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.

项目摘要这笔赠款的目的是阐明Cu进口商的硫醇氧化还原修饰为CTR1 在Cu转运蛋白之间，活性氧（ROS）依赖性VEGFR2信号传导和修复血管生成。源自NADPH氧化酶（NOX）的ROS充当信号分子，以促进VEGF诱导的内皮细胞（EC）和修复新血管的血管生成。基本问题仍然是可扩散的ROS如何激活特定的氧化还原信号传导以增强热血管生成？信号传导 ROS的功能通过氧化反应性Cys保留在蛋白质中以产生“半胱氨酸硫酸（cys-OH）”（磺苯基），参与二硫键形成和氧化还原信号传导。铜（Cu），A 必需的微量营养素，通过未知机制在血管生成中也起着重要作用。主要的铜进入途径是通过CU进口商CTR1，该进口途径在高度保守的C-中只有一个胞质Cys189 终端三合会，HCH190。该赠款的共同研究人员报告说，HCH190三合会是一个松散的“插头” CU进入，对于CU诱导的CTR1内在化（调节性内吞作用）至关重要过量的Cu诱导的HEK细胞毒性。但是，CTR1和VEGF诱导的机械链接 ROS信号在EC中介导的血管生成及其体内作用完全未知。基于我们初步数据，我们假设VEGF通过NOX衍生的ROS在Cys189处诱导Ctr1的磺苯基化，驱动器：1）Ctr1与VEGFR2结合及其随后的共内部化激活所需的共同内部化持续的VEGFR2信号传导以Cu传输独立的方式传导； 2）CU进入依赖性 EC中Cu靶蛋白的激活。这反过来促进了全血管生成和新血管形成在缺血性疾病中。 AIM 1将表征VEGF引起的CTR1的CYS氧化并确定其作用在人和小鼠EC中的血管生成反应中。 AIM 2将确定Cys氧化的机制 CTR1激活EC中Cu靶标的VEGFR2信号传导和Cu进入依赖性激活。 AIM 3将确定内皮CTR1功能在ROS依赖性的修复新生血管和的体内意义使用小鼠后肢缺血和伤口愈合模型来解决潜在的机制。我们将使用各种创新试剂，包括生物素标记的Cys-OH捕获探针；基于BIFC的蛋白质 - 蛋白质相互作用原位和活细胞成像，细胞表面生物素化；和靶向各种CTR1突变体的基因转移；新开发的诱导EC特异性CTR1 - / - 小鼠和CRISPR/CAS9生成的CTR1 CYS氧化 - 有缺陷的敲入突变小鼠。高度创新的ICP质量规格，X射线荧光显微镜，Cu 荧光探针将用于分析细胞和组织中的细胞内CU。我们的建议将提供小说对CYS的洞察力将CTR1氧化为缺血性心血管疾病的潜在治疗靶点。