Nox3 regulation by NOXO1, p47phox, and p67phox

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 approximately 30% 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.     

Tyrosine phosphorylation of HoxA10 decreases DNA binding and transcriptional repression during interferon gamma -induced differentiation of myeloid leukemia cell lines

The DNA binding affinity of HoxA10 is increased by partnering with Pbx proteins. A consensus sequence for Pbx1-HoxA10 DNA binding has been derived, but genuine target genes have not been identified. We noted that the derived Pbx-HoxA10 DNA-binding consensus is similar to a repressor element in the CYBB promoter. The CYBB gene, which encodes the respiratory burst oxidase component gp91(phox), is expressed only in myeloid cells that have differentiated beyond the promyelocyte stage. In these studies, we demonstrate that interferon gamma (IFN-gamma)-induced differentiation of myeloid cell lines abolishes in vitro Pbx-HoxA10 binding to either the derived consensus or the similar CYBB sequence. We also demonstrate that HoxA10, overexpressed in myeloid cell lines, represses reporter gene expression from artificial promoter constructs with Pbx-HoxA10 binding sites. We determine that HoxA10 has endogenous repression domains that are not functionally altered by IFN-gamma treatment. However, IFN-gamma-induced differentiation of myeloid cell lines leads to HoxA10 tyrosine phosphorylation, which decreases in vitro DNA binding to Pbx-HoxA10 binding sites. Therefore, these investigations identify the CYBB gene as a potential target for HoxA10 and define repression of genes expressed in mature myeloid cells as a novel role for HoxA10 during myeloid differentiation.     

Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity

The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox. Antibody binding studies indicate that the extreme N terminus of p22phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22phox extend into the cytoplasm, anchored by two membrane-embedded regions.     

Activation of the flavoprotein domain of gp91phox upon interaction with N-terminal p67phox (1-210) and the Rac complex

A series of truncated forms of His(6)-tagged gp91phox were expressed, solubilized, and purified in the presence of 30 microM FAD. The truncated gp91phox with the longest sequence in the C-terminal region (221-570) (gp91C) showed the highest activity (turnover rate, 0.92) for NADPH diaphorase in the presence of either 0.3% Triton X-100 or 0.5% Genapol X-80. Activity was not inhibited by superoxide dismutase but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium. The flavinated gp91C contained approximately 0.9 mol of FAD/mol of protein (MW 46 kDa) and 12% alpha-helix content. In the absence of p47phox, p67phox showed considerable activation of gp91C in the presence of Rac. Carboxyl-terminal truncated p67phox (1-210) (p67N), which is the minimal active fragment, was fused with Rac or Q61LRac. The fusion protein p67N-Rac (or p67N-Q61LRac) showed a 2-fold higher stimulatory effect on NBT reductase activity of gp91C than the combination of the individual cytosolic p67N and Rac proteins. In contrast, Rac-p67N, a fusion with the opposite orientation, showed a smaller significant effect on the enzyme activity. The EC(50) values for p67phox, p67N, p67N-Rac, and Rac-p67N were 8.00. 4.35, 2.56, and 15.2 microM, respectively, while the K(m) value for NADPH in the presence and absence of the cytosolic components was almost the same (40-55 microM). In the presence of Rac, p67N or p67phox bound to gp91C with a molar ratio of approximately 1:1 but neither p67N nor Rac alone showed significant binding.     

Rotenone activates phagocyte NADPH oxidase by binding to its membrane subunit gp91phox

Rotenone, a widely used pesticide, reproduces parkinsonism in rodents and associates with increased risk for Parkinson disease. We previously reported that rotenone increased superoxide production by stimulating the microglial phagocyte NADPH oxidase (PHOX). This study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91(phox), the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91(phox). Functional studies showed that both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91(phox)/p22(phox)) and cytosolic subunits (p67(phox) and p47(phox)). Rotenone-elicited extracellular superoxide release in p47(phox)-deficient macrophages suggested that rotenone enabled activation of PHOX through a p47(phox)-independent mechanism. Increased membrane translocation of p67(phox), elevated binding of p67(phox) to rotenone-treated membrane fractions, and coimmunoprecipitation of p67(phox) and gp91(phox) in rotenone-treated wild-type and p47(phox)-deficient macrophages indicated that p67(phox) played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91(phox). Rac1, a Rho-like small GTPase, enhanced p67(phox)-gp91(phox) interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91(phox); such an interaction triggered membrane translocation of p67(phox), leading to PHOX activation and superoxide production.     

The X+ chronic granulomatous disease as a fabulous model to study the NADPH oxidase complex activation

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. Patients with CGD suffer from recurrent bacterial and fungal infections because of the absence of superoxide anions (O2- degrees ) generatingsystem. The NADPH oxidase complex is composed of a membranous cytochrome b558, cytosolic proteins p67phox, p47phox, p40phox and two small GTPases Rac2 and Rap1A. Cytochrome b558 consists of two sub-units gp91phox and p22phox. The most common form of CGD is due to mutations in CYBB gene encoding gp91phox. In some rare cases, the mutated gp91phox is normally expressed but is devoided of oxidase activity. These variants called X+ CGD, have provided interesting informations about oxidase activation mechanisms. However modelization of such variants is necessary to obtain enough biological material for studies at the molecular level. A cellular model (knock-out PLB-985 cells) has been developed for expressing recombinant mutated gp91phox for functional analysis of the oxidase complex. Recent works demonstrated that this cell line genetically deficient in gp91phox is a powerful tool for functional analysis of the NADPH oxidase complex activation.     

Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases

The catalytic core of a superoxide-producing NADPH oxidase (Nox) in phagocytes is gp91phox/Nox2, a membrane-integrated protein that forms a heterodimer with p22phox to constitute flavocytochrome b558. The cytochrome becomes activated by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. Here we describe the cloning of human cDNAs for novel proteins homologous to p47phox and p67phox, designated p41nox and p51nox, respectively; the former is encoded by NOXO1 (Nox organizer 1), and the latter is encoded by NOXA1 (Nox activator 1). The novel homologue p41nox interacts with p22phox via the two tandem SH3 domains, as does p47phox. The protein p51nox as well as p67phox can form a complex with p47phox and with p41nox via the C-terminal SH3 domain and binds to GTP-bound Rac via the N-terminal domain containing four tetratricopeptide repeat motifs. These bindings seem to play important roles, since p47phox and p67phox activate the phagocyte oxidase via the same interactions. Indeed, p41nox and p51nox are capable of replacing the corresponding classical homologue in activation of gp91phox. Nox1, a homologue of gp91phox, also can be activated in cells, when it is coexpressed with p41nox and p51nox, with p41nox and p67phox, or with p47phox and p51nox; in the former two cases, Nox1 is partially activated without any stimulants added, suggesting that p41nox is normally in an active state. Thus, the novel homologues p41nox and p51nox likely function together or in combination with a classical one, thereby activating the two Nox family oxidases.     

PU.1 is dominant and HAF-1 supplementary for activation of the gp91(phox) promoter in human monocytic PLB-985 cells

Gp91(phox) is a key component of the phagocyte NADPH oxidase. Mutations of its promoter found in patients with chronic granulomatous disease cause deficient binding of PU.1 and HAF-1. Because the two factors bind to the same site (Pu box) of the promoter, we attempted to clarify their relative in vivo contributions to activation of the gp91(phox) promoter in monocytically differentiated PLB-985 cells using a dual luciferase reporter assay and a gel shift competition assay. We found that the activity of a series of single-point-mutated promoters increases or decreases according to an increase or decrease, respectively, in the affinity of the promoters to PU.1 but not to HAF-1. Two of 7 mutants showing weak binding affinity to PU.1 exhibited moderate promoter activity and normal binding affinity for HAF-1. These results suggest PU.1 is the dominant activator and HAF-1 is supplementary. The increased promoter activity of single-, double-, and triple-point-mutated constructs with sequences closer to that of the Ets-binding element correlates with their binding affinity to PU.1 but not to HAF-1, supporting that PU.1 is a more efficient activator than HAF-1. In contrast to co-expressed wild-type PU.1, dominant-negative PU.1 significantly inhibited the activity of a PU.1-optimised gp91(phox) promoter construct. Therefore, we conclude that PU.1 and HAF-1 binding to the Pu box is dominant and supplementary, respectively, for activation of the gp91(phox) promoter in human monocytic cells.     

Adventitial delivery of dominant-negative p67phox attenuates neointimal hyperplasia of the rat carotid artery

Several essential components of NADPH oxidase, including p22phox, gp91phox (nox2) and its homologs nox1 and nox4, p47phox, p67phox, and rac1, are present in the vasculature. We previously reported that p67phox is essential for adventitial fibroblast NADPH oxidase O2- production. Thus we postulated that inhibition of adventitial p67phox activity would attenuate angioplasty-induced hyperplasia. To test this hypothesis, we treated the adventitia of carotid arteries with a control adenovirus (Ad-control), a virus expressing dominant-negative p67phox (Ad-p67dn), or a virus expressing a competitive peptide (gp91ds) targeting the p47phox-gp91phox interaction (Ad-gp91ds). Common carotid arteries (CCAs) from male Sprague-Dawley rats were transfected with Ad-control, Ad-p67dn, or Ad-gp91ds in pluronic gel. After 2 days, a 2-F (Fogarty) catheter was used to injure CCAs in vivo. After 14 days, CCAs were perfusion-fixed and analyzed. In 13 experiments, digital morphometry suggested a reduction of neointimal hyperplasia with Ad-p67dn compared with Ad-control; however, the reduction did not reach statistical significance (P = 0.058). In contrast, a significant reduction was achieved with Ad-gp91ds (P = 0.006). No changes in medial area or remodeling were observed with either treatment. Moreover, adventitial fibroblast proliferation in vitro was inhibited by Ad-gp91ds but not by Ad-p67dn, despite confirmation that Ad-p67dn inhibits NADPH oxidase in fibroblasts. These data appear to suggest that a multicomponent vascular NADPH oxidase plays a role in neointimal hyperplasia. However, inhibition of p47phox may be more effective than inhibition of p67phox at attenuating neointimal growth.