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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：
10488226
负责人：
CHINNASWAMY TIRUPPATHI
金额：
$ 3.2万
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 2022-09-19
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10488226
关键词：
AKT1 gene Acute Acute Respiratory Distress Syndrome Address Adherens Junction 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.

这个修改后的响应性应用程序将从机制上调查新发现的情况