Protein-Protein Interaction Assay to Analyze NOX4/p22^phox Heterodimerization

The stabilization and activation of NOX4 through its binding with p22^phox are well documented; however little is known of the precise manner by which these two proteins interact. In recent years, the field of proteomics has undergone tremendous development with the introduction of many novel methods for the identification and characterization of protein-protein interactions (PPIs). To enhance our understanding of structural determinants leading to the association between NOX4 and p22^phox, we developed a binary luciferase reporter assay (NanoBiT^®) to quantitatively assess NOX4-p22^phox heterodimerization. The complementation reporter quantitatively determines the accurate, reduced, or failed complex assembly, which can be confirmed and further interrogated by analyzing NOX4 catalytic activity (H₂O₂ release), protein expression, and dimer localization. This association-based PPI technique represents both a much-needed expansion of the NOX4 lead discovery tool box and a versatile method to probe the architecture of NOX and DUOX complexes in the future.