Novel E3 Ubiquitin Ligase CHFR Regulates Endothelial Barrier Integrity and Innate Immune Function

新型 E3 泛素连接酶 CHFR 调节内皮屏障完整性和先天免疫功能

基本信息

批准号：
10297258
负责人：
CHINNASWAMY TIRUPPATHI
金额：
$ 38.38万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-15 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10297258
关键词：
AKT1 gene Acute Address Adherens Junction Adult Respiratory Distress Syndrome Angiopoietin-2 Biochemical Biological Assay Blood Vessels Cell surface Cells Coupled Data Down-Regulation Edema Endothelial Cells Endothelium Event Extravasation FOXO1A gene Feasibility Studies Gatekeeping Generations Genetic Genetic Transcription Head Host Defense Host Defense Mechanism Human Inflammatory Injury Innate Immune Response Knock-out Knockout Mice Life Link Lung Mediating Mediator of activation protein Molecular Mus Nodal Nuclear Translocation P-Cadherin Pathogenicity Pathway interactions Permeability Phagocytes Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Physiological Plasma Polyubiquitin Process Proteins Pseudomonas aeruginosa Pseudomonas aeruginosa pneumonia Pulmonary Edema Ring Finger Domain Role Signal Transduction TIE-2 Receptor TLR4 gene Testing Therapeutic Time Tissues Ubiquitin Ubiquitination Up-Regulation Vascular Endothelial Cell bactericide base cadherin 5 defense response in vivo innate immune function intravital imaging lung injury macromolecule migration mouse model neutrophil new therapeutic target novel novel therapeutic intervention pathogen prevent protein degradation pulmonary function respiratory response tissue injury transcription factor ubiquitin-protein ligase vascular inflammation vascular injury

项目摘要

This revised and responsive application will investigate mechanistically how newly discovered endothelial cell expressed ubiquitin E3 ligase CHFR (checkpoint with fork-head and ring finger domain) regulates the barrier integrity and the innate immune function of vascular endothelial cells. VE-cadherin expressed at endothelial adherens junctions (AJs) functions as a “gatekeeper” to restrict extravasation of plasma macromolecules and influx of phagocytic neutrophils (PMNs) into tissue. However, the key biochemical mechanisms triggering the loss of VE-cad expression at AJs have remained elusive. Our Supporting Data show: 1) CHFR-mediated ubiquitylation through K48-linked polyubiquitin (poly-Ub) chains induced VE-cadherin degradation, 2) genetic deletion of CHFR in human lung endothelial cells (ECs) or mouse ECs in vivo prevented ubiquitylation and degradation of VE-cadherin; 3) EC-specific deletion of Chfr in (ChfrDEC) mice also reduced the generation of the potent endothelial barrier-disrupting mediator angiopoietin-2 (Ang-2) which was coupled to reduction in pulmonary edema; 4) CHFR additionally ubiquitylates AKT1 via K48-linked poly-Ub in ECs, which reduced AKT1 expression and led to increased FoxO1 nuclear translocation and activation; 5) ECspecific deletion of FoxO1 (FoxO1DEC) in mice prevented expression of CHFR and Ang-2, and disruption of VEcadherin barrier; and 6) EC-specific deletion of Chfr in mice also enhanced the ability of PMNs to phagocytose and eliminate Pseudomonas aeruginosa. Based on these exciting Supporting Data, in Aim 1, we will test the hypothesis that expression of CHFR in lung ECs, downstream of TLR4 signaling, causes the loss of VE-cad expression at AJs by ubiquitylation of VE-cad through K48-linked polyubiquitin chains. In Aim 2, we will test the hypothesis that TLR4-induced CHFR expression increases FoxO1-mediated Ang-2 generation to injure the endothelial barrier subsequent to the degradation of the FoxO1 negative regulator through the ubiquitylation of AKT1 via K48-linked polyubiquitin chains. In Aim 3, we will test the hypothesis that CHFR-mediated loss of VE-cadherin at AJs induces transendothelial migration of PMNs and is an essential host-defense mechanism regulating bacterial elimination capacity of transmigrated PMNs. These studies will employ rigorous biochemical, molecular, in vivo real-time intravital imaging, and functional assays to define how CHFR mediates the degradation of VE-cadherin and AKT1 through the ubiquitylation-dependent pathway and its consequences on endothelial barrier integrity and innate immune function of the endothelium. We will use ECrestricted knockout (Chfr􀀀EC and FoxO1􀀀EC) mouse models generated by us to accomplish the above aims. The intent of these studies is to identify and develop novel therapeutic approaches targeting ARDS via the manipulation of CHFR.

这个经过修改的响应式应用程序将机械地调查新发现的内皮细胞表达的泛素 E3 连接酶 CHFR（具有叉头和环指结构域的检查点）调节血管内皮细胞的屏障完整性和先天免疫功能，血管内皮细胞在内皮粘附连接 (AJ) 上表达，发挥“看门人”的作用。限制血浆大分子的外渗和吞噬性中性粒细胞（PMN）流入组织然而，关键的生化作用。触发 AJ 处 VE-cad 表达缺失的机制仍然难以捉摸。我们的支持数据显示：1) CHFR 通过 K48 连接的多聚泛素 (poly-Ub) 诱导的 VE-cadherin 链介导的泛素化。 2) 体内人肺内皮细胞 (EC) 或小鼠 EC 中 CHFR 的基因缺失可阻止 VE-钙粘蛋白的泛素化和降解；3) (ChfrDEC) 小鼠中 Chfr 的 EC 特异性缺失也减少了强效蛋白的产生。内皮屏障破坏介质血管生成素-2 (Ang-2) 与肺水肿的减轻有关 4) CHFR 另外； EC 中通过 K48 连接的聚泛素泛素化 AKT1，从而减少 AKT1 表达并导致 FoxO1 核易位和激活增加；5) 小鼠中 EC 特异性删除 FoxO1 (FoxO1DEC) 可阻止 CHFR 和 Ang-2 的表达以及 VEcadherin 的破坏6) 小鼠中 EC 特异性删除 Chfr 也增强了 PMN 的吞噬和消除能力基于这些令人兴奋的支持数据，在目标 1 中，我们将测试以下假设：肺 EC 中 TLR4 信号下游的 CHFR 表达通过 K48 泛素化 VE-cad 导致 AJ 处 VE-cad 表达缺失。在目标 2 中，我们将检验 TLR4 诱导的 CHFR 表达增加 FoxO1 介导的 Ang-2 生成的假设。伤害在目标 3 中，我们将测试以下假设：CHFR 介导的 AJ 处 VE-钙粘蛋白的丢失会诱导 PMN 的跨内皮迁移，并且是通过 K48 连接的多聚泛素链泛素化 FoxO1 负调节因子降解后的内皮屏障。调节迁移的 PMN 的细菌消除能力的重要宿主防御机制。这些研究将采用严格的生物化学、分子、体内方法。实时活体成像和功能测定，以确定 CHFR 如何通过泛素化依赖性途径介导 VE-钙粘蛋白和 AKT1 的降解及其对内皮屏障完整性和内皮先天免疫功能的影响EC 和 FoxO1EC）小鼠模型是我们为实现上述目标而创建的，这些研究的目的是确定和开发新的治疗靶向方法。 ARDS 通过 CHFR 的操纵。