NADPH Oxidases pp 139-151 | Cite as

Insights into the NOX NADPH Oxidases Using Heterologous Whole Cell Assays

  • Mary C. DinauerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)


Assays based on ectopic expression of NOX NADPH oxidase subunits in heterologous mammalian cells are an important approach for investigating features of this family of enzymes. These model systems have been used to analyze the biosynthesis and functional domains of NOX enzyme components as well as their regulation and cellular activities. This chapter provides an overview of the basic principles and applications of heterologous whole cell assays in studying NOX NADPH oxidases.

Key words

NOX Flavocytochrome b COS-7 Expression phox 



MCD is funded by the Children’s Discovery Institute at Washington University and St. Louis Children’s Hospital and NIH grants R01HL045635 and R01AR072212. I thank Tina McGrath and Diane Jensen for assistance with preparation of the manuscript. I apologize to colleagues whose work could not be cited due to space limitations.


  1. 1.
    Nunes P, Demaurex N, Dinauer MC (2013) Regulation of the NADPH oxidase and associated ion fluxes during phagocytosis. Traffic 14(11):1118–1131. CrossRefPubMedGoogle Scholar
  2. 2.
    Leto TL, Lavigne MC, Homoyounpour N, Lekstrom K, Linton G, Malech HL, de Mendez I (2007) The K-562 cell model for analysis of neutrophil NADPH oxidase function. In: Quinn MT, DeLeo FR, Bokoch GM (eds) Neutrophil methods and protocols. Humana Press, Totowa, NJ, pp 365–383. CrossRefGoogle Scholar
  3. 3.
    Miyano K, Sumimoto H (2012) Assessment of the role for rho family GTPases in NADPH oxidase activation. In: Rivero F (ed) Rho GTPases: methods and protocols. Springer New York, New York, NY, pp 195–212. CrossRefGoogle Scholar
  4. 4.
    Banfi B, Clark RA, Steger K, Krause KH (2003) Two novel proteins activate superoxide generation by the NADPH oxidase NOX1. J Biol Chem 278(6):3510–3513CrossRefGoogle Scholar
  5. 5.
    Takeya R, Ueno N, Kami K, Taura M, Kohjima M, Izaki T, Nunoi H, Sumimoto H (2003) Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases. J Biol Chem 278(27):25234–25246CrossRefGoogle Scholar
  6. 6.
    Yu L, Zhen L, Dinauer MC (1997) Biosynthesis of the phagocyte NADPH oxidase cytochrome b558. Role of heme incorporation and heterodimer formation in maturation and stability of gp91phox and p22phox subunits. J Biol Chem 272(43):27288–27294CrossRefGoogle Scholar
  7. 7.
    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):35273–35282. CrossRefGoogle Scholar
  8. 8.
    Suh CI, Stull ND, Li XJ, Tian W, Price MO, Grinstein S, Yaffe MB, Atkinson S, Dinauer MC (2006) The phosphoinositide-binding protein p40phox activates the NADPH oxidase during FcgammaIIA receptor-induced phagocytosis. J Exp Med 203(8):1915–1925. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Casbon AJ, Allen LA, Dunn KW, Dinauer MC (2009) Macrophage NADPH oxidase flavocytochrome B localizes to the plasma membrane and Rab11-positive recycling endosomes. J Immunol 182(4):2325–2339. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Matono R, Miyano K, Kiyohara T, Sumimoto H (2014) Arachidonic acid induces direct interaction of the p67phox-Rac complex with the phagocyte oxidase Nox2, leading to superoxide production. J Biol Chem 289(36):24874–24884. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    He R, Nanamori M, Sang H, Yin H, Dinauer MC, Ye RD (2004) Reconstitution of chemotactic peptide-induced nicotinamide adenine dinucleotide phosphate (reduced) oxidase activation in transgenic COS-phox cells. J Immunol 173(12):7462–7470CrossRefGoogle Scholar
  12. 12.
