Redox Regulation of Cu Importer CTR1 in Angiogenesis

项目总结 这项资助的目的是阐明铜进口商CTR1的硫醇氧化还原修饰是一个至关重要的环节 铜转运体、活性氧依赖的VEGFR2信号转导与修复 血管生成。NADPH氧化酶(NOX)产生的ROS作为信号分子促进血管内皮生长因子的诱导 血管内皮细胞(ECs)的血管生成和修复性新生血管。根本的问题仍然存在， 可扩散的ROS如何激活特定的氧化还原信号以促进治疗性血管生成？信令 ROS的作用是通过氧化蛋白质中的活性半胱氨酸残基生成“半胱氨酸” (Cys-OH)“(亚磺化)，参与二硫键的形成和氧化还原信号的传递。铜(铜)、An 必需微量营养素也通过未知的机制在血管生成中发挥重要作用。主要的铜 进入途径是通过铜导入蛋白CTR1，它在高度保守的C- 终端三合会，HCH190。这笔赠款的联合调查员报告说，HCH190三合会充当了一个松动的“插头” 铜进入，对于铜诱导的CTR1内化(调节性内吞作用)是必不可少的，它可以防止 过量铜对HEK细胞的毒性作用。然而，CTR1和血管内皮生长因子之间的机制联系诱导了 ROS信号在内皮细胞血管生成中的作用及其在体内的作用尚不清楚。基于我们的 初步数据，我们假设血管内皮生长因子通过NOX衍生的ROS诱导Cys189位CTR1的硫苯基化， 哪些驱动因素：1)CTR1与VEGFR2的结合及其随后的共同内化是激活所需的 以铜转运非依赖的方式持续的VEGFR2信号；以及2)铜进入依赖 内皮细胞中铜靶蛋白的激活。这反过来又促进了充分的血管生成和新生血管。 在缺血性疾病中。目的1研究血管内皮细胞生长因子诱导的CTR1半胱氨酸氧化及其作用 在人和小鼠内皮细胞的血管生成反应中。目标2将确定Cys-氧化的机制 CTR1激活血管内皮细胞生长因子受体2信号通路，并依赖于铜进入激活内皮细胞中的铜靶。目标3将决定 内皮细胞CTR1功能在ROS依赖的修复性新生血管中的体内意义 使用小鼠后肢缺血和伤口愈合模型解决潜在的机制。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；EC靶向的各种CTR1突变体的基因转移； 新研制的可诱导EC特异性CTR1-/-小鼠及CRISPR/Cas9产生的CTR1半胱氨酸氧化- 有缺陷的敲入突变小鼠。极具创新性的电感耦合等离子体质谱、X射线荧光显微镜、铜 荧光探针将用于分析细胞和组织中的细胞内铜。我们的提案将提供新颖的 对半胱氨酸氧化CTR1作为治疗缺血性心血管疾病的潜在靶点的见解。

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.