SUMOylation of Cu transport protein Atox1 in Atherosclerosis

这项资助的目的是证明一种新的依赖于氧化还原的铜(铜)伴侣的SUMO化 抗氧化剂-1(Atox1)作为促进铜依赖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 未知的机制。我们先前发现，铜伴侣蛋白Atox1具有铜依赖转录的功能 P47Phox促进炎症反应中ROS依赖的炎症反应的因子 内皮细胞(ECs)。然而，控制Atox1开关的分子机制从其作为铜的作用 伴侣蛋白在炎症内皮细胞中作为铜依赖转录因子的作用及内皮Atox1在其中的作用 动脉粥样硬化是完全未知的。相思甲基化在转录因子调控中起着重要作用 定位和DNA结合活性。我们的初步数据显示，Atox1定位于动脉粥样硬化的细胞核中。 容易积聚铜的大动脉。EC特异性Atox1/-小鼠表现出显著的减轻炎症和 动脉硬化。从机制上讲，肿瘤坏死因子α刺激人内皮细胞可迅速诱导Atox1在Lys发生苏氨酸化 (K)3以ROS依赖的方式，这是铜进口商CTR1/依赖铜的Atox1核所必需的 胞浆易位、炎性基因表达和EC通透性。根据我们的初步数据， 我们推测，Atox1通过半胱氨酸氧化脱糖基酶SENP1的糖基化是关键 Check Point作为铜依赖的转录因子促进EC炎症和屏障 功能障碍，参与动脉粥样硬化的发病机制。目标1将描述时间- 促炎症细胞因子对Atox1 SUMO化反应的空间关系，并检查ROS是否- 依赖于铜的Atox1核转位需要胞浆中依赖的Atox1 SUMO化， 炎症内皮细胞的炎症反应和内皮屏障功能障碍。目标2将确定半胱氨酸氧化(Cys- ··· 转位和转录因子功能，导致炎症内皮细胞的内皮功能障碍。生物素标记 将使用CyS-OH捕集探头。目的3确定血管内皮细胞Atox1的体内意义 EC-Atox1-/-和EC-CTR1-/-在铜依赖的血管炎症和动脉粥样硬化中的苏莫化作用 小鼠，以及CRISPR-Cas9介导的Atox1 K3R敲入突变小鼠。我们将使用创新的电感耦合等离子体质谱； X射线荧光显微镜；铜荧光探针分析细胞和组织中的铜。我们的 该提案将为相扑-Atox1作为潜在的治疗靶点提供新的见解 依赖炎症的心血管疾病，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.