Dual Oxidase-2 Has an Intrinsic Ca2+-dependent H2O2-generating Activity

Abstract

Duox2 (and probablyDuox1Reactive oxygen species (ROS) 1 have emerged as important molecules involved in regulating essential cell functions, such as growth and differentiation (1). NAD(P)H oxidases are a major source of ROS. Phagocyte oxidase is the oxidase that has been investigated most thoroughly (2). It catalyzes the production of superoxide by the one-electron reduction of oxygen, using NADPH as the electron donor. The catalytic moiety of the phagocyte NADPH oxidase is gp91phox , a plasma membraneassociated flavohemoprotein. Recently, it was discovered that gp91 phox belongs to a family consisting of several very similar oxidases. Seven NOX (NADPH oxidase) and DUOX/ThOX (dual oxidase/thyroid oxidase) genes have been identified that encode different NADPH oxidases with differing mRNA tissue expression. The Nox family comprises gp91phox , now known as Nox2; Nox1, which is predominantly expressed in the colon (3); Nox3, cloned from fetal kidney (4); Nox4 found in the kidney cortex (5, 6); and Nox5, expressed in the testis, spleen, and lymph nodes (7). In addition to the basic structure of gp91 phox , Nox5 has a long intracellular N-terminal domain with four calcium binding sites implicated in its Ca 2ϩ -dependent activation (8).The biological functions of these Nox proteins are now under investigation. They are involved in signal transduction related to cell growth and cancer (9 -11) and to angiogenesis (12).Duox1 and Duox2 are large homologues of Nox2 with an N-terminal extension comprising two EF-hand motifs, an additional transmembrane helix, and a peroxidase homology ectodomain (see Fig. 4A) (13,14). DUOX genes have been identified in the thyroid gland, where they are strongly expressed (13,14). However, the DUOX are also expressed on the mucosal surfaces of the trachea and the bronchi (15) and in the airway epithelial cells (16,17), where it has been suggested that Duox1 is the isoform responsible for acid production and secretion in airways (16) and plays a critical role in mucin expression (18). DUOX2 was also expressed throughout the digestive tract, where it was found to be functional (19,20), in addition to the salivary gland and rectum (15).It has been suggested that Duox2, which was identified by purifying thyroid NADPH oxidase, may constitute the catalytic core of this enzyme and generate the H 2 O 2 used by Tpo to catalyze the biosynthesis of thyroid hormones at the apical surface of the thyrocytes (13). Although no functional Duoxbased H 2 O 2 -generating system has yet been reconstituted (21), this proposal is corroborated by a recent report of permanent and severe congenital hypothyroidism in a patient with a bial-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.¶ Recipient of a fellowship from the Commissariat à l'Energie Atomique (Paris, France).ʈ Recipients of a fellowship from the National Education, Research, an...

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…NOX5 and DUOX1/2 differ from the other NOX homologs and contain additional intracellular Ca 2+ -binding EF-hand domain regions. Consequently, these homologs can be activated by Ca 2+ -mobilizing stimuli and do not rely on association with p22phox or with cytosolic co-factors (9)(10)(11)(12). In this regard, NOX5 and DUOX1/2 relate closely to NADPH oxidases in plants and in protists (Fig.…”

“…phagosomes) (Fig. 2) (1,10,17). The biological actions of NOX/DUOX are therefore thought to be mediated by either H 2 O 2 or O 2 •− (or indirect secondary ROS production), through their ability to directly (in)activate specific oxidant-sensitive cellular or extracellular targets, thereby affecting various cellular signaling pathways or biological processes.…”

“…While mature N-glycosylated DUOX proteins are localized to the plasma membrane, substantial amounts of DUOX protein are also found intracellularly (27,32), presumably in association with the ER. Heterologous DUOX1/2 expression in non-epithelial cell types typically does not result in a functional H 2 O 2 -generating enzyme at the plasma membrane, presumably because these proteins are not fully glycosylated and are retained in the ER (10,93). This was recently resolved by the discovery of maturation factors of DUOX (DUOXA1/2), which are ER-resident transmembrane proteins that facilitate ER-to-Golgi transition and translocation of DUOX proteins to the plasma membrane (93).…”

“…2+ binding domains within DUOX suggests that it can be directly activated by Ca 2+ -mobilizing stimuli. Indeed, Ca 2+ mobilization by various diverse stimuli were been found to promote apical H 2 O 2 production in airway epithelial cells (10,27,84,133). Moreover, in analogy to activation of NOX2 in phagocytes, H 2 O 2 production by airway epithelial cells or thyrocytes was also stimulated in response to activation of protein kinase C (PKC), and Ca 2+ and PKC stimulation appear to act synergistically in promoting H 2 O 2 production (82,105,134,135).…”

Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps

“…The stability of their superoxide adducts in vivo or in the presence of biological material remains low and these adducts are easily transformed to diamagnetic compounds. However, most publications dedicated to spin trapping in enzymatic systems, cells or organs make use of these spin traps [10,[17][18][19][20][21][22][23][24] . Recently, cyclic nitrones bearing a triphenylphosphonium group (Mito-DEPMPO and Mito-DIPPMPO, Scheme 1) [25,26] , or conjugated to a -cyclodextrin ring (CDNMPO, CD-DEPMPO, CD-DIPPMPO) [27,28] (Scheme 1) have been synthesized.…”