的扩展）调节在之间调节肌球蛋白分子之间的交换 游泳池。 AIM 3将利用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 假设调节肌球蛋白折叠，影响肌球蛋白与细胞质的有效浓度，并且 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更新的研究，并且 在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 完全发育的肌肉的概念创新以这种方式设计 肌球蛋白在分钟到分钟的时间尺度上重新排列。机械发现有可能添加 目前有关较厚细丝结构的范式，并解释如何从 单个分子到整个器官水平。获得的新知识可能使我们能够利用这一点 整个动物中调节肌肉结构和/或功能的机制。