Involvement of Rac1 in activation of multicomponent Nox1- and Nox3-based NADPH oxidases

Several Nox family NADPH oxidases function as multicomponent enzyme systems. We explored determinants of assembly of the multicomponent oxidases Nox1 and Nox3 and examined the involvement of Rac1 in their regulation. Both enzymes are supported by p47phox and p67phox or homologous regulators called Noxo1 and Noxa1, although Nox3 is less dependent on these cofactors for activity. Plasma membrane targeting of Noxa1 depends on Noxo1, through tail-to-tail interactions between these proteins. Noxa1 can support Nox1 without Noxo1, when targeted to the plasma membrane by fusing membrane-binding sequences from Rac1 (amino acids 183 to 192) to the C terminus of Noxa1. However, membrane targeting of Noxa1 is not sufficient for activation of Nox1. Both the Noxo1-independent and -dependent Nox1 systems involve Rac1, since they are affected by Rac1 mutants or Noxa1 mutants defective in Rac binding or short interfering RNA-mediated Rac1 silencing. Nox1 or Nox3 expression promotes p22phox transport to the plasma membrane, and both oxidases are inhibited by mutations in the p22phox binding sites (SH3 domains) of the Nox organizers (p47phox or Noxo1). Regulation of Nox3 by Rac1 was also evident from the effects of mutant Rac1 or mutant Nox3 activators (p67phox or Noxa1) or Rac1 silencing. In the absence of Nox organizers, the Nox activators (p67phox or Noxa1) colocalize with Rac1 within ruffling membranes, independently of their ability to bind Rac1. Thus, Rac1 regulates both oxidases through the Nox activators, although it does not appear to direct the subcellular localization of these activators.     

Role of the small GTPase Rac in p22phox-dependent NADPH oxidases

The superoxide-producing phagocyte NADPH oxidase gp91(phox)/Nox2 and the non-phagocytic oxidases Nox1 and Nox3 each form a complex in the membrane with p22(phox), which provides both stabilization and a docking site for organizer proteins. The p22(phox)-complexed Nox2 and Nox1 are dormant on their own, and their activation requires soluble supportive proteins such as a Nox organizer (p47(phox) or Noxo1) and a Nox activator (p67(phox) or Noxa1). The small GTPase Rac directly binds to the activators, and thus plays an essential role in the Nox2-based oxidase containing p47(phox) and p67(phox) or a positive role in Nox1 activity supported by Noxo1 and Noxa1. Although Nox3 complexed with p22(phox) constitutively produce superoxide, the production can be enhanced by supportive proteins. Here we compare the roles of Rac in these p22(phox)-dependent oxidases using the organizer and activator in different combinations. Expression of constitutively active Rac1(Q61L) is essential for activation of the Nox2- or Nox1-based oxidase containing the organizer p47(phox) and either p67(phox) or Noxa1. When these oxidases use Noxo1 as an organizer instead of p47(phox), they produce a small but significant amount of superoxide without expression of Rac1(Q61L), although the production is enhanced by Rac1(Q61L). Thus p47(phox) is likely related to strict dependence on Rac. The Nox3-based oxidase has a similar tendency in the change of the dependence: Rac plays a positive role in Nox3 activation in the presence of p47(phox) and either p67(phox) or Noxa1, whereas Rac fails to upregulate Nox3 activity when p47(phox) is replaced with Noxo1. We also demonstrate that, in the Nox3-based oxidase containing solely p67(phox) as supportive protein, expression of Rac1(Q61L) enhances not only superoxide production but also membrane translocation of p67(phox). Since the enhancements are not observed with a mutant p67(phox) defective in binding to Rac, this GTPase appear to directly recruit p67(phox) to the membrane.     

Noxa1 as a moderate activator of Nox2-based NADPH oxidase

Noxa1 was discovered as an activating factor for Nox1, an O(2)(-)-generating enzyme. Subsequent studies have shown that Noxa1 is colocalized with Nox2 in several cell types, including vascular cells. Nox2 activation by Noxa1 has been examined in reconstituted model cells. However, little is known about the kinetic properties of Noxa1 in Nox2 activation. In the present study, we used purified cyt.b(558) (Nox2 plus p22(phox)), Rac(Q61L), and Noxo1 to examine the ability of Noxa1 to activate Nox2. In the pure reconstitution system, Noxa1 activated Nox2 with lower efficiency than p67(phox), a canonical activator of Nox2. The EC(50) value of Noxa1 was considerably higher than that of p67(phox). The V(max) value with Noxa1 and Noxo1 was one-third of that with p67(phox) and p47(phox). The EC(50) value of Noxo1 or Rac(Q61L) was also higher when Noxa1 was used. The affinity of FAD for the oxidase and the stability of the active complex were remarkably low when Noxa1 and Noxo1 were used compared with p67(phox) and p47(phox). The stability was not improved by fusion of Noxa1 with Rac(Q61L). These findings show that Noxa1 has quite different kinetic properties from p67(phox) and suggest that Noxa1 may function as a moderate activator of Nox2.     

