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Global Analysis of Redox Modification on Protein Kinase Function

氧化还原修饰对蛋白激酶功能的整体分析

基本信息

批准号：
9272407
负责人：
Robert Howard Newman
金额：
$ 11.52万
依托单位：
NORTH CAROLINA AGRI & TECH ST UNIV
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-05-01 至 2018-09-09
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9272407
关键词：
Affect Antioxidants Biological Assay Cardiovascular Diseases Cell Proliferation Cell Respiration Cells Complement Coupled Cultured Cells Cyclic AMP-Dependent Protein Kinases Diabetes Mellitus Differentiated Gene Disease Docking Enzymes Event Family Family member Foundations Gene Expression Generations Goals Health Human Hydrogen Peroxide MAPK1 gene Malignant Neoplasms Mediating Modeling Modification Molecular Oxidases Oxidation-Reduction Oxidative Stress Oxides Pathologic Pathologic Processes Pathway interactions Peptides Phosphorylation Phosphotransferases Physiological Platelet-Derived Growth Factor Positioning Attribute Principal Investigator Process Protein Kinase Protein Microchips Protein phosphatase Proteins Proteome Reactive Oxygen Species Regulation Research Role Second Messenger Systems Signal Pathway Signal Transduction Site Site-Directed Mutagenesis Substrate Specificity Testing Therapeutic Time Western Blotting antioxidant enzyme catalase cell growth regulation insight member migration novel oxidation preference programs protein protein interaction public health relevance spatiotemporal

项目摘要

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) are emerging as critical second messengers in many signaling pathways related to health and disease. While much progress has been made in understanding the mechanisms by which ROS levels are regulated inside cells, less is known about the molecular signaling events that occur downstream of ROS generation. Protein phosphatases are perhaps the best-characterized ROS effectors, suggesting that crosstalk readily occurs between ROS- and phosphorylation-dependent pathways. This notion is supported by recent studies demonstrating that several protein kinases are also directly regulated by ROS modification. Indeed, the reversible oxidation of specific Cys residues in redox-sensitive kinases has been shown to influence their activity (either positively or negatively), subcellular localization, and protein-protein interactions. In mny cases, the modified Cys in the affected kinase is conserved among other members in the same kinase family. This raises the possibility that reversible oxidation may be a general means of regulating kinase function inside cells. Therefore, in Aim 1, we will examine the redox sensitivity of kinases containing conserved Cys residues at positions known to be oxidized in other representative family members. In Aim 2, we will explore the impact of oxidation on substrate selection by redox-sensitive kinases using functional "HuProt" protein microarrays composed of 19,000 unique human proteins (representing ~90% of the human proteome). Our hypothesis is that oxidation will lead to differential phosphorylation of proteins on the microarrays and may even shift the substrate preference of a given kinase such that distinct sets of substrates are targeted by the oxidized and reduced forms of the kinase. Not only will these studies offer unique insights into oxidation-induced changes in substrate specificity but, due to the large number of proteins on the HuProt microarrays, they also promise to dramatically expand the number of known substrates for each kinase in the reference state (i.e., under reducing conditions). We will build upon our findings in Aims 1 and 2 through targeted analysis of redox-sensitive kinases and their substrates following activation of physiologically-relevant redox signaling pathways in cultured cells.

描述（由申请人提供）：活性氧（ROS）正在成为许多与健康和疾病相关的信号传导途径中的关键第二信使。虽然在理解细胞内 ROS 水平调节机制方面已经取得了很大进展，但人们对 ROS 生成下游发生的分子信号转导事件知之甚少。蛋白磷酸酶可能是最具特征的 ROS 效应子，这表明 ROS 依赖性途径和磷酸化依赖性途径之间很容易发生串扰。最近的研究证明了这一观点，表明几种蛋白激酶也受到 ROS 修饰的直接调节。事实上，氧化还原敏感激酶中特定半胱氨酸残基的可逆氧化已被证明会影响其活性（正面或负面）、亚细胞定位和蛋白质-蛋白质相互作用。在许多情况下，受影响激酶中修饰的半胱氨酸在同一激酶家族的其他成员中是保守的。这提出了可逆氧化可能是调节细胞内激酶功能的一般手段的可能性。因此，在目标 1 中，我们将检查氧化还原敏感性已知在其他代表性家族成员中被氧化的位置含有保守的 Cys 残基的激酶。在目标 2 中，我们将使用由 19,000 个独特的人类蛋白质（代表约 90% 的人类蛋白质组）组成的功能性“HuProt”蛋白质微阵列，探索氧化对氧化还原敏感激酶底物选择的影响。我们的假设是，氧化将导致微阵列上蛋白质的差异磷酸化，甚至可能改变给定激酶的底物偏好，使得氧化和还原形式的激酶靶向不同的底物组。这些研究不仅将为氧化诱导的底物特异性变化提供独特的见解，而且由于 HuProt 微阵列上有大量蛋白质，它们还有望显着增加参考状态（即还原条件下）每种激酶的已知底物数量。我们将在目标 1 和 2 的发现基础上，通过在培养细胞中激活生理相关氧化还原信号通路后，对氧化还原敏感激酶及其底物进行有针对性的分析。