Structural and functional effects of oxidative modification of myosin

肌球蛋白氧化修饰的结构和功能影响

• 批准号: 8268410
• 负责人: Rebecca J Moen
• 金额: $ 3.64万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-19 至 2014-07-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8268410
• 关键词: Actins; Age; Aging; Atrophic; Binding; Biochemical; Biochemistry; Biological Assay; Biophysics; Catalytic Domain; Cell Culture Techniques; Coupled; Coupling; Cysteine; Degenerative Disorder; Dictyostelium; Doctor of Philosophy; Elderly; Electron Spin Resonance Spectroscopy; Enzymes; Future; Glutamine; Goals; Health; Human; Hydrogen Peroxide; Label; Leucine; Magnetic Resonance; Measures; Methionine; Methods; Modeling; Modification; Molecular; Muscle; Muscle Proteins; Muscle Weakness; Mutagenesis; Mutation; Myosin ATPase; Myosin Type II; Oxidative Stress; Peroxides; Physiological; Physiology; Play; Proteins; Reporting; Research; Research Personnel; Role; Sedimentation process; Site; Site-Directed Mutagenesis; Spectrum Analysis; Spin Labels; Structural Protein; Techniques; Testing; Training; Training Programs; Work; age effect; age related; base; biological systems; career; experience; functional mimics; insight; muscle strength; mutant; novel; oxidation; prevent; programs; response

The primary goals of the training program are (a) to provide me with extensive training in muscle biochemistry, physiology, and biophysics, toward completion of a PhD thesis in the molecular biophysics of muscle protein oxidation, and (b) to prepare me for the next step along the path to becoming an independent investigator. The long-term goal of the present research program in the Thomas lab is to understand the structural and functional basis of oxidative modification of muscle proteins, in order to determine the molecular basis of decline in muscle strength due to aging and degenerative diseases. Previous work in this lab has shown that (a) aging produces a decline in muscle force, independent of atrophy, accompanied by oxidative modification of myosin, decreasing actin-activated ATPase activity, and (b) peroxide treatment of muscle mimics the functional effects of aging and results primarily in modification of Met residues. I hypothesize that specific Met residues in myosin are responsible for these effects, which result from specific structural perturbations in the catalytic domain of myosin. I will test this hypothesis by performing site-directed Met mutagenesis on myosin in Dictyostelium cell culture, then performing functional and structural tests to determine the correlation between structural dynamics and function. The principal structural approach is site-directed spin labeling (SDSL), followed by electron paramagnetic resonance (EPR). I will pursue three aims. (1) Functional impact of methionine oxidation on myosin catalytic domain. (2) Structural dynamics of myosin catalytic domain. (3) Structural impact of methionine oxidation on the myosin catalytic domain. There is considerable scientific evidence that protein oxidation plays a major role in degenerative disorders associated with aging, but there is very little information about the biochemical and structural effects of oxidation on specific proteins. This project aims to provide a site-specific protein structural explanation for the decline in muscle strength with oxidation, which is hypothesized to play a major role in the loss of muscle strength with age in humans. This advance in fundamental understanding of oxidative mechanisms is important for making progress in treatment of age-related health problems. The approach of using site-directed mutagenesis and spectroscopy to probe the structural basis of oxidative modification in the muscle protein myosin is quite novel. Thus, in addition to providing new structural insight into muscle protein oxidation, this work can serve as a general model for future studies that focus on the specific structural consequences of oxidative stress. This project will build on my past experience in magnetic resonance and aging, while introducing me to a new biological system (muscle) for which the mechanisms of oxidative stress are intensely studied and a new spectroscopic technique (EPR) that is more sensitive than NMR and can be applied under more physiological conditions (e.g., myosin bound to actin).

培训计划的主要目标是 (a) 为我提供肌肉生物化学、生理学和生物物理学方面的广泛培训，以完成肌肉蛋白氧化分子生物物理学的博士论文，以及 (b) 为我成为一名独立研究者的下一步做好准备。托马斯实验室目前研究计划的长期目标是了解肌肉蛋白氧化修饰的结构和功能基础，以确定因衰老和退行性疾病而导致肌肉力量下降的分子基础。该实验室之前的工作表明，(a) 衰老会导致肌肉力量下降，与萎缩无关，并伴有肌球蛋白的氧化修饰，降低肌动蛋白激活的 ATP 酶活性，(b) 肌肉的过氧化物处理模拟衰老的功能效应，主要导致 Met 残基的修饰。我假设肌球蛋白中的特定 Met 残基造成了这些效应，这是由肌球蛋白催化结构域中的特定结构扰动造成的。我将通过对盘基网柄菌细胞培养物中的肌球蛋白进行定点 Met 诱变来检验这一假设，然后进行功能和结构测试以确定结构动力学和功能之间的相关性。主要的结构方法是定点自旋标记（SDSL），然后是电子顺磁共振（EPR）。我将追求三个目标。 (1)蛋氨酸氧化对肌球蛋白催化结构域的功能影响。 (2)肌球蛋白催化结构域的结构动力学。 (3)蛋氨酸氧化对肌球蛋白催化结构域的结构影响。有大量科学证据表明蛋白质氧化在与衰老相关的退行性疾病中起着重要作用，但有关氧化对特定蛋白质的生化和结构影响的信息却很少。该项目旨在为氧化引起的肌肉力量下降提供位点特异性蛋白质结构解释，假设氧化在人类肌肉力量随着年龄增长而丧失的过程中发挥着重要作用。对氧化机制的基本理解的进步对于在治疗与年龄相关的健康问题方面取得进展非常重要。使用定点诱变和光谱学来探测肌肉蛋白肌球蛋白氧化修饰的结构基础的方法是相当新颖的。因此，除了提供对肌肉蛋白质氧化的新结构见解之外，这项工作还可以作为未来研究的通用模型，重点关注氧化应激的特定结构后果。该项目将建立在我过去在磁共振和衰老方面的经验的基础上，同时向我介绍一种新的生物系统（肌肉），该系统的氧化应激机制得到了深入研究，以及一种新的光谱技术（EPR），该技术比核磁共振更灵敏，可以在更多的生理条件下应用（例如肌球蛋白与肌动蛋白结合）。