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Protein–Protein Interaction Assay to Analyze NOX4/p22phox Heterodimerization

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NADPH Oxidases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1982))

Abstract

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.

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References

  1. DeLeo FR, Burritt JB, Yu L, Jesaitis AJ, Dinauer MC, Nauseef WM (2000) Processing and maturation of flavocytochrome b558 include incorporation of heme as a prerequisite for heterodimer assembly. J Biol Chem 275(18):13,986–13,993

    Article  CAS  Google Scholar 

  2. Biberstine-Kinkade KJ, DeLeo FR, Epstein RI, LeRoy BA, Nauseef WM, Dinauer MC (2001) Heme-ligating histidines in flavocytochrome b(558): identification of specific histidines in gp91(phox). J Biol Chem 276(33):31,105–31,112

    Article  CAS  Google Scholar 

  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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Groemping Y, Rittinger K (2005) Activation and assembly of the NADPH oxidase: a structural perspective. Biochem J 386. (Pt 3:401–416. https://doi.org/10.1042/BJ20041835

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Sumimoto H (2008) Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species. FEBS J 275(13):3249–3277. https://doi.org/10.1111/j.1742-4658.2008.06488.x

    Article  CAS  PubMed  Google Scholar 

  6. Luxen S, Noack D, Frausto M, Davanture S, Torbett BE, Knaus UG (2009) Heterodimerization controls localization of Duox-DuoxA NADPH oxidases in airway cells. J Cell Sci 122. (Pt 8:1238–1247. https://doi.org/10.1242/jcs.044123

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ameziane-El-Hassani R, Morand S, Boucher JL, Frapart YM, Apostolou D, Agnandji D, Gnidehou S, Ohayon R, Noel-Hudson MS, Francon J, Lalaoui K, Virion A, Dupuy C (2005) Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity. J Biol Chem 280(34):30,046–30,054

    Article  CAS  Google Scholar 

  8. Ueyama T, Sakuma M, Ninoyu Y, Hamada T, Dupuy C, Geiszt M, Leto TL, Saito N (2015) The extracellular A-loop of dual oxidases affects the specificity of reactive oxygen species release. J Biol Chem 290(10):6495–6506. https://doi.org/10.1074/jbc.M114.592717

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Takac I, Schroder K, Zhang L, Lardy B, Anilkumar N, Lambeth JD, Shah AM, Morel F, Brandes RP (2011) The E-loop is involved in hydrogen peroxide formation by the NADPH oxidase Nox4. J Biol Chem 286(15):13,304–13,313. https://doi.org/10.1074/jbc.M110.192138

    Article  CAS  Google Scholar 

  10. von Lohneysen K, Noack D, Jesaitis AJ, Dinauer MC, Knaus UG (2008) Mutational analysis reveals distinct features of the Nox4-p22 phox complex. J Biol Chem 283(50):35,273–35,282. https://doi.org/10.1074/jbc.M804200200

    Article  CAS  Google 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Xing S, Wallmeroth N, Berendzen KW, Grefen C (2016) Techniques for the analysis of protein-protein interactions in vivo. Plant Physiol 171(2):727–758. https://doi.org/10.1104/pp.16.00470

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ambasta RK, Kumar P, Griendling KK, Schmidt HH, Busse R, Brandes RP (2004) Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J Biol Chem 279(44):45,935–45,941

    Article  CAS  Google 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

    Article  CAS  PubMed  PubMed Central  Google 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Remy I, Michnick SW (2006) A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nat Methods 3(12):977–979. https://doi.org/10.1038/nmeth979

    Article  CAS  PubMed  Google 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Mo XL, Fu H (2016) BRET: nanoluc-based bioluminescence resonance energy transfer platform to monitor protein-protein interactions in live cells. Methods Mol Biol 1439:263–271. https://doi.org/10.1007/978-1-4939-3673-1_17

    Article  CAS  PubMed  Google 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

    Article  CAS  PubMed  Google Scholar 

  20. Oh-Hashi K, Hirata Y, Kiuchi K (2016) SOD1 dimerization monitoring using a novel split NanoLuc, NanoBit. Cell Biochem Funct 34(7):497–504. https://doi.org/10.1002/cbf.3222

    Article  CAS  PubMed  Google Scholar 

  21. Duellman SJ, Machleidt T, Cali JJ, Vidugiriene J (2017) Cell-based, bioluminescent assay for monitoring the interaction between PCSK9 and the LDL receptor. J Lipid Res 58(8):1722–1729. https://doi.org/10.1194/jlr.D074658

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ruch W, Cooper PH, Baggiolini M (1983) Assay of H2O2 production by macrophages and neutrophils with homovanillic acid and horse-radish peroxidase. J Immunol Methods 63(3):347–357

    Article  CAS  PubMed  Google Scholar 

  23. Martyn KD, Frederick LM, von Loehneysen K, Dinauer MC, Knaus UG (2006) Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases. Cell Signal 18(1):69–82. https://doi.org/10.1016/j.cellsig.2005.03.023

    Article  CAS  PubMed  Google Scholar 

  24. Czupryna J, Tsourkas A (2011) Firefly luciferase and RLuc8 exhibit differential sensitivity to oxidative stress in apoptotic cells. PLoS One 6(5):e20073. https://doi.org/10.1371/journal.pone.0020073

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

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.

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Authors and Affiliations

  1. Conway Institute, School of Medicine, University College Dublin, Dublin, Ireland

    Sharon O’Neill & Ulla G. Knaus

Authors
  1. Sharon O’Neill
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  2. Ulla G. Knaus
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Correspondence to Ulla G. Knaus .

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Editors and Affiliations

  1. Conway Institute, School of Medicine, University College Dublin, Dublin, Ireland

    Ulla G. Knaus

  2. Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Thomas L. Leto

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O’Neill, S., Knaus, U.G. (2019). Protein–Protein Interaction Assay to Analyze NOX4/p22phox Heterodimerization. In: Knaus, U., Leto, T. (eds) NADPH Oxidases. Methods in Molecular Biology, vol 1982. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9424-3_26

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  • DOI: https://doi.org/10.1007/978-1-4939-9424-3_26

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9423-6

  • Online ISBN: 978-1-4939-9424-3

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