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NADPH Oxidases pp 173-190 | Cite as

Functional Characterization of DUOX Enzymes in Reconstituted Cell Models

  • Agnieszka Korzeniowska
  • Ágnes P. Donkó
  • Stanislas Morand
  • Thomas L. LetoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)

Abstract

Biosynthesis of active human dual oxidases (DUOX1 and DUOX2) requires maturation factors, a.k.a. DUOX activator proteins (DUOXA1 and DUOXA2), that form covalent complexes with DUOX; both chains together represent the mature catalytic unit that functions as a dedicated hydrogen peroxide-generating enzyme. Genetic defects in DUOX2 or DUOXA2 can result in congenital hypothyroidism, whereas partial defects in DUOX2 activity also have been associated with very early-onset inflammatory bowel disease. Our understanding of the links between DUOX dysfunction and these diseases remains incomplete. An important challenge in developing a better understanding of the pathogenic roles of DUOX defects requires robust and reliable DUOX reconstitution cell models to examine the functional consequences of candidate DUOX missense mutations and polymorphisms. Here, we describe methods for efficient heterologous DUOX/DUOXA co-expression and functional characterization, including detailed assessments of posttranslational processing and subcellular translocation of DUOX that accompanies the maturation of these enzymes into catalytically active NADPH oxidases.

Key words

NADPH oxidases Dual oxidases DUOX DUOX activator DUOXA NOX Hydrogen peroxide Cell surface targeting 

Notes

Acknowledgments

This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, NIH.

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Copyright information

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

Authors and Affiliations

  • Agnieszka Korzeniowska
    • 1
  • Ágnes P. Donkó
    • 1
  • Stanislas Morand
    • 1
    • 2
  • Thomas L. Leto
    • 1
    Email author
  1. 1.Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of HealthBethesdaUSA
  2. 2.L’Oreal Advanced Research, Aulnay-Sous-BoisParisFrance

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