SUMOylation of Cu transport protein Atox1 in Atherosclerosis

该基金的目的是证明一种新的铜（Cu）分子伴侣的氧化还原依赖性SUMO化 “抗氧化剂-1（Atox 1）”作为促进Cu依赖性转录因子功能的关键开关与 血管炎症和动脉粥样硬化。铜与炎症和动脉粥样硬化有关， 未知机制我们以前发现铜分子伴侣Atox 1作为铜依赖性转录因子发挥作用， p47 phox因子促进炎症性组织中活性氧（ROS）依赖性炎症反应 内皮细胞（EC）。然而，控制Atox 1从其作为Cu的作用转换的分子机制， 伴侣蛋白作为炎症内皮细胞中铜依赖性转录因子的作用以及内皮Atox 1在炎症中的作用 动脉粥样硬化是完全未知的。SUMO化在转录调控中起重要作用 定位和DNA结合活性。我们的初步数据显示Atox 1定位于动脉粥样硬化的细胞核， 其中Cu积累的俯卧主动脉。EC特异性Atox 1-/-小鼠显示炎症显著减少， 动脉粥样硬化从机制上讲，TNFα刺激人内皮细胞可快速诱导Atox 1在Lys的SUMO化 (K)3在ROS依赖的方式，这是所需的铜进口商CTR 1/铜依赖Atox 1核 从胞浆易位，炎症基因表达和EC通透性。根据我们的初步数据， 我们假设Atox 1的SUMO化通过去SUMO化酶SENP 1的Cys氧化是一个关键， 作为Cu依赖性转录因子发挥作用的检查点促进EC炎症和屏障 功能障碍，这有助于动脉粥样硬化的发病机制。目标1将描述时间- Atox 1 SUMO化对促炎细胞因子应答的空间关系，并检测ROS- 细胞溶质中的依赖性Atox 1 SUMO化是Cu依赖性Atox 1核转位所必需的， 炎症反应和EC屏障功能障碍。目标2将确定Cys氧化（Cys-1） OH形成）将增加Atox 1在细胞质、Atox 1核 易位和转录因子的功能，导致炎症EC中的内皮功能障碍。生物素 将使用Cys-OH捕获探针。目的3将确定内皮Atox 1在体内的意义， 使用EC-Atox 1-/-和EC-CTR 1-/-研究铜依赖性血管炎症和动脉粥样硬化中的SUMO化 小鼠和CRISPR-Cas9介导的Atox 1 K3 R敲入突变小鼠。我们将使用创新的ICP-质谱; X射线荧光显微镜; Cu荧光探针，用于分析细胞和组织中的细胞内Cu。我们 该提案将为SUMO-Atox 1作为治疗以下疾病的潜在治疗靶点提供新的见解： 炎症依赖性心血管疾病如动脉粥样硬化。

The aim of this grant is to demonstrate a novel redox-dependent SUMOylation of Copper (Cu) chaperone “Antioxidant-1 (Atox1)” as a key switch to promote Cu-dependent transcription factor function linked to vascular inflammation and atherosclerosis. Cu is implicated in inflammation and atherosclerosis with unknown mechanism. We previously found that Cu-chaperone Atox1 functions as a Cu-dependent transcription factor for p47phox to promote reactive oxygen species (ROS)-dependent inflammatory responses in inflamed endothelial cells (ECs). However, molecular mechanisms that control the switch of Atox1 from its role as a Cu chaperone to function as a Cu-dependent transcription factor in inflamed ECs and role of endothelial Atox1 in atherosclerosis are entirely unknown. SUMOylation plays an important role in regulating transcription factor localization and DNA binding activity. Our preliminary data show that Atox1 is localized in the nuclei of athero- prone aorta in which Cu is accumulated. EC-specific Atox1-/- mice show significant reduction of inflammation and atherosclerosis. Mechanistically, TNFα stimulation in human ECs rapidly induced Atox1 SUMOylation at Lys (K)3 in ROS-dependent manner, which was required for Cu importer CTR1/Cu-dependent Atox1 nuclear translocation from cytosol, inflammatory gene expression and EC permeability. Based on our preliminary data, we hypothesize that SUMOylation of Atox1 via Cys oxidation of deSUMOylation enzyme SENP1 is a key check point to function as a Cu-dependent transcription factor to promote EC inflammation and barrier dysfunction, which contributes to pathogenesis of atherosclerosis. Aim 1 will characterize the temporal- spatial relationships of Atox1 SUMOylation in response to proinflammatory cytokines and examine if ROS- dependent Atox1 SUMOylation in the cytosol is required for Cu-dependent Atox1 nuclear translocation, inflammatory responses and EC barrier dysfunction in inflamed ECs. Aim 2 will determine if Cys oxidation (Cys- OH formation) of deSUMOylating enzyme SENP1 will increase Atox1 SUMOylation in the cytosol, Atox1 nuclear translocation and transcription factor function, leading to endothelial dysfunction in inflamed ECs. Biotin-labeled Cys-OH trapping probe will be used. Aim 3 will determine the in vivo significance of endothelial Atox1 SUMOylation in Cu-dependent vascular inflammation and atherosclerosis using EC-Atox1-/- and EC-CTR1-/- mice, and CRISPR-Cas9-mediated Atox1 K3R knock-in mutant mice. We will use innovative ICP-Mass Spec; X-ray fluorescence microscopy; Cu fluorescence probe to analyze intracellular Cu in cells and tissues. Our proposal will provide novel insights into SUMO-Atox1 as a potential therapeutic target for treatment of inflammation-dependent cardiovascular disease such as atherosclerosis.