Protein–Protein Interaction Assay to Analyze NOX4/p22phox Heterodimerization
The stabilization and activation of NOX4 through its binding with p22phox 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 p22phox, we developed a binary luciferase reporter assay (NanoBiT®) to quantitatively assess NOX4-p22phox 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.
Key wordsNADPH oxidase NOX4 p22phox Protein–protein interaction (PPI) Heterodimerization NanoBiT® Bioluminescence
This work was supported by Science Foundation Ireland (UGK) and the MolCellBiol Programme (Programme for Research in Third-Level Institutions, co-funded under the EU Regional Development Fund) (SON). We thank M. Mathis and R. Bouhelal of the Novartis Institutes for Biomedical Research, Switzerland, and Promega Corporation, Wisconsin, USA, for the collaborative effort in establishing this assay and for providing reagents.
- 3.O’Neill S, Mathis M, Kovacic L, Zhang S, Reinhardt J, Scholz D, Schopfer U, Bouhelal R, Knaus UG (2018) Quantitative interaction analysis permits molecular insights into functional NOX4 NADPH oxidase heterodimer assembly. J Biol Chem 293(23):8750–8760. https://doi.org/10.1074/jbc.RA117.001045 CrossRefGoogle Scholar
- 11.Stynen B, Tournu H, Tavernier J, Van Dijck P (2012) Diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian split-luciferase system. Microbiol Mol Biol Rev 76(2):331–382. https://doi.org/10.1128/MMBR.05021-11 CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Prior KK, Wittig I, Leisegang MS, Groenendyk J, Weissmann N, Michalak M, Jansen-Durr P, Shah AM, Brandes RP (2016) The endoplasmic reticulum chaperone calnexin is a NADPH oxidase NOX4 interacting protein. J Biol Chem 291(13):7045–7059. https://doi.org/10.1074/jbc.M115.710772 CrossRefPubMedPubMedCentralGoogle Scholar
- 15.Al Ghouleh I, Meijles DN, Mutchler S, Zhang Q, Sahoo S, Gorelova A, Henrich Amaral J, Rodriguez AI, Mamonova T, Song GJ, Bisello A, Friedman PA, Cifuentes-Pagano ME, Pagano PJ (2016) Binding of EBP50 to Nox organizing subunit p47phox is pivotal to cellular reactive species generation and altered vascular phenotype. Proc Natl Acad Sci U S A 113(36):E5308–E5317. https://doi.org/10.1073/pnas.1514161113 CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7(11):1848–1857. https://doi.org/10.1021/cb3002478 CrossRefPubMedPubMedCentralGoogle Scholar
- 19.Dixon AS, Schwinn MK, Hall MP, Zimmerman K, Otto P, Lubben TH, Butler BL, Binkowski BF, Machleidt T, Kirkland TA, Wood MG, Eggers CT, Encell LP, Wood KV (2016) NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem Biol 11(2):400–408. https://doi.org/10.1021/acschembio.5b00753 CrossRefPubMedGoogle Scholar