Selective Recapitulation of Conserved and Nonconserved Regions of Putative NOXA1 Protein Activation Domain Confers Isoform-specific Inhibition of Nox1 Oxidase and Attenuation of Endothelial Cell Migration

Excessive vascular and colon epithelial reactive oxygen species production by NADPH oxidase isoform 1 (Nox1) has been implicated in a number of disease states, including hypertension, atherosclerosis, and neoplasia. A peptide that mimics a putative activation domain of the Nox1 activator subunit NOXA1 (NOXA1 docking sequence, also known as NoxA1ds) potently inhibited Nox1-derived superoxide anion (O₂^⨪) production in a reconstituted Nox1 cell-free system, with no effect on Nox2-, Nox4-, Nox5-, or xanthine oxidase-derived reactive oxygen species production as measured by cytochrome c reduction, Amplex Red fluorescence, and electron paramagnetic resonance. The ability of NoxA1ds to cross the plasma membrane was tested by confocal microscopy in a human colon cancer cell line exclusively expressing Nox1 (HT-29) using FITC-labeled NoxA1ds. NoxA1ds significantly inhibited whole HT-29 carcinoma cell-derived O₂^⨪ generation. ELISA and fluorescence recovery after photobleaching experiments indicate that NoxA1ds, but not its scrambled control, binds Nox1. FRET experiments conducted using Nox1-YFP and NOXA1-CFP illustrate that NoxA1ds disrupts the binding interaction between Nox1 and NOXA1, whereas a control peptide did not. Moreover, hypoxia-induced human pulmonary artery endothelial cell O₂^⨪ production was completely inhibited by NoxA1ds. Human pulmonary artery endothelial cell migration under hypoxic conditions was also reduced by pretreatment with NoxA1ds. Our data indicate that a peptide recapitulating a putative activation subdomain of NOXA1 (NoxA1ds) is a highly efficacious and selective inhibitor of Nox1 activity and establishes a critical interaction site for Nox1-NOXA1 binding required for enzyme activation.