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.
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Dupuy C, Ohayon R, Valent A et al (1999) Purification of a novel flavoprotein involved in the thyroid NADPH oxidase. Cloning of the porcine and human cdnas. J Biol Chem 274:37265–37269
De Deken X, Wang D, Many MC et al (2000) Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family. J Biol Chem 275:23227–23233
Moreno JC, Visser TJ (2007) New phenotypes in thyroid dyshormonogenesis: hypothyroidism due to DUOX2 mutations. Endocr Dev 10:99–117
Grasberger H (2010) Defects of thyroidal hydrogen peroxide generation in congenital hypothyroidism. Mol Cell Endocrinol 322:99–106
Ohye H, Sugawara M (2010) Dual oxidase, hydrogen peroxide and thyroid diseases. Exp Biol Med 235:424–433
Muzza M, Fugazzola L (2017) Disorders of H2O2 generation. Best Pract Res Clin Endocrinol Metab 31:225–240
Grasberger H, Refetoff S (2006) Identification of the maturation factor for dual oxidase. Evolution of an eukaryotic operon equivalent. J Biol Chem 281:18269–18272
Morand S, Ueyama T, Tsujibe S et al (2009) Duox maturation factors form cell surface complexes with Duox affecting the specificity of reactive oxygen species generation. FASEB J 23:1205–1218
Luxen S, Noack D, Frausto M et al (2009) Heterodimerization controls localization of Duox-DuoxA NADPH oxidases in airway cells. J Cell Sci 122:1238–1247
Carre A, Louzada RA, Fortunato RS et al (2015) When an Intramolecular Disulfide Bridge Governs the Interaction of DUOX2 with Its Partner DUOXA2. Antioxid Redox Signal 23:724–733
Maruo Y, Takahashi H, Soeda I et al (2008) Transient congenital hypothyroidism caused by biallelic mutations of the dual oxidase 2 gene in Japanese patients detected by a neonatal screening program. J Clin Endocrinol Metab 93:4261–4267
Zamproni I, Grasberger H, Cortinovis F et al (2008) Biallelic inactivation of the dual oxidase maturation factor 2 (DUOXA2) gene as a novel cause of congenital hypothyroidism. J Clin Endocrinol Metab 93:605–610
Hayes P, Dhillon S, O’Neill K et al (2015) Defects in NADPH oxidase genes NOX1 and DUOX2 in very early onset inflammatory bowel disease. Cell Mol Gastroenterol Hepatol 1:489–502
Parlato M, Charbit-Henrion F, Hayes P et al (2017) First identification of biallelic inherited DUOX2 inactivating mutations as a cause of very early onset inflammatory bowel disease. Gastroenterology 153:609–611
Grasberger H, Noureldin M, Kao TD et al (2018) Increased risk for inflammatory bowel disease in congenital hypothyroidism supports the existence of a shared susceptibility factor. Sci Rep 8:10158
Donko A, Orient A, Szabo PT et al (2009) Detection of hydrogen peroxide by lactoperoxidase-mediated dityrosine formation. Free Radic Res 43:440–445
Donko A, Morand S, Korzeniowska A et al (2014) Hypothyroidism-associated missense mutation impairs NADPH oxidase activity and intracellular trafficking of Duox2. Free Radic Biol Med 73:190–200
Rigutto S, Hoste C, Grasberger H et al (2009) Activation of dual oxidases Duox1 and Duox2: differential regulation mediated by camp-dependent protein kinase and protein kinase C-dependent phosphorylation. J Biol Chem 284:6725–6734
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This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, NIH.
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Korzeniowska, A., Donkó, Á.P., Morand, S., Leto, T.L. (2019). Functional Characterization of DUOX Enzymes in Reconstituted Cell Models. 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_11
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DOI: https://doi.org/10.1007/978-1-4939-9424-3_11
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