Functional Characterization of DUOX Enzymes in Reconstituted Cell Models

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.