Chemical Methods for Dissecting Protein Glutathionylation in Sarcomere

摘要/摘要 本申请描述了确定蛋白质半胱氨酸谷胱甘肽基化在 肉骨节。肌节是肌肉中肌原纤维的基本单位。肌节含有大量的肌节 肌球蛋白包括肌动蛋白、肌动蛋白和肌球蛋白在内的蛋白质，它们形成一种高度有组织的结构，使细胞持续收缩 肌肉。在肌肉细胞中，活性氧物种(ROS)正在成为关键的信号分子， 对与心脏功能相关的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的ROS靶蛋白及其氧化还原调节机制 仍然不为人所知。谷胱甘肽是主要的蛋白质半胱氨酸氧化修饰之一，它介导 ROS在氧化还原信号和氧化应激中的作用。这一应用是基于我们最近开发的化学品 方法，即可点击的谷胱甘肽，用于鉴定和表征谷胱甘肽。SET和MyND 含结构域蛋白2(SMYD2)是一种在心肌和骨骼肌中含量丰富的蛋白质。使用可点击功能 我们发现SMYD2在C13位选择性地发生谷胱甘肽基化，SMYD2 C13谷胱甘肽基化是一种 ROS导致心肌细胞肌节不稳定的关键机制。的主要目标是 应用是确定包括SMYD2在内的对谷胱甘肽基化敏感的肌瘤蛋白。 对ROS的反应和表征蛋白质谷胱甘肽基化在调节肌节稳定性中的功能作用。 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相结合，以确定 SMYD2和其他肌瘤蛋白在缺血条件下的谷胱甘肽基化反应。可点击 谷胱甘肽方法将用于同位素标记叠氮丙氨酸和可裂解生物素-炔的H9c2细胞系 用于氧-糖剥夺条件下谷胱甘肽基化蛋白的定量质量分析。第二，我们将 确定SMYD2 C13谷胱甘肽基化所产生的肌节稳定性和完整性。我们将决定 通过肌节蛋白的荧光成像研究ROS对心肌细胞肌节稳定性的影响 肌球蛋白和肌动蛋白。此外，我们将结合可点击的谷胱甘肽和邻近结扎，以可视化的定位 谷胱甘肽标记的SMYD2。第三，我们计划确定SMYD2的分子机制 谷胱甘肽的作用导致肌节不稳定。我们将合成定位于谷胱甘肽的SMYD2， 它将被用来表征SMYD2 C13谷胱甘肽基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和 ROS促进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.