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Chemical Methods for Dissecting Protein Glutathionylation in Sarcomere

解剖肌节中蛋白质谷胱甘肽化的化学方法

基本信息

批准号：
10171883
负责人：
Young-Hoon Ahn
金额：
$ 36.39万
依托单位：
WAYNE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-15 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10171883
关键词：
Accounting Actins Affect Alkynes Americas Anabolism Biotin Calcium Signaling Cardiac Cardiac Myocytes Cell Line Cells Cessation of life Chemicals Complex Coronary Arteriosclerosis Cysteine Data Detection Disease Engineering Enzymes Functional disorder Glucose Glutathione Goals Heart Diseases Heart failure Heat-Shock Proteins 90 Individual Isotope Labeling Lead Ligation Mass Spectrum Analysis Mediating Metabolic Methods Mitochondria Modification Molecular Molecular Target Muscle Muscle Cells Muscle Contraction Myocardial Ischemia Myocardial dysfunction Myocardium Myofibrils Myosin ATPase Oxidation-Reduction Oxidative Stress Oxygen Pathology Pathway interactions Physiology Protein Analysis Proteins Reaction Reactive Oxygen Species Research Role Sarcomeres Signal Transduction Signaling Molecule Site Skeletal Muscle Structure Work base connectin cysteinylglycine deprivation disulfide bond fluorescence imaging fluorophore glutathione synthase heart function innovation insight mortality mutant novel novel diagnostics novel therapeutic intervention protein function response

项目摘要

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.

摘要/摘要本申请描述了用于确定蛋白质半胱氨酸谷胱甘肽化作用的化学方法肌节。肌节是肌肉中肌原纤维的基本单位。肌节含有大量肌节蛋白质，包括肌联蛋白、肌动蛋白和肌球蛋白，形成高度组织化的结构，用于持续收缩肌肉。在肌肉细胞中，活性氧 (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 ROS 的靶蛋白及其基于氧化还原的调节机制影响肌节的稳定性和完整性仍然未知。谷胱甘肽化是介导蛋白质半胱氨酸氧化修饰的主要方式之一 ROS 在氧化还原信号和氧化应激中的作用。该应用程序基于我们最近开发的化学品方法，即可点击的谷胱甘肽，用于谷胱甘肽化的识别和表征。设置和MYND 含结构域蛋白 2 (SMYD2) 是心脏和骨骼肌中丰富的蛋白质。带有可点击的谷胱甘肽，我们发现SMYD2在C13处选择性谷胱甘肽化，并且SMYD2 C13谷胱甘肽化是一种 ROS 诱导心肌细胞肌节不稳定的关键机制。主要目标应用是鉴定肌节蛋白，包括 SMYD2，它们在对 ROS 的反应并表征蛋白质谷胱甘肽化在调节肌节稳定性中的功能作用。具体目标有三个。首先，我们计划将可点击的谷胱甘肽与质量分析结合起来，以确定 SMYD2 和其他肌节蛋白响应缺血条件而发生谷胱甘肽化。可点击谷胱甘肽方法将用于具有同位素标记的叠氮基-丙氨酸和可裂解的生物素-炔的 H9c2 细胞系用于缺氧葡萄糖剥夺下谷胱甘肽化蛋白质的定量质量分析。其次，我们将确定 SMYD2 C13 谷胱甘肽化产生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观察肌细胞中肌节对 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结合起来，以可视化定位谷胱甘肽化 SMYD2。第三，我们计划确定SMYD2的分子机制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SMYD2，它将用于表征 SMYD2 C13 谷胱甘肽化的结构和功能变化随后的细胞研究。总而言之，这些研究将揭示关键的分子靶蛋白和 ROS 导致肌肉功能障碍的分子机制。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Titin N2A Domain and Its Interactions at the Sarcomere.

Titin N2A域及其在肌节的相互作用。

DOI：
10.3390/ijms22147563
发表时间：
2021-07-15
期刊：
International journal of molecular sciences
影响因子：
5.6
作者：
Adewale AO;Ahn YH
通讯作者：
Ahn YH

Redox-Inactive Peptide Disrupting Trx1-Ask1 Interaction for Selective Activation of Stress Signaling.

DOI：
10.1021/acs.biochem.7b01083
发表时间：
2018-02-06
期刊：
Biochemistry
影响因子：
2.9
作者：
Kekulandara DN;Nagi S;Seo H;Chow CS;Ahn YH
通讯作者：
Ahn YH

Synthesis and evaluation of tiaprofenic acid-derived UCHL5 deubiquitinase inhibitors.

DOI：
10.1016/j.bmc.2020.115931
发表时间：
2021-01-15
期刊：
Bioorganic & medicinal chemistry
影响因子：
3.5
作者：
Gurusingha Arachchige HS;Herath Mudiyanselage PDH;VanHecke GC;Patel K;Cheaito HA;Dou QP;Ahn YH
通讯作者：
Ahn YH

Emerging chemistry and biology in protein glutathionylation.