Chemical Methods for Dissecting Protein Glutathionylation in Sarcomere

总结/摘要 本申请描述了用于确定蛋白质半胱氨酸谷胱甘肽化在蛋白质合成中的作用的化学方法。 肌节肌节是肌肉中肌原纤维的基本单位。肌节包含许多肌节 蛋白质，包括肌联蛋白、肌动蛋白和肌球蛋白，它们形成一个高度组织化的结构，使肌肉连续收缩。 肌肉.在肌肉细胞中，活性氧（ROS）作为关键的信号分子出现， 对与心脏功能相关生理学和病理学有很大贡献。然而，精确的分子 影响肌节稳定性和完整性ROS靶蛋白及其基于氧化还原的调节机制 仍然未知。谷胱甘肽化是蛋白质半胱氨酸氧化修饰的主要途径之一， ROS在氧化还原信号和氧化应激中的作用。该应用程序是基于我们最近开发的化学 方法，即用于谷胱甘肽化鉴定和表征的可点击谷胱甘肽。SET & MYND 含结构域蛋白2（SMYD 2）是心脏和骨骼肌中丰富的蛋白质。关于clickable 我们发现SMYD 2在C13处选择性地谷胱甘肽化，并且SMYD 2 C13谷胱甘肽化是一种选择性的谷胱甘肽化。 ROS诱导心肌细胞肌节不稳定的重要机制。的主要目标 本申请的目的是鉴定肌节蛋白，包括SMYD 2，其在哺乳动物中对谷胱甘肽化敏感， 反应活性氧和表征功能作用的蛋白谷胱甘肽化在调节肌节稳定性。 有三个具体目标。首先，我们计划将可点击的谷胱甘肽与质量分析结合起来， SMYD 2和其他肌节蛋白的谷胱甘肽化对缺血状况的响应。可点击 谷胱甘肽方法将用于H9 c2细胞系，具有同位素标记的叠氮基-Ala和可裂解的生物素-炔 用于在氧-葡萄糖剥夺下谷胱甘肽化蛋白质的定量质量分析。二是 测定SMYD 2 C13谷胱甘肽化导致的肌节稳定性和完整性。我们将确定 通过肌节蛋白的荧光成像，肌细胞中肌节对ROS的稳定性，包括 肌球蛋白和肌动蛋白。此外，我们还将可点击的谷胱甘肽与邻近连接偶联，用于可视化谷胱甘肽的定位。 谷胱甘肽化SMYD 2。第三，我们计划确定SMYD 2 谷胱甘肽化导致肌节不稳定。我们将合成位点特异性谷胱甘肽化的SMYD 2， 其将用于表征SMYD 2 C13谷胱甘肽化的结构和功能变化， 随后的细胞研究。总之，这些研究将揭示关键的分子靶蛋白， ROS导致肌肉功能障碍的分子机制。

Summary/Abstract This application describes chemical approaches for determining the role of protein cysteine glutathionylation in the sarcomere. Sarcomere is a basic unit of myofibrils in muscle. Sarcomere contains numerous sarcomeric proteins, including titin, actin and myosin, that form a highly organized structure for continuous contraction of muscle. In muscle cell, the reactive oxygen species (ROS) are emerging as critical signaling molecules that strongly contribute to physiology and pathology associated with heart function. However, the precise molecular target proteins of ROS and their redox-based regulatory mechanisms that affect sarcomere stability and integrity remain unknown. Glutathionylation is one of the major protein cysteine oxidative modifications that mediate the role of ROS in redox signaling and oxidative stress. This application is based on our recently developed chemical approach, i.e. clickable glutathione for identification and characterization of glutathionylation. SET and MYND domain-containing protein 2 (SMYD2) is an abundant protein in heart and skeletal muscle. With clickable glutathione, we found that SMYD2 is selectively glutathionylated at C13, and SMYD2 C13 glutathionylation is a crucial mechanism by which ROS induce sarcomere destabilization in cardiomyocytes. The main goal of application is to identify sarcomeric proteins, including SMYD2, that are susceptible to glutathionylation in response to ROS and to characterize functional roles of protein glutathionylation in regulating sarcomere stability. There are three specific aims. First, we plan to couple clickable glutathione with mass analysis to identify glutathionylation of SMYD2 and other sarcomeric proteins in response to ischemic conditions. Clickable glutathione approach will be used in H9c2 cell line with isotopic-labelled azido-Ala and cleavable biotin-alkyne for quantitative mass analysis of glutathionylated proteins under oxygen-glucose-deprivation. Second, we will determine sarcomere stability and integrity resulting from SMYD2 C13 glutathionylation. We will determine sarcomere stability in myocytes in response to ROS by fluorescence imaging of sarcomeric proteins, including myosin and actin. Also, we will couple clickable glutathione with proximity ligation for visualizing localization of glutathionylated SMYD2. Third, we plan to determine the molecular mechanism by which SMYD2 glutathionylation leads to sarcomere destabilization. We will synthesize site-specifically glutathionylated SMYD2, which will be used for characterizing structural and functional changes of SMYD2 C13 glutathionylation with subsequent cellular studies. Taken together, these studies will uncover the key molecular target protein and molecular mechanisms by which ROS contribute to muscle dysfunction.