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: 10375545
• 负责人: Michael Joseph Previs
• 金额: $ 45.02万
• 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/10375545
• 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; 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.

摘要 横纹肌肌球蛋白被高度组织成粗丝，这些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收缩性至关重要，但 允许充分发育的肌肉取代肌球蛋白分子，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的时间动力学（即周转） 以及如何选择降解的分子？横纹肌球蛋白分子是否处于动态平衡状态 用粗纤维来交换纤维如果是粗丝结构， 动态的，什么是分子机制，管理这个平衡？最重要的是， 可调的修改横纹肌结构和/或功能？我们将在一只成年小鼠身上解决这些问题 模型有三个目标。我们的总体假设是，肌球蛋白周转是一个随机过程， 单个肌球蛋白分子在细胞溶质单体池和粗肌丝之间的交换， 由胞质溶胶内单体的折叠控制的机制。目标1将确定 心肌肌球蛋白在我们的模型，并确定是否发生肌球蛋白降解通过随机（即随机） 通过使用同位素标记策略和质谱法的组合来研究其机制。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， 体内荧光标记，并使用多光子荧光检测心肌内肌球蛋白的迁移率 光漂白后恢复。目的3将测试结构构象（即折叠与折叠）的假设。 细胞质中单个肌球蛋白分子的（延伸）调节肌球蛋白分子之间的交换， 池.目标3将利用一种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，影响肌球蛋白与胞质溶胶的有效浓度， 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和 在任何完整的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，充分发展的肌肉是以这样的方式设计，以允许结构 肌球蛋白在一分钟到一分钟的时间尺度上的重排。机械的发现有可能增加 到目前的范式关于粗丝结构，并解释如何横纹肌是维持从 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 调节整个动物横纹肌结构和/或功能的机制。