Regulation of thrombin-induced p38 MAPK pro-inflammatory signaling by deubiquitinases

去泛素酶对凝血酶诱导的 p38 MAPK 促炎信号的调节

• 批准号: 10794926
• 负责人: Norton Cheng
• 金额: $ 3.98万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10794926
• 关键词: Actins; Adaptor Signaling Protein; Adherens Junction; Automobile Driving; Binding; Binding Proteins; Biochemical; Biological Assay; Blood Vessels; Cardiovascular Diseases; Cause of Death; Cell membrane; Cells; Centers for Disease Control and Prevention (U.S.); Complex; Cytoskeleton; Deubiquitination; Development; Early Endosome; Endosomes; Endothelial Cells; Endothelium; Enzymes; Extravasation; Functional disorder; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GPR3 gene; Generations; Goals; Human; In Vitro; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Knowledge; Libraries; Link; MAP3K7 gene; MAP3K7IP1 gene; Mediating; Methods; Microscopic; Microscopy; Mitogen-Activated Protein Kinases; Molecular; Mus; Myosin Light Chains; PAR-1 Receptor; Pathway interactions; Permeability; Phosphotransferases; Process; RNA interference screen; Regulation; Research; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Testing; Thrombin; Time; Ubiquitin; Ubiquitination; United States; Work; endothelial dysfunction; genome-wide; in vivo; mouse model; novel therapeutic intervention; p38 Mitogen Activated Protein Kinase; protein complex; recruit; response; therapeutic development; ubiquitin isopeptidase; ubiquitin-protein ligase; vascular inflammation; vascular injury

PROJECT SUMMARY / ABSTRACT Cardiovascular disease (CVD) is a leading cause of deaths in the United States, however the underlying inflammatory pathways that contribute to CVD are poorly understood. Endothelial dysfunction is a hallmark of CVD and induced by various inflammatory mediators, many of which signal through G-protein coupled receptor (GPCRs). Thrombin, an inflammatory mediator, is generated in response to vascular injury and disrupts endothelial barrier integrity and promotes vascular leakage, a hallmark of inflammation. Thrombin activates endothelial protease-activated receptor-1 (PAR1) to disrupt endothelial barrier through the RhoA/myosin light chain (MLC) pathway. However, thrombin/PAR1-stimulated p38 mitogen-activated protein kinase (MAPK) signaling also promotes barrier permeability through a pathway that does not integrate with the RhoA/MLC signaling, suggesting that p38 acts through an uncharacterized pathway to promote inflammatory responses in endothelial cells. Our previous work showed that thrombin-activated PAR1 ubiquitination drives the recruitment of TAB2-TAB1 protein complexes on endosomes to trigger non-canonical p38 activation and endothelial barrier disruption. The mechanisms that regulate PAR1 ubiquitination/de-ubiquitination are not known. This proposal aims to understand the molecular processes that regulate PAR1-ubiquitin-driven p38 signaling and most importantly its impact on endothelial barrier integrity. The central hypothesis is that de-ubiquitination of PAR1 leads to termination of p38-mediated pro-inflammatory signaling and will be tested with two specific aims: 1) To delineate the function of PAR1-specific deubiquitinases (DUBs) enzymes in the regulation of ubiquitin-driven p38 MAPK inflammatory signaling and 2) To study the functional impact of ubiquitination / de-ubiquitination of PAR1 on endothelial barrier permeability. Using advanced biochemical, microscopic and genome-wide RNAi screening methods, the proposed research plan will allow for the first time to investigate molecular determinants that regulate the dynamics of PAR1-ubiquitination and the impact on endothelial proinflammatory responses in vitro using human endothelial cells and in vivo using mice. The contribution of this work is expected to be significant as this study will reveal new information regarding the function of PAR1 ubiquitination in driving non-canonical endosomal p38 MAPK signaling in endothelial barrier disruption, a hallmark of inflammation and will further reveal a new signaling pathway that controls endothelial barrier function, which will provide an opportunity for the development new strategies for the treatment of inflammatory responses associated with CVD.

项目总结/摘要 心血管疾病（CVD）是美国死亡的主要原因，然而， 导致CVD的炎症途径知之甚少。内皮功能障碍是 CVD由多种炎症介质诱导，其中许多炎症介质通过G蛋白偶联受体（G-protein coupled receptor，G-PC）信号传导 （GPCR）。凝血酶是一种炎症介质，在血管损伤时产生， 内皮屏障完整性和促进血管渗漏，炎症的标志。凝血酶活化 内皮蛋白酶激活受体-1（PAR 1）通过RhoA/肌球蛋白光破坏内皮屏障 链（MLC）途径。然而，凝血酶/PAR 1刺激的p38丝裂原活化蛋白激酶（MAPK） 信号传导还通过不与RhoA/MLC整合的途径促进屏障通透性 信号，表明p38通过一个未表征的途径，以促进炎症反应， 内皮细胞我们以前的工作表明，凝血酶激活的PAR 1泛素化驱动了细胞的募集， TAB 2-TAB 1蛋白复合物在核内体上引发非典型p38激活和内皮屏障 破坏调节PAR 1泛素化/去泛素化的机制尚不清楚。这项建议 旨在了解调节PAR 1-泛素驱动的p38信号传导的分子过程， 重要的是其对内皮屏障完整性的影响。中心假设是PAR 1的去泛素化 导致p38介导的促炎信号传导的终止，并将以两个特定目的进行测试：1） 描述PAR 1特异性去泛素化酶（DUBs）在调节泛素驱动的p38中的功能 MAPK炎症信号传导和2）研究PAR 1的泛素化/去泛素化的功能影响 对内皮屏障通透性的影响。利用先进的生物化学、显微镜和全基因组RNAi筛选 方法，拟议的研究计划将首次允许调查分子决定因素， 调节PAR 1-泛素化的动力学和对体外内皮促炎反应的影响 使用人内皮细胞和在体内使用小鼠。预计这项工作的贡献将是巨大的 因为这项研究将揭示关于PAR 1泛素化在驱动非典型细胞中的功能的新信息。 内体p38 MAPK信号在内皮屏障破坏中的作用，是炎症的标志， 揭示了一种控制内皮屏障功能的新信号通路，这将为 开发治疗CVD相关炎症反应的新策略。