Cu importer CTR1 Cys189 oxidation in atherosclerosis

Charlie Norwood Va Medical Center, Augusta GA

Investigators

Linked publications & trials

Abstract

The aim of this grant is to elucidate the novel role of endothelial Cu importer CTR1 Cys189 oxidation to protect against Cu-dependent senescence and cuproptosis involved in inflammatory vascular disease. Oxidative stress, inflammation, mitochondrial (mito) ROS/dysfunction in endothelial cells (ECs) contribute to atherosclerosis, which is the major cause of mortality in the Veteran population. However, current anti- inflammatory and antioxidants therapy have mixed results. Evidence suggests that senescent cells provide a chronic source of pro-inflammatory cytokines, and thus become potential therapeutic targets for atherosclerosis. Copper (Cu), an essential micronutrient, is increased in human atherosclerotic plaques, while Cu promotes, and Cu chelators inhibit atherosclerosis in mice via unknown mechanisms. Excess Cu and mitoROS are shown to promote senescence. Since excess Cu is toxic, the bioavailability of intracellular Cu ([Cu]i) is tightly controlled by the Cu importer CTR1 that promotes Cu entry to provide Cu not only to the cytosolic Cu chaperone/nuclear Cu-dependent transcription factor Atox1 to promote inflammation, but also to the mito matrix to maintain mito function. We reported that growth factor VEGF-induced H2O2 stimulates rapid oxidation of CTR1 at Cys189, which promotes VEGFR2 signaling and angiogenesis in ECs. In contrast, the highly conserved CTR1-HCH190 triad in C-terminal is known to be involved in Cu-induced CTR1 regulatory endocytosis which prevents excess Cu entry-driven Cu toxicity; however, the role of endothelial CTR1-Cys189 in Cu- and ROS-dependent senescence and atherosclerosis is entirely unknown. In addition to senescence, recent study reported that excess Cu induces a new type of programmed cell death, termed “cuproptosis”, characterized by decrease in lipoylated TCA cycle proteins and Fe-S cluster proteins, resulting in mito dysfunction and cell death. Role of cuproptosis in atherosclerosis and its relation to endothelial CTR1-Cys189 remain elusive. Our preliminary data suggest that inducible EC-specific mCtr1-/-/ApoE-/- with high fat diet (HFD) mice aorta exhibited reduced atherosclerosis vs. atherosclerotic ApoE-/-/HFD mice aorta which showed Ctr1-CysOH formation vs. control ApoE-/-/chow diet mice. Unexpectedly, inhibiting Ctr1 Cys oxidation using our CRISPR/Cas9-generated “redox- dead and internalization defective” CTR1-Cys189Ala knock-in mutant (mCtr1-C/A)/ApoE-/-/HFD mice showed accelerated atherosclerosis vs. ApoE-/-/HFD mice, which was due to increasing mito[Cu]i and inhibited by Cu chelator TTM. These in vivo data were recapitulated by using human ECs stimulated with proatherogenic cytokine TNF. With other preliminary data, we will test the novel hypothesis that endothelial CTR1-Cys189 oxidation limits excess Cu entry to protect against pro-atherogenic phenotype of CTR1/Cu. Preventing CTR1-Cys189-mediated internalization increases mitoCu following CTR1 binding to mitoCu transporter, leading to mitoROS-senescence/inflammation axis as well as mito dysfunction/cuproptosis axis, which accelerates atherosclerosis. Aim 1 will establish the role of CTR1-Cys189 oxidation in limiting excess Cu- induced senescence and cuproptosis in inflamed ECs stimulated with TNF. Aim 2 will examine if preventing CTR1-Cys189 oxidation-mediated internalization increases mitoCu following CTR1 binding to mitoCu transporter in ECs stimulated with TNF. Aim 3 will determine whether endothelial CTR1-Cys189 oxidation protects against atherosclerosis by limiting excess Cu-induced senescence and cuptoptosis in vivo. We will use inducible EC- Ctr1-/- or -SLC25a3-/- mice or innovative CRISPR/Cas9-generated mCtr1-C/A mice with HFD or partial carotic ligation atherosclerosis models. We will also use compartment-specific redox-sensitive biosensors; biotin- labeled CysOH trapping probe; scRNAseq and scATACseq; newly developed, mito-targeted Cu-depleting nanoparticle (mitoCDN); highly innovative ICP-Mass Spec, X-ray fluorescence microscopy and mito-targeted Cu fluorescence probes to measure Cu levels. Our proposal will provide new insights into mitoCu or endothelial CTR1 Cys189 oxidation as a potential therapeutic target for Cu-dependent inflammatory vascular disease.