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, TNF? 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.
Atherosclerosis is the major cause of mortality in Veteran population. Copper, an essential nutrient, has been shown to have critical roles in vascular inflammation and atherosclerosis with unknown mechanism. The current studies will not only advance the field to understand the molecular mechanism through which copper transporters and copper are involved in the progression of atherosclerotic process, but also provide a basis to discover the novel therapeutic targets and strategies for treatment for atherosclerosis and various vascular diseases.
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