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Redox Regulation of Cu Importer CTR1 in Angiogenesis

Cu 进口商 CTR1 在血管生成中的氧化还原调节

基本信息

批准号：
9916528
负责人：
TOHRU FUKAI
金额：
$ 74.27万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-15 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9916528
关键词：
Address Adenoviruses Binding Biological Assay Biosensor Biotin Biotinylation Blood Vessels C-terminal CRISPR/Cas technology Cardiovascular Diseases Caveolae Cell Fractionation Cell surface Cells Clustered Regularly Interspaced Short Palindromic Repeats Complex Copper Cysteine Data Diffuse Disease Endocytosis Endosomes Endothelial Cells Endothelium Event Fluorescence Fluorescence Microscopy Fluorescent Probes Fractionation Future Gene Transfer Grant Growth Hindlimb Human Hydrogen Peroxide Impairment In Situ Inductively Coupled Plasma Mass Spectrometry Inflammatory Ischemia KDR gene Knock-in Knock-in Mouse Knockout Mice Label Link Measures Mediating Membrane Microdomains Micronutrients Modeling Modification Molecular Molecular Chaperones Mus Mutant Strains Mice NADPH Oxidase Oxidation-Reduction Oxides Pathway interactions Patients Peripheral arterial disease Play Process Production Protein-Lysine 6-Oxidase Proteins Reactive Oxygen Species Reagent Regulation Reporting Research Personnel Roentgen Rays Role Sampling Signal Transduction Signaling Molecule Skin wound healing Sulfhydryl Compounds Synchrotrons TXNIP gene Testing Tissues Toxic effect Triad Acrylic Resin Vascular Endothelial Growth Factors Wound models angiogenesis base cell motility cysteinesulfenic acid disulfide bond in vivo in vivo evaluation innovation insight live cell imaging mutant neovascularization new therapeutic target novel overexpression oxidation prevent promoter protein protein interaction response therapeutic angiogenesis therapeutic target uptake wound

项目摘要

PROJECT SUMMARY The aim of this grant is to elucidate the thiol redox modification of the Cu importer, CTR1 as a vital link between Cu transporters, reactive oxygen species (ROS)-dependent VEGFR2 signaling and reparative angiogenesis. ROS derived from NADPH oxidase (NOX) act as signaling molecules to promote VEGF-induced angiogenesis in endothelial cells (ECs) and reparative neovascularization. The fundamental question remains, how diffusible ROS can activate specific redox signaling to enhance therapeutic angiogenesis? The signaling function of ROS acts through oxidation of reactive Cys residues in proteins to generate “Cysteine sulfenic acid (Cys-OH)” (sulfenylation), which is involved in disulfide bond formation and redox signaling. Copper (Cu), an essential micronutrient, also plays an important role in angiogenesis via unknown mechanisms. The major Cu entry pathway is via the Cu importer, CTR1, which has only one cytosolic Cys189 in the highly conserved C- terminal triad, HCH190. The co-investigator of this grant reported that the HCH190 triad acts as a loose “plug” for Cu entry, and is essential for Cu-induced CTR1 internalization (regulatory endocytosis) which protects against excess Cu-induced toxicity in HEK cells. However, the mechanistic linkage between CTR1 and VEGF-induced ROS signaling in mediating angiogenesis in ECs and its in vivo role are entirely unknown. Based on our preliminary data, we hypothesize that VEGF induces sulfenylation of CTR1 at Cys189 via NOX-derived ROS, which drives: 1) CTR1 binding to VEGFR2 and their subsequent co-internalization required for activating sustained VEGFR2 signaling in a Cu transport-independent manner; and 2) Cu entry-dependent activation of Cu target proteins in ECs. This in turn promotes full angiogenesis and neovascularization in ischemic diseases. Aim 1 will characterize the VEGF-induced Cys oxidation of CTR1 and determine its role in angiogenic responses in human and mouse ECs. Aim 2 will determine the mechanisms by which Cys-oxidized CTR1 activates VEGFR2 signaling and Cu entry-dependent activation of Cu targets in ECs. Aim 3 will determine the in vivo significance of endothelial CTR1 function in ROS-dependent reparative neovascularization and address underlying mechanisms using mice hindlimb ischemia and wound healing models. We will use various innovative reagents, including biotin-labelled Cys-OH trapping probe; BiFC-based protein-protein interaction in situ and live cell imaging, cell surface biotinylation; and gene transfer of EC-targeted various CTR1 mutants; newly-developed inducible EC-specific CTR1-/- mice and CRISPR/Cas9-generated CTR1 Cys oxidation- defective knock-in mutant mice. Highly innovative ICP-Mass Spec, X-ray fluorescence microscopy, Cu fluorescence probe will be used to analyze intracellular Cu in cells and tissues. Our proposal will provide novel insights into Cys oxidized CTR1 as a potential therapeutic target for ischemic cardiovascular diseases.

项目总结