Redox Regulation of Cu Importer CTR1 in Angiogenesis

项目摘要 这项研究的目的是阐明铜进口商的巯基氧化还原修饰，CTR 1作为一个重要的环节 铜转运蛋白，活性氧（ROS）依赖性VEGFR 2信号转导和修复性 血管生成来源于NADPH氧化酶（NOX）的ROS作为信号分子促进VEGF诱导的细胞凋亡。 内皮细胞（EC）中的血管生成和修复性新血管形成。最根本的问题仍然是， 可扩散ROS如何激活特异性氧化还原信号以增强治疗性血管生成？信令 活性氧的功能是通过氧化蛋白质中的活性半胱氨酸残基产生“半胱氨酸次磺酸”而起作用的 （Cys-OH）-（亚磺酰化），其参与二硫键形成和氧化还原信号传导。铜（Cu），一种 必需的微量营养素，也通过未知的机制在血管生成中发挥重要作用。主要Cu 进入途径是通过铜输入者，CTR 1，它只有一个胞质Cys 189在高度保守的C- 末端三联体，HCH 190。这项研究的合作研究者报告说，HCH 190三联体作为一个松散的“插头”， Cu进入，并且对于Cu诱导的CTR 1内化（调节性内吞作用）是必需的，其保护免受 过量Cu诱导的HEK细胞毒性。然而，CTR 1和VEGF诱导的细胞凋亡之间的机制联系， ROS信号在介导内皮细胞血管生成及其在体内的作用是完全未知的。基于我们 根据初步数据，我们假设VEGF通过NOX衍生的ROS诱导Cys 189处CTR 1的磺酰化， 其驱动：1）CTR 1与VEGFR 2的结合以及其随后的共内化，所述共内化是激活VEGFR 2所需的。 以Cu转运非依赖性方式持续VEGFR 2信号传导;和2）Cu进入依赖性 EC中Cu靶蛋白的活化。这反过来又促进了全面的血管生成和新血管形成 在缺血性疾病中。目的1将表征VEGF诱导的CTR 1的Cys氧化并确定其作用 在人类和小鼠EC的血管生成反应中。目的2将确定半胱氨酸氧化的机制， CTR 1激活VEGFR 2信号传导和EC中Cu靶点的Cu进入依赖性激活。目标3将决定 内皮细胞CTR 1功能在ROS依赖性修复性新生血管形成中的体内意义， 使用小鼠后肢缺血和伤口愈合模型来阐明潜在的机制。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; BiFC基蛋白质-蛋白质相互作用， 原位和活细胞成像、细胞表面生物素化;以及EC靶向的各种CTR 1突变体的基因转移; 新开发的诱导型EC特异性CTR 1-/-小鼠和CRISPR/Cas9产生的CTR 1 Cys氧化- 缺陷基因敲入突变小鼠。高度创新的ICP-质谱仪、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铜。我们的建议将提供新颖的 Cys氧化CTR 1作为缺血性心血管疾病的潜在治疗靶点。

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.