Nox3 Regulation by NOXO1, p47 , and p67

Abstract

gp91 phox (Nox2), the catalytic subunit of the superoxide-generating respiratory burst oxidase, is regulated by subunits p47 phox and p67 phox . Nox1, a homolog of gp91 phox , is regulated by NOXO1 and NOXA1, homologs of p47 phox and p67 phox , respectively. For both Nox1 and gp91 phox , an organizer protein (NOXO1 or p47 phox ) cooperates with an activator protein (NOXA1 or p67 phox ) to regulate the catalytic subunit. Herein, we investigate the subunit regulation of Nox3 compared with that of other Nox enzymes. Nox3, like gp91 phox , was activated by p47 phox plus p67 phox . Whereas gp91 phox activity required the protein kinase C activator phorbol myristate acetate (PMA), Nox3 activity was already high without PMA, but was further stimulated ϳ30% by PMA. gp91 phox was also activated by NOXO1/NOXA1 and required PMA for high activity. gp91 phox regulation required an intact activation domain in the activator protein, as neither p67 phox (V204A) nor NOXA1(V205A) were effective. In contrast, p67 phox (V204A) was effective (along with p47 phox ) in activating Nox3. Unexpectedly, Nox3 was strongly activated by NOXO1 in the absence of NOXA1 or p67 phox . Nox3 activity was regulated by PMA only when p47 phox but not NOXO1 was present, consistent with the phosphorylation-regulated autoinhibitory region in p47 phox but not in NOXO1. Deletion of the autoinhibitory region from p47 phox rendered this subunit highly active in the absence of PMA toward both gp91 phox and Nox3, and high activity required an activator subunit. The unique regulation of Nox3 supports a model in which multiple interactions with regulatory subunits stabilize an active conformation of the catalytic subunit.The Nox family of NAD(P)H oxidases has recently been described (1-10). These enzymes are structural homologs of gp91 phox (a.k.a. Nox2), the catalytic subunit of the phagocyte NADPH oxidase, and are distributed in a variety of non-phagocytic tissues such as colon, kidney, vascular smooth muscle, and brain. Reactive oxygen species generated by these novel enzymes are proposed to function in signal transduction related to cell growth and cancer (1,(11)(12)(13)(14)(15)(16)(17), angiogenesis (18), and in innate immunity (15), e.g. in barrier cells such as epithelium. In addition, reactive oxygen species from these enzymes are proposed to be causally linked to pathological states including atherosclerosis (19), cancer (11,15,20,21), and diabetes (22).The human Nox enzymes are encoded by at least 7 genes. NOX1, NOX3, and NOX4 encode proteins that are similar in size and domain structure to gp91 phox . These consist of a Cterminal flavoprotein domain containing both an FAD-binding site and an NADPH-binding site, and an N-terminal membrane-associated hydrophobic domain consisting of 6 transmembrane ␣ helices that provide binding sites for 2 hemes (3). Nox5 consists of these same domains along with an N-terminal calcium-binding domain (9); this enzyme is dormant when expressed in cells, but is activated by calcium. Duox1 and Duox2 build upon the Nox5 str...

Involvement of Rac1 in Activation of Multicomponent Nox1- and Nox3-Based NADPH Oxidases

“…7). Consistent with previous work (8,15,55), transfected Nox3 exhibits detectable activity in the absence of any cotransfected organizer or activator protein. Interestingly, the activity of Nox3 alone was enhanced by Rac1(Q61L).…”

“…Nox3 also appears to function as a multicomponent system involving p22 phox and either the Nox or phox supportive cofactors (8,15,55). We examined assembly and activation mechanisms of the Nox3 system with a focus on potential involvement of Rac1.…”

“…Both the Nox1 and Nox3 oxidases appear to function as multicomponent enzymes similar to the phox (Nox2-based) system (7,8,14,15,21,22,29,54,55). Nox1 expressed alone produces little superoxide, although its activity is dramatically enhanced in the presence of two cofactors (Noxo1 and Noxa1) that are detected in colon epithelium (7,21,54).…”

“…Both the human and murine Nox1 systems (comprising Nox1, Noxo1, and Noxa1) exhibit high constitutive activity, while activity of the human system is significantly enhanced by cellular activation in several cell types (7,21,28,54). Recently, several groups examined involvement of the Nox1 or Nox2 (phox) regulators in Nox3 activity and showed that this enzyme is less subject to the tight cofactordependent controlling mechanisms observed with Nox1 or Nox2 (8,15,55). Furthermore, there has been no direct evidence for Rac-dependent regulation of Nox3 activity (55).…”

Subcellular localization and function of alternatively spliced Noxo1 isoforms

“…These structural and functional similarities between the Nox and phox regulators may explain why Noxo1 and Noxa1 can function in partially compensating for p47 phox and p67 phox in supporting Nox2 activity [8,[13][14][15]. Noxo1, Noxa1, and Rac1 also support Nox3 activity, which appears to function critically in the inner ear [17,[20][21][22]; mice with lesions in either the NOX3 or NOXO1 gene have defects in sensing gravity resulting from impaired otoconia formation [23,24]. Nox3 activity can be supported by p47 phox and p67 phox , although mice deficient in p47 phox or chronic granulomatous disease patients deficient in either protein show no deficits in balance.…”