Interaction between the SH3 domains and C-terminal proline-rich region in NADPH oxidase organizer 1 (Noxo1)

NADPH oxidase organizer 1 (Noxo1), harboring a PX domain, two SH3 domains, and a proline-rich region (PRR), participates in activation of superoxide-producing Nox-family NADPH oxidases. Here, we show that Noxo1 supports superoxide production in a cell-free system for gp91(phox)/Nox2 activation by arachidonic acid. This lipid enhances an SH3-mediated binding of Noxo1 to p22(phox), a protein complexed with Nox oxidases; the binding is known to be required for Nox activation. We also demonstrate that the bis-SH3 domain directly interacts with the Noxo1 PRR. The interaction appears to prevent the bis-SH3 domain and PRR from binding to their target proteins; disruption of the intramolecular interaction facilitates Noxo1 binding to p22(phox) and also allows the PRR to associate with the Nox activator Noxa1, which association is crucial for Nox activation as well. These findings suggest that Nox activation involves a conformational change leading to disruption of the bis-SH3-PRR interaction in Noxo1.     

The NADPH oxidase Nox3 constitutively produces superoxide in a p22phox-dependent manner: its regulation by oxidase organizers and activators

Nox3, a member of the superoxide-producing NADPH oxidase (Nox) family, participates in otoconia formation in mouse inner ears, which is required for perception of balance and gravity. The activity of other Nox enzymes such as gp91(phox)/Nox2 and Nox1 is known to absolutely require both an organizer protein (p47(phox) or Noxo1) andanactivatorprotein (p67(phox) or Noxa1); for the p47(phox)-dependent activation of these oxidases, treatment of cells with stimulants such as phorbol 12-myristate 13-acetate is also indispensable. Here we show that ectopic expression of Nox3 in various types of cells leads to phorbol 12-myristate 13-acetate-independent constitutive production of a substantial amount of superoxide under the conditions where gp91(phox) and Nox1 fail to generate superoxide, i.e. in the absence of the oxidase organizers and activators. Nox3 likely forms a functional complex with p22(phox); Nox3 physically interacts with and stabilizes p22(phox), and the Nox3-dependent superoxide production is totally dependent on p22(phox). The organizers p47(phox) and Noxo1 are capable of enhancing the superoxide production by Nox3 in the absence of the activators, and the enhancement requires the interaction of the organizers with p22(phox), further indicating a link between Nox3 and p22(phox). The p47(phox)-enhanced Nox3 activity is further facilitated by p67(phox) or Noxa1, whereas the activators cancel the Noxo1-induced enhancement. On the other hand, the small GTPase Rac, essential for the gp91(phox) activity, is likely dispensable to the Nox3 system. Thus Nox3 functions together with p22(phox) as an enzyme constitutively producing superoxide, which can be distinctly regulated by combinatorial use of the organizers and activators.     

Expression and function of Noxo1gamma, an alternative splicing form of the NADPH oxidase organizer 1

Activation of the superoxide-producing NADPH oxidase Nox1 requires both the organizer protein Noxo1 and the activator protein Noxa1. Here we describe an alternative splicing form of Noxo1, Noxo1gamma, which is expressed in the testis and fetal brain. The Noxo1gamma protein contains an additional five amino acids in the N-terminal PX domain, a phosphoinositide-binding module; the domain plays an essential role in supporting superoxide production by NADPH oxidase (Nox) family oxidases including Nox1, gp91(phox)/Nox2, and Nox3, as shown in this study. The PX domain isolated from Noxo1gamma shows a lower affinity for phosphoinositides than that from the classical splicing form Noxo1beta. Consistent with this, in resting cells, Noxo1gamma is poorly localized to the membrane, and thus less effective in activating Nox1 than Noxo1beta, which is constitutively present at the membrane. On the other hand, cell stimulation with phorbol 12-myristate 13-acetate (PMA), an activator of Nox1-3, facilitates membrane translocation of Noxo1gamma; as a result, Noxo1gamma is equivalent to Noxo1beta in Nox1 activation in PMA-stimulated cells. The effect of the five-amino-acid insertion in the Noxo1 PX domain appears to depend on the type of Nox; in activation of gp91(phox)/Nox2, Noxo1gamma is less active than Noxo1beta even in the presence of PMA, whereas Noxo1gamma and Noxo1beta support the superoxide-producing activity of Nox3 to the same extent in a manner independent of cell stimulation.     

Subcellular localization and function of alternatively spliced Noxo1 isoforms

Nox organizer 1 (Noxo1), a p47(phox) homolog, is produced as four isoforms with unique N-terminal PX domains derived by alternative mRNA splicing. We compared the subcellular distribution of these isoforms or