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: 9893026
• 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 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9893026
• 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.

项目概要 我们最近证明泛素特异性蛋白酶 20 (USP20) 由​​接头支架构成 称为 β-arrestin2 (βarr2) 的蛋白质，USP20 使泛素依赖性信号通路脱敏 Toll 样受体 4 (TLR4) 通过去泛素化 TRAF6 和 βarr2 来激活 NFκB。使用转基因 在平滑肌细胞中表达显性失活 USP20 的小鼠中，我们发现 USP20 降低 动脉损伤后新生内膜增生以及 SMC 中的 USP20 活性可减少动脉粥样硬化 Ldlr-/- 小鼠。确定系统表达的 USP20 的抗动脉粥样硬化作用，并阐明 USP20 预防动脉粥样硬化的进一步分子机制，该项目将测试 USP20 通过使几种底物蛋白去泛素化来减轻动脉粥样硬化的假设 以前与 USP20 无关，但在激活的信号通路中很重要 NFκB：βarr1、TRAF6、TRAF2 和 RIPK1。此外，由于USP20采用βarr2作为支架 为了促进与不同蛋白质的结合，并且由于 βarr1 减少血管炎症，这 该项目将测试 USP20 的抗动脉粥样硬化活性是否涉及 βarr1 介导的支架。到 为此，本项目将通过比较 Usp20-/- 来研究 USP20 对动脉粥样硬化的全身影响 /Ldlr-/- 与 Ldlr-/- 小鼠相比，以 βarr1+/+ 或 βarr1-/+ 为背景。确定内皮细胞的影响 USP20 关于动脉粥样硬化，该项目将比较 VECad-Cre- 之间的动脉粥样硬化 ERT2/Usp20flox/flox/Ldlr-/- 与 Usp20flox/flox/Ldlr-/- 治疗小鼠±他莫昔芬；此外，我们将调查 原代主动脉内皮细胞中细胞因子的分泌和信号蛋白的动态泛素化 分别是 WT、Usp20-/-、Usp20-/-/βarr1-/+ 或 βarr1-/-。确定内皮细胞中的激酶 在 Ser333 上磷酸化 USP20（从而消除 USP20 去泛素酶活性），该项目 将使用多种功能丧失方法（包括 USP20）测试 IRAK1、PAK1 和 ROCK1 原代内皮细胞中的小基因。这些研究共同可能确定 USP20 磷酸化 作为动脉粥样硬化的新治疗靶点。