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

Abstract

Reactive oxygen species (ROS) are produced in phagocytic cells by NADPH oxidase, a complex that transports electrons across membranes and generates superoxide anion from molecular oxygen. This enzyme is assembled from a membranespanning flavocytochrome b 558 , composed of Nox2 (also called gp91 phox ) and p22 phox and four cytosolic factors (p47 phox

Subcellular localization and function of alternatively spliced Noxo1 isoforms

“…Noxa1 was designated as a "Nox activator" based on its homology to p67 phox , which binds to Rac1 or Rac2 and plays a more direct role in modulating oxidase activity. Recent evidence suggests that Rac1 also regulates Nox1 most likely through GTP-dependent interactions with the Nox activator, Noxa1 [16][17][18][19] We showed that the association of Noxa1 with the plasma membrane depends on its interaction with Noxo1 [17]. 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].…”

“…The pcDNA3.1 plasmids (Invitrogen) containing the complete coding sequence of human Nox1, p41 nox (Noxo1α and Noxo1β), p51 nox (Noxa1), and p22 phox were described previously [5,8,17]. Human Noxo1γ in pcDNA3.1 and Noxo1δ in pcDNA3.1were made from Noxo1β and Noxo1α using the QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene), respectively.…”

“…The cells were fed 5 h post-transfection with complete medium, and were assayed 48 h after transfection. Trypsinized cells (250,000 cells) were assayed for ROS release (with or without 2 μg/ml PMA) by SOD-inhibitable chemiluminescence methods using the Diogenes reagent (National Diagnotics), as described previously [17].…”

“…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.…”

“…AY255768) [14]. Most reports studied Noxo1β (human or mouse), the most common isoform [9,13,15,17,25]. Noxo1β and Noxo1γ isoforms were compared recently for their abilities to support Nox1 and Nox3-based oxidases [26].…”