Regulation of Vascular Inflammatory Signaling by the Deubiquitinase USP20

去泛素酶 USP20 对血管炎症信号的调节

• 批准号: 10349573
• 负责人: NEIL J. FREEDMAN
• 金额: $ 53.46万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-15 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10349573
• 关键词: Adaptor Signaling Protein; Adenoviruses; Amino Acids; Anti-Inflammatory Agents; Antiatherogenic; Arrestins; Arterial Injury; Atherosclerosis; Attenuated; Binding; Biological Assay; Blood Vessels; CCL2 gene; CRISPR/Cas technology; Cause of Death; Cell Adhesion Molecules; Chronic; Cytokine Receptors; Deubiquitination; Dominant-Negative Mutation; Endothelial Cells; Endothelium; Family; Goals; Human; Hyperplasia; I Kappa B-Alpha; IRAK1 gene; In Vitro; Industrialization; Inflammation; Inflammatory; Interleukin 4 Receptor; Interleukin-1 Receptors; LOX gene; Link; Lipopolysaccharides; Mediating; Molecular; Mus; Myocardial Infarction; Partner in relationship; Phosphorylation; Phosphotransferases; Polyubiquitination; Post-Translational Protein Processing; Protein Kinase; Proteins; RIPK1 gene; ROCK1 gene; Regulation; Role; Signal Pathway; Signal Transduction; Signaling Protein; Smooth Muscle Myocytes; Stroke; TLR2 gene; TLR4 gene; TNF gene; TNFRSF1A gene; TRAF2 gene; TRAF6 gene; Tamoxifen; Testing; Transgenic Mice; Transgenic Organisms; Tumor Necrosis Factor Receptor; Ubiquitin; Ubiquitination; atherogenesis; cellular transduction; congenic; cytokine; desensitization; in vivo; interleukin-1 receptor-associated kinase; loss of function; lysophosphatidic acid; macrophage; mutant; new therapeutic target; novel; protein B; response; scaffold; transcription factor; ubiquitin-protein ligase; ubiquitin-specific protease; vascular inflammation; western diet

PROJECT SUMMARY We recently demonstrated that ubiquitin-specific protease-20 (USP20) is scaffolded by the adaptor protein known as β-arrestin2 (βarr2), and that USP20 desensitizes ubiquitin-dependent signaling from Toll-like receptor-4 (TLR4) to NFκB activation by deubiquitinating TRAF6 and βarr2. Using transgenic mice expressing dominant-negative USP20 in smooth muscle cells, we found that USP20 reduces neointimal hyperplasia after arterial injury and that USP20 activity in SMCs reduces atherosclerosis in Ldlr-/- mice. To establish anti-atherogenic effects of systemically expressed USP20, and to elucidate further molecular mechanisms by which USP20 protects against atherosclerosis, this project will test the hypothesis that USP20 attenuates atherosclerosis by deubiquitinating several substrate proteins that were previously unassociated with USP20 but that are important in signaling pathways that activate NFκB: βarr1, TRAF6, TRAF2, and RIPK1. Furthermore, because USP20 employs βarr2 as a scaffold to facilitate association with distinct proteins, and because βarr1 reduces vascular inflammation, this project will test whether USP20’s anti-atherogenic activity involves βarr1-mediated scaffolding. To these ends, this project will study systemic effects of USP20 on atherosclerosis by comparing Usp20-/- /Ldlr-/- versus Ldlr-/- mice, on a background of βarr1+/+ or βarr1-/+. To determine the effects of endothelial USP20 on atherosclerosis, this project will compare atherosclerosis among VECad-Cre- ERT2/Usp20flox/flox/Ldlr-/- vs. Usp20flox/flox/Ldlr-/- mice treated ± tamoxifen; furthermore, we will investigate cytokine secretion, and dynamic ubiquitination of signaling proteins in primary aortic endothelial cells that are WT, Usp20-/-, Usp20-/-/βarr1-/+, or βarr1-/-. To determine what kinase in endothelial cells phosphorylates USP20 on Ser333 (and thereby abrogates USP20 deubiquitinase activity), this project will test IRAK1, PAK1, and ROCK1 with several loss-of function approaches, including a USP20 minigene, in primary endothelial cells. These studies collectively may identify USP20 phosphorylation as novel therapeutic target for atherosclerosis.

项目摘要 我们最近证明，泛素特异性蛋白酶-20（USP 20）是由接头支架 一种称为β-arrestin 2（β arr 2）的蛋白质，USP 20使泛素依赖性信号转导脱敏， Toll样受体4（TLR 4）通过去泛素化TRAF 6和β arr 2激活NFκB。利用转基因 在平滑肌细胞中表达显性阴性USP 20的小鼠中，我们发现USP 20降低了 动脉损伤后新生内膜增生和SMC中USP 20活性降低动脉粥样硬化， LDLR-/-小鼠。确定全身表达的USP 20的抗动脉粥样硬化作用，并阐明 USP 20预防动脉粥样硬化的进一步分子机制，该项目将测试 USP 20通过去遍在化几种底物蛋白来减轻动脉粥样硬化的假设 以前与USP 20无关，但在激活的信号通路中很重要 NFκB：βarr1、TRAF 6、TRAF 2和RIPK 1。此外，由于USP 20采用β arr 2作为支架， 为了促进与不同蛋白质的结合，并且因为β arr 1减少血管炎症， 该项目将测试USP 20的抗动脉粥样硬化活性是否涉及β arr 1介导的支架。到 为此，本项目将通过比较USP 20-/- /Ldlr-/-与Ldlr-/-小鼠，以β arr 1 +/+或β arr 1-/+为背景。为了确定内皮细胞的作用， USP 20关于动脉粥样硬化，该项目将比较VECad-Cre- ERT 2/Usp 20 flox/flox/Ldlr-/-与±他莫昔芬处理的Usp 20 flox/flox/Ldlr-/-小鼠;此外，我们将研究 原代主动脉内皮细胞中细胞因子分泌和信号蛋白的动态泛素化 即WT、Usp 20-/-、Usp 20-/-/β arr 1-/+或β arr 1-/-。为了确定内皮细胞中 使USP 20在Ser 333上磷酸化（从而消除USP 20去泛素化酶活性），该项目 我将使用几种功能丧失方法测试IRAK 1、PAK 1和ROCK 1，包括USP 20 minigene，在原代内皮细胞。这些研究可以共同确定USP 20磷酸化 作为动脉粥样硬化的新治疗靶点。