Abstract 883: Nadph Oxidase Nox1-dependent Endocytosis and Activation is Regulated by a Conserved Trafficking Motif in Vascular Smooth Muscle Cells

Abstract

We previously reported that TNF-α-mediated Nox1-dependent redox signaling of vascular smooth muscle cells (SMC) requires endocytosis. However, the molecular mechanisms regulating Nox1 endocytosis are not known. The goal of this study was to identify motifs in the Nox1 protein sequence necessary for Nox1 endocytosis and reactive oxygen species (ROS) generation. Immunoprecipitation of Nox1, after treatment of SMC with TNF-α, showed it to be associated with Early Endosome Antigen 1 (EEA1) and Ras-related protein Rab-5A (Rab5), markers of early endosomes. Analysis of the Nox1 protein sequence identified two conserved putative trafficking motifs, Leucine-Leucine (LL), and Valine-Leucine-Valine (VLV). Biotinylation of SMC plasma membrane proteins under non-stimulated conditions suggested increased levels of Nox1 VLV mutant in the plasma membrane, whereas levels of Nox1 LL mutant where comparable to wildtype (WT) Nox1. Furthermore, cells expressing the VLV mutant showed impaired endocytosis of Nox1 following TNF-α as compared to cells expressing WT Nox1 or the LL mutant. SMC were treated with TNF-α in the presence of Oxyburst, a non-cell permeable redox-sensitive fluorophore whose internalization requires endocytosis. Cells expressing WT or Nox1 LL, but not Nox1 VLV, exhibited punctate intracellular fluorescence, indicating endosomal ROS production. Next, we examined whether the VLV motif is required for Nox1-dependent SMC migration. Transwell migration of SMC was similar in cells expressing WT or the LL Nox1 mutant; however, migration was decreased in cells expressing the Nox1 VLV mutant. Finally, we identified proteins whose association with Nox1 during endocytosis were dependent on the VLV motif. Nox1 WT and VLV mutant were cloned in frame with the biotin ligase BioID2. Following stimulation with TNF-α, Western blotting for anti-biotin, and subsequent mass spectrometry, identified several proteins directly associated with WT Nox1 but not Nox1 VLV. These findings suggest that endosomal compartmentalization of Nox1 and its redox signaling is regulated by a specific motif on Nox1 that is necessary for protein interactions. Targeted inhibition of Nox1 activation is a novel therapeutic strategy in the prevention of vascular disease.