Nox5 forms a functional oligomer mediated by self-association of its dehydrogenase domain.

The family of human reactive oxygen species (ROS)-generating NADPH-oxidases (Nox) consists of seven members that include Nox1, Nox2, Nox3, Nox4 and Nox5 plus the Dual oxidases Duox1 and Duox2 (1). Structural features and experimental data have been used to classify the Nox and Duox enzymes into two broad groups: those regulated by subunits including a membrane-associated subunit p22phox (Nox1-Nox4) and those that are regulated by calcium ion (Nox5 and Duox1 and 2) (2–4). In the former subgroup, Nox forms a tightly associated, mutually stabilizing heterodimer with p22phox. Nox1-3 enzymes require additional regulatory subunits such as p47phox, p67phox, p40phox, NOXO1, NOXA1, and the small GTPase Rac (5). The calcium-regulated subgroup possesses a calcium regulatory domain that contains single or multiple EF-hand calcium-binding motif(s). While Duox1 and Duox2 require the maturation factors DuoxA1 and DuoxA2, respectively, to facilitate their movement to the plasma membrane (6, 7), Nox5 apparently functions independently of subunits [(8) and vide infra]. Human Nox5 contains four EF-hand motifs in its calcium-binding domain, which is located at the N-terminus (9). Rapid activation of Nox5 by calcium ion has been observed in Nox5-overexpressing HEK293 cells (8, 9), human aortic smooth muscle cells (10), prostate cells (11) and vascular endothelial cells (12, 13). In addition to calcium, other co-activating mechanisms cooperate by lowering the concentration of calcium needed to trigger activation: these include phorbol 12-myristate 13-acetate-dependent phosphorylation of Nox5 (14), calmodulin (13, 15), and agonist-generated lipids including phosphatidic acid and arachidonate (Kawahara and Lambeth, manuscript submitted). The physiological role of Nox5 has been extensively studied using Drosophila melanogaster where Nox5 (termed d-Nox) generates a ROS signal in smooth muscle to mediate hormone-induced smooth muscle contraction and egg laying (16). In human, Nox5 expression is abundant in the spleen, testis, and vascular tissue, cells of the gastrointestinal tract, reproductive systems, and is also seen in various cancers (17). Nox5 has been lost in rodent genomes (2), making it impossible to investigate its role in mouse knockout models. Results from in vitro studies in human cells indicate that Nox5-derived ROS enhances cell growth of prostate cancer cells (11), hairy cell leukemia cells (18), aortic smooth muscle cells (10), and microvascular endothelial cell (12). Recently, studies of human biopsy samples of coronary arteries demonstrated increased expression of Nox5 in coronary artery disease, using immunoblotting, immunofluorescence, and quantitative PCR (19). The present studies were undertaken based on the observation (vide infra) that co-transfection of catalytically inactive point-mutated forms of several Nox isoforms along with the same wild-type isoform resulted in inhibition of wild-type enzyme activity, meeting the definition of a “dominant negative” genetic effect. There are at least two possible explanations for this type of phenomenon. One involves sequestering of a needed regulatory subunit or other component by the catalytically inactive enzyme, thereby removing a factor that is essential for the activity of the wild-type enzyme. A second class of explanations involves catalytically essential dimerization or higher-order oligomerization. According to the latter scenario, the inclusion of a catalytically inactive unit in the oligomer renders the whole inactive or partially active, for example, by creating a poorly active conformation of the whole. Because Nox5 does not require the participation of heterologous subunits, interpretation of data are more straightforward than for other Nox isoforms, and this isoform was therefore selected for an in-depth investigation of the possibility of homo-oligomerization. A variety of methods were used to demonstrate oligomerization, and to show that this interaction is essential for Nox5 function. In addition, several approaches were used to identify the region of Nox5 that mediates oligomerization.