Novel Interaction of Antioxidant-1 with p47phox and TRAF4: Role in TNFalpha-induced ROS signaling and atherosclerosis

Atherosclerosis is a chronic inflammatory disease through NADPH oxidase (Nox) derived ROS and its downstream adhesion molecule expression in endothelial cells (ECs). Proinflammatory cytokine TNFalpha plays a key role in progression of atherosclerosis and activates Nox via promoting p47pox membrane translocation and binding to adaptor protein TRAF4 in ECs. Copper (Cu), an essential micronutrient, is involved in inflammation and atherogenesis with unknown mechanism. We reported that Cu-chaperone antioxidant-1(Atox1) functions as a Cu-dependent transcription factor to regulate cell growth. Recently we found that Atox1 increases p47phox transcription in a Cu-dependent manner in ECs. However, the role of Atox1 in p47phox membrane translocation and mechanism of Atox1 nuclear translocation in response to TNFalpha in ECs, and role of Atox1 in atherosclerosis are entirely unknown. Preliminary studies identified p47phox and TRAF4 as novel Atox1 binding proteins required for TNFalpha-induced biphasic ROS production in ECs. In vivo, Atox1-/-ApoE-/- mice showed reduced atherosclerotic lesions compared to ApoE-/- mice. Together with other preliminary data, we hypothesize that 'Atox1 binding to p47phox is required for Cu-independent Atox1/p47phox membrane translocation to produce early phase ROS, and subsequent Atox1 binding to TRAF4 is required for Cu-dependent Atox1 nuclear translocation for increasing p47phox expression to produce late phase ROS in TNFalpha;-stimulated ECs. This in turn promotes inflammatory cell recruitment and atherosclerosis'. Aim 1 will determine the molecular mechanisms by which Atox1 translocates to membrane and subsequently to nucleus to regulate p47phox function involved in biphasic ROS production and inflammatory responses in TNFalpha-stimulated ECs. We will perform in vitro pull-down and in vivo co-transfection assays to characterize the Atox1 binding to p47phox and TRAF4, and use ECs derived from Atox1-/- mice transfected with Atox1 mutated at p47phox or TRAF4 binding sites. Aim 2 will assess the functional role of endothelial Atox1 in atherosclerosis using EC-specific conditional Atox1-/-mice crossed with ApoE-/- mice. Innovative Cu imaging using X-ray fluorescence microscopy will be used. Our studies should provide a new insight to Atox1 as a potential therapeutic target for inflammatory diseases such as atherosclerosis. (AHA Program: Postdoctoral Fellowship)