Heterodimerization controls localization of Duox-DuoxA NADPH oxidases in airway cells

Abstract

Duox NADPH oxidases generate hydrogen peroxide at the air-liquid interface of the respiratory tract and at apical membranes of thyroid follicular cells. Inactivating mutations of Duox2 have been linked to congenital hypothyroidism, and epigenetic silencing of Duox is frequently observed in lung cancer. To study Duox regulation by maturation factors in detail, its association with these factors, differential use of subunits and localization was analyzed in a lung cancer cell line and undifferentiated or polarized lung epithelial cells. We show here that Duox proteins form functional heterodimers with their respective DuoxA subunits, in close analogy to the phagocyte NADPH oxidase. Characterization of novel DuoxA1 isoforms and mispaired Duox-DuoxA complexes revealed that heterodimerization is a prerequisite for reactive oxygen species production. Functional Duox1 and Duox2 localize to the leading edge of migrating cells, augmenting motility and wound healing. DuoxA subunits are responsible for targeting functional oxidases to distinct cellular compartments in lung epithelial cells, including Duox2 expression in ciliated cells in an ex vivo differentiated lung epithelium. As these locations probably define signaling specificity of Duox1 versus Duox2, these findings will facilitate monitoring Duox isoform expression in lung disease, a first step for early screening procedures and rational drug development.

Structural Insights into Nox4 and Nox2: Motifs Involved in Function and Cellular Localization

“…In contrast, chimeric replacement of the Nox2 N terminus with the Nox4 sequence or N-terminal sequence additions in the form of a V5 epitope tag reduced Nox2-mediated ROS generation but did not alter localization of these oxidases. V5-tagged, inducible Nox4 backbone chimeras (Nox4 (20,23), and thus migration rates of Cos cells stably expressing Nox2, Nox4, or catalytically inactive Nox4 were compared. While Nox4 activity doubled migration rates, PMA-stimulated Nox2 activity inhibited cell motility.…”

“…Human H661 lung carcinoma cells (ATCC HTB-183) were cultivated in RPMI 1640, and Cos-p22phox cells (36), Cos-phox cells (27), and Cos-Nox4/p22phox cells were cultivated in Dulbecco modified Eagle medium (DMEM). Cos-Nox4/p22phox and Cos-Nox4 P437H/p22phox cells were generated by lentiviral transduction and sorted as described previously (20,35). All growth media were obtained from Invitrogen, CA, and supplemented with 10% fetal calf serum (FCS).…”

NADPH Oxidases: A Perspective on Reactive Oxygen Species Production in Tumor Biology

“…DUOX proteins have a unique protein association with a maturation factor (DUOXA) required to afford trafficking from the endoplasmic reticulum (ER) to the plasma membrane, where a functional membrane complex is formed (46). This association is known to be preferential for DUOX1-DUOXA1 and DUOX2-DUOXA2; DUOXA can cross-function in supporting DUOX membrane transport, however, mismatched associations promote cell line-dependent variability in the level and specificity of membrane targeting and ROS production (superoxide vs. H 2 O 2 ) (83,95). Complex formation, organization, and characterization of individual association partners, briefly outlined above, have previously been extensively reviewed (13,77).…”

Lifespan extension by peroxidase and dual oxidase-mediated ROS signaling through pyrroloquinoline quinone in C. elegans

“…Stable transfectants expressing HA-tagged human DUOX1 and FLAG-tagged human DUOXA1α (D1A1), or HA-tagged human DUOX2 and FLAG-tagged human DUOXA2 (D2A2), were established because DUOX1-DUOXA1 and DUOX2-DUOXA2 were reported to be the best partner pairings for proper H 2 O 2 production (De Deken et al, 2014). The HA tag was inserted between Ala 21 and Gln 22 of DUOX1 and between Asp 27 and Ala 28 of DUOX2 (Grasberger and Refetoff, 2006;Luxen et al, 2009;Morand et al, 2009). The FLAG tag was attached to the C-terminus of DUOXA1α and DUOXA2.…”