    de Souza SM, Salomon D, Orth K (2017) T3SS effector VopL inhibits the host ROS response, promoting the intracellular survival of Vibrio parahaemolyticus. PLoS Pathog 13(6):e1006438. CrossRefGoogle Scholar
  13. 13.
    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):31105–31112. CrossRefPubMedGoogle Scholar
  14. 14.
    Yu L, Quinn MT, Cross AR, Dinauer MC (1998) Gp91(phox) is the heme binding subunit of the superoxide-generating NADPH oxidase. Proc Natl Acad Sci U S A 95(14):7993–7998CrossRefGoogle Scholar
  15. 15.
    Biberstine-Kinkade KJ, Yu L, Stull N, LeRoy BA, Bennett S, Cross AR, Dinauer M (2002) Mutagenesis of p22phox Histidine 94. J Biol Chem 277(33):30368–30374CrossRefGoogle Scholar
  16. 16.
    von Löhneysen K, Noack D, Wood MR, Friedman JS, Knaus UG (2010) Structural insights into Nox4 and Nox2: motifs involved in function and cellular localization. Mol Cell Biol 30(4):961–975. CrossRefGoogle Scholar
  17. 17.
    O'Neill S, Mathis M, Kovačič 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. CrossRefPubMedGoogle Scholar
  18. 18.
    Leto TL, Adams AG, de Mendez I (1994) Assembly of the phagocyte NADPH oxidase: binding of Src homology 3 domains to proline-rich targets. Proc Natl Acad Sci U S A 91(22):10650–10654CrossRefGoogle Scholar
  19. 19.
    Koga H, Terasawa H, Nunoi H, Takeshige K, Inagaki F, Sumimoto H (1999) Tetratricopeptide repeat (TPR) motifs of p67(phox) participate in interaction with the small GTPase Rac and activation of the phagocyte NADPH oxidase. J Biol Chem 274(35):25051–25060CrossRefGoogle Scholar
  20. 20.
    Ago T, Nunoi H, Ito T, Sumimoto H (1999) Mechanism for phosphorylation-induced activation of the phagocyte NADPH oxidase protein p47(phox). Triple replacement of serines 303, 304, and 328 with aspartates disrupts the SH3 domain-mediated intramolecular interaction in p47(phox), thereby activating the oxidase. J Biol Chem 274(47):33644–33653CrossRefGoogle Scholar
  21. 21.
    Kanai F, Liu H, Field SJ, Akbary H, Matsuo T, Brown GE, Cantley LC, Yaffe MB (2001) The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nat Cell Biol 3(7):675–678CrossRefGoogle Scholar
  22. 22.
    Ellson CD, Gobert-Gosse S, Anderson KE, Davidson K, Erdjument-Bromage H, Tempst P, Thuring JW, Cooper MA, Lim ZY, Holmes AB, Gaffney PR, Coadwell J, Chilvers ER, Hawkins PT, Stephens LR (2001) PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40(phox). Nat Cell Biol 3(7):679–682CrossRefGoogle Scholar
  23. 23.
    Ellson CD, Davidson K, Ferguson GJ, O'Connor R, Stephens LR, Hawkins PT (2006) Neutrophils from p40phox−/− mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing. J Exp Med 203(8):1927–1937CrossRefGoogle Scholar
  24. 24.
    Ellson C, Davidson K, Anderson K, Stephens LR, Hawkins PT (2006) PtdIns3P binding to the PX domain of p40phox is a physiological signal in NADPH oxidase activation. EMBO J 25(19):4468–4478. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Matute JD, Arias AA, Wright NA, Wrobel I, Waterhouse CC, Li XJ, Marchal CC, Stull ND, Lewis DB, Steele M, Kellner JD, Yu W, Meroueh SO, Nauseef WM, Dinauer MC (2009) A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity. Blood 114(15):3309–3315. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    van de Geer A, Nieto-Patlán A, Kuhns DB, Tool ATJ, Arias AA, Bouaziz M, de Boer M, Franco JL, Gazendam RP, van Hamme JL, van Houdt M, van Leeuwen K, Verkuijlen PJH, van den Berg TK, Alzate JF, Arango-Franco CA, Batura V, Bernasconi AR, Boardman B, Booth C, Burns SO, Cabarcas F, Cerf Bensussan N, Charbit-Henrion F, Corveleyn A, Deswarte C, Esnaola Azcoiti M, Foell D, Gallin JI, Garcés C, Guedes M, Hinze CH, Holland SM, Hughes SM, Ibañez P, Malech HL, Meyts I, Moncada-Velez M, Moriya K, Neves E, Oleastro M, Perez L, Rattina V, Oleaga-Quintas C, Warner N, Muise AM, Serafin López J, Trindade E, Vasconselos J, Vermeire S, Wittkowski H, Worth A, Abel L, Dinauer MC, Arkwright PD, Roos D, Casanova J-L, Kuijpers TW, Bustamante J (2018) Inherited p40phox deficiency differs from classic chronic granulomatous disease. J Clin Invest 128(9):3957–3975. [Epub ahead of print]CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Li XJ, Marchal CC, Stull ND, Stahelin RV, Dinauer MC (2010) p47phox Phox homology domain regulates plasma membrane but not phagosome neutrophil NADPH oxidase activation. J Biol Chem 285(45):35169–35179. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Zhao J, Ma J, Deng Y, Kelly JA, Kim K, Bang SY, Lee HS, Li QZ, Wakeland EK, Qiu R, Liu M, Guo J, Li Z, Tan W, Rasmussen A, Lessard CJ, Sivils KL, Hahn BH, Grossman JM, Kamen DL, Gilkeson GS, Bae SC, Gaffney PM, Shen N, Tsao BP (2017) A missense variant in NCF1 is associated with susceptibility to multiple autoimmune diseases. Nat Genet 49(3):433–437. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Olsson LM, Johansson AC, Gullstrand B, Jonsen A, Saevarsdottir S, Ronnblom L, Leonard D, Wettero J, Sjowall C, Svenungsson E, Gunnarsson I, Bengtsson AA, Holmdahl R (2017) A single nucleotide polymorphism in the NCF1 gene leading to reduced oxidative burst is associated with systemic lupus erythematosus. Ann Rheum Dis 76(9):1607–1613. CrossRefPubMedGoogle Scholar
  30. 30.
    Geiszt M, Lekstrom K, Witta J, Leto TL (2003) Proteins homologous to p47phox and p67phox support superoxide production by NAD(P)H oxidase 1 in colon epithelial cells. J Biol Chem 278(22):20006–20012. CrossRefPubMedGoogle Scholar
  31. 31.
    Miyano K, Ueno N, Takeya R, Sumimoto H (2006) Direct involvement of the small GTPase Rac in activation of the superoxide-producing NADPH oxidase Nox1. J Biol Chem 281(31):21857–21868. CrossRefPubMedGoogle Scholar
  32. 32.
    Cheng G, Ritsick D, Lambeth JD (2004) Nox3 regulation by NOXO1, p47phox, and p67phox. J Biol Chem 279(33):34250–34255. CrossRefPubMedGoogle Scholar
  33. 33.
    Ueno N, Takeya R, Miyano K, Kikuchi H, Sumimoto H (2005) The NADPH oxidase Nox3 constitutively produces superoxide in a p22phox-dependent manner: its regulation by oxidase organizers and activators. J Biol Chem 280(24):23328–23339CrossRefGoogle Scholar
  34. 34.
    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. CrossRefPubMedGoogle Scholar
  35. 35.
    von Löhneysen K, Noack D, Hayes P, Friedman JS, Knaus UG (2012) Constitutive NADPH oxidase 4 activity resides in the composition of the B-loop and the penultimate C terminus. J Biol Chem 287(12):8737–8745. CrossRefGoogle Scholar
  36. 36.
    Al Ghouleh I, Khoo NK, Knaus UG, Griendling KK, Touyz RM, Thannickal VJ, Barchowsky A, Nauseef WM, Kelley EE, Bauer PM, Darley-Usmar V, Shiva S, Cifuentes-Pagano E, Freeman BA, Gladwin MT, Pagano PJ (2011) Oxidases and peroxidases in cardiovascular and lung disease: new concepts in reactive oxygen species signaling. Free Radic Biol Med 51(7):1271–1288. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Abo A, Pick E, Hall A, Totty N, Teahan CG, Segal AW (1991) Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature 353(6345):668–670CrossRefGoogle Scholar
  38. 38.
    Knaus UG, Heyworth PG, Evans T, Curnutte JT, Bokoch GM (1991) Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science 254(5037):1512–1515CrossRefGoogle Scholar
  39. 39.
    Cheng G, Diebold BA, Hughes Y, Lambeth JD (2006) Nox1-dependent reactive oxygen generation is regulated by Rac1. J Biol Chem 281(26):17718–17726CrossRefGoogle Scholar
  40. 40.
    Price MO, Atkinson SJ, Knaus UG, Dinauer MC (2002) Rac activation induces NADPH oxidase activity in transgenic COSphox cells, and the level of superoxide production is exchange factor-dependent. J Biol Chem 277(21):19220–19228. CrossRefPubMedGoogle Scholar
  41. 41.
    Ming W, Li S, Billadeau DD, Quilliam LA, Dinauer MC (2007) The Rac effector p67phox regulates phagocyte NADPH oxidase by stimulating Vav1 guanine nucleotide exchange activity. Mol Cell Biol 27(1):312–323. CrossRefPubMedGoogle Scholar
  42. 42.
    Graham DB, Stephenson LM, Lam SK, Brim K, Lee HM, Bautista J, Gilfillan S, Akilesh S, Fujikawa K, Swat W (2007) An ITAM-signaling pathway controls cross-presentation of particulate but not soluble antigens in dendritic cells. J Exp Med 204(12):2889–2897. CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Jacob CO, Eisenstein M, Dinauer MC, Ming W, Liu Q, John S, Quismorio FP Jr, Reiff A, Myones BL, Kaufman KM, McCurdy D, Harley JB, Silverman E, Kimberly RP, Vyse TJ, Gaffney PM, Moser KL, Klein-Gitelman M, Wagner-Weiner L, Langefeld CD, Armstrong DL, Zidovetzki R (2012) Lupus-associated causal mutation in neutrophil cytosolic factor 2 (NCF2) brings unique insights to the structure and function of NADPH oxidase. Proc Natl Acad Sci U S A 109(2):E59–E67. CrossRefPubMedGoogle Scholar
  44. 44.
    Cheng N, He R, Tian J, Dinauer MC, Ye RD (2007) A critical role of protein kinase C delta activation loop phosphorylation in formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of p47(phox) and rapid activation of nicotinamide adenine dinucleotide phosphate oxidase. J Immunol 179(11):7720–7728CrossRefGoogle Scholar
  45. 45.
    Nauseef WM (2004) Assembly of the phagocyte NADPH oxidase. Histochem Cell Biol 122(4):277–291CrossRefGoogle Scholar
  46. 46.
    Chen J, He R, Minshall RD, Dinauer MC, Ye RD (2007) Characterization of a mutation in the Phox homology domain of the NADPH oxidase component p40phox identifies a mechanism for negative regulation of superoxide production. J Biol Chem 282(41):30273–30284. CrossRefPubMedGoogle Scholar
  47. 47.
    Al Ghouleh I, Meijles DN, Mutchler S, Zhang Q, Sahoo S, Gorelova A, Henrich Amaral J, Rodríguez 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. CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    DeCoursey TE, Morgan D, Cherny VV (2003) The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels. Nature 422(6931):531–534CrossRefGoogle Scholar
  49. 49.
    Huang J, Canadien V, Lam GY, Steinberg BE, Dinauer MC, Magalhaes MA, Glogauer M, Grinstein S, Brumell JH (2009) Activation of antibacterial autophagy by NADPH oxidases. Proc Natl Acad Sci U S A 106(15):6226–6231. CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Martinez J, Almendinger J, Oberst A, Ness R, Dillon CP, Fitzgerald P, Hengartner MO, Green DR (2011) Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells. Proc Natl Acad Sci U S A 108(42):17396–17401. CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Bagaitkar J, Huang J, Zeng MY, Pech NK, Monlish DA, Perez-Zapata LJ, Miralda I, Schuettpelz LG, Dinauer MC (2018) NADPH oxidase activation regulates apoptotic neutrophil clearance by murine macrophages. Blood 131(21):2367–2378. CrossRefPubMedPubMedCentralGoogle Scholar

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Authors and Affiliations

  1. 1.Department of PediatricsWashington University in St. Louis School of Medicine St. LouisUSA
  2. 2.Department of Pathology and ImmunologyWashington University in St. Louis School of MedicineSt. LouisUSA

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