Redox Regulation of Cu Importer CTR1 in Angiogenesis

项目概要 这笔资助的目的是阐明铜输入端 CTR1 的硫醇氧化还原修饰是一个重要的环节 Cu 转运蛋白、活性氧 (ROS) 依赖性 VEGFR2 信号传导和修复之间 血管生成。 NADPH 氧化酶 (NOX) 衍生的 ROS 作为信号分子促进 VEGF 诱导 内皮细胞（EC）中的血管生成和修复性新血管形成。根本问题依然存在， 扩散性ROS如何激活特定的氧化还原信号以增强治疗性血管生成？信令 ROS的功能是通过氧化蛋白质中的反应性半胱氨酸残基来产生“半胱氨酸亚磺酸” (Cys-OH)”（磺酰化），参与二硫键形成和氧化还原信号传导。铜 (Cu) 必需的微量营养素，还通过未知的机制在血管生成中发挥重要作用。主要铜 进入途径是通过 Cu 输入器 CTR1，它在高度保守的 C- 中只有一个胞质 Cys189 末端三联体，HCH190。这项资助的联合研究员报告说，HCH190 三联体充当了松散的“插头” Cu 进入，对于 Cu 诱导的 CTR1 内化（调节性内吞作用）至关重要，可防止 HEK 细胞中过量的 Cu 诱导的毒性。然而，CTR1 和 VEGF 诱导的细胞因子之间的机制联系 ROS 信号在介导 EC 血管生成中的作用及其体内作用尚不清楚。基于我们的 初步数据，我们假设 VEGF 通过 NOX 衍生的 ROS 诱导 CTR1 在 Cys189 处的磺酰化， 它驱动：1) CTR1 与 VEGFR2 结合以及激活所需的后续共内化 以不依赖 Cu 转运的方式维持 VEGFR2 信号传导； 2) Cu 条目依赖 EC 中 Cu 靶蛋白的激活。这反过来又促进充分的血管生成和新血管形成 在缺血性疾病中。目标 1 将表征 VEGF 诱导的 CTR1 Cys 氧化并确定其作用 人类和小鼠 EC 的血管生成反应。目标 2 将确定 Cys 氧化的机制 CTR1 激活 VEGFR2 信号传导以及 EC 中 Cu 靶标的 Cu 进入依赖性激活。目标 3 将决定 内皮 CTR1 功能在 ROS 依赖性修复性新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解决潜在机制。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；以及 EC 靶向的各种 CTR1 突变体的基因转移； 新开发的诱导型 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 质谱、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 深入了解 Cys 氧化 CTR1 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.