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NADPH Oxidases pp 461-472 | Cite as

Isolation of Redox-Active Endosomes (Redoxosomes) and Assessment of NOX Activity

  • Weam S. Shahin
  • John F. EngelhardtEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)

Abstract

Reactive oxygen species (ROS) convey signals essential for proliferation, maintenance, and senescence of a growing list of cell types. Compartmentalization of these signals is integral to cell viability as well as the signaling pathways ROS direct. Redox-active endosomes (redoxosomes) are formed downstream of several ligand-activated receptors. NADPH oxidase (NOX) is a main component of redoxosomes, which recruits multiple proteins (Rac1, NOX2, p67phox, SOD1). Isolation of redoxosomes and evaluation of how superoxide (O2˙) production directs receptor signaling at the level of the endosome have enabled a better understanding of biologic processes controlled by ROS. In this chapter, we will first review the major signaling pathways that utilize redoxosomes and components that control its redox-dependent functions. We will then outline biochemical and biophysical methods for the isolation and characterization of redoxosome properties.

Key words

ROS Redoxosomes Iodixanol Immuno-affinity isolation Lucigenin EPR NOX Rac1 TRAF 

Notes

Acknowledgments

This work was supported by NIH grant R24 DK096518 (to J.F.E.).

References

  1. 1.
    Oakley FD, Abbott D, Li Q, Engelhardt JF (2009) Signaling components of redox active endosomes: the redoxosomes. Antioxid Redox Signal 11(6):1313–1333.  https://doi.org/10.1089/ARS.2008.2363 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Klomsiri C, Rogers LC, Soito L, McCauley AK, King SB, Nelson KJ, Poole LB, Daniel LW (2014) Endosomal H2O2 production leads to localized cysteine sulfenic acid formation on proteins during lysophosphatidic acid-mediated cell signaling. Free Radic Biol Med 71:49–60.  https://doi.org/10.1016/j.freeradbiomed.2014.03.017 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Li Q, Harraz MM, Zhou W, Zhang LN, Ding W, Zhang Y, Eggleston T, Yeaman C, Banfi B, Engelhardt JF (2006) Nox2 and Rac1 regulate H2O2-dependent recruitment of TRAF6 to endosomal interleukin-1 receptor complexes. Mol Cell Biol 26(1):140–154.   https://doi.org/10.1128/MCB.26.1.140-154.2006 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Li Q, Spencer NY, Oakley FD, Buettner GR, Engelhardt JF (2009) Endosomal Nox2 facilitates redox-dependent induction of NF-kappaB by TNF-alpha. Antioxid Redox Signal 11(6):1249–1263.  https://doi.org/10.1089/ARS.2008.2407 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Oakley FD, Smith RL, Engelhardt JF (2009) Lipid rafts and caveolin-1 coordinate interleukin-1beta (IL-1beta)-dependent activation of NFkappaB by controlling endocytosis of Nox2 and IL-1beta receptor 1 from the plasma membrane. J Biol Chem 284(48):33,255–33,264.  https://doi.org/10.1074/jbc.M109.042127 CrossRefGoogle Scholar
  6. 6.
    Miller FJ Jr, Filali M, Huss GJ, Stanic B, Chamseddine A, Barna TJ, Lamb FS (2007) Cytokine activation of nuclear factor kappa B in vascular smooth muscle cells requires signaling endosomes containing Nox1 and ClC-3. Circ Res 101(7):663–671.  https://doi.org/10.1161/CIRCRESAHA.107.151076 CrossRefPubMedGoogle Scholar
  7. 7.
    Li Q, Zhang Y, Marden JJ, Banfi B, Engelhardt JF (2008) Endosomal NADPH oxidase regulates c-Src activation following hypoxia/reoxygenation injury. Biochem J 411(3):531–541.  https://doi.org/10.1042/BJ20071534 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    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):3510–3513.  https://doi.org/10.1074/jbc.C200613200 CrossRefPubMedGoogle Scholar
  9. 9.
    Lambeth JD (2004) NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 4(3):181–189.  https://doi.org/10.1038/nri1312 CrossRefPubMedGoogle Scholar
  10. 10.
    Mumbengegwi DR, Li Q, Li C, Bear CE, Engelhardt JF (2008) Evidence for a superoxide permeability pathway in endosomal membranes. Mol Cell Biol 28(11):3700–3712.  https://doi.org/10.1128/MCB.02038-07 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Lynch RE, Fridovich I (1978) Permeation of the erythrocyte stroma by superoxide radical. J Biol Chem 253(13):4697–4699PubMedGoogle Scholar
  12. 12.
    Salvador A, Sousa J, Pinto RE (2001) Hydroperoxyl, superoxide and pH gradients in the mitochondrial matrix: a theoretical assessment. Free Radic Biol Med 31(10):1208–1215CrossRefGoogle Scholar
  13. 13.
    Harraz MM, Marden JJ, Zhou W, Zhang Y, Williams A, Sharov VS, Nelson K, Luo M, Paulson H, Schoneich C, Engelhardt JF (2008) SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model. J Clin Invest 118(2):659–670.  https://doi.org/10.1172/JCI34060 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Mizuno T, Kaibuchi K, Ando S, Musha T, Hiraoka K, Takaishi K, Asada M, Nunoi H, Matsuda I, Takai Y (1992) Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins. J Biol Chem 267(15):10,215–10,218Google Scholar
  15. 15.
    Kopani M, Celec P, Danisovic L, Michalka P, Biro C (2006) Oxidative stress and electron spin resonance. Clin Chim Acta 364(1-2):61–66.  https://doi.org/10.1016/j.cca.2005.05.016 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Anatomy and Cell BiologyCarver College of Medicine, University of IowaIowa CityUSA
  2. 2.Department of Internal MedicineCarver College of Medicine, University of IowaIowa CityUSA
  3. 3.Center for Gene TherapyCarver College of Medicine, University of IowaIowa CityUSA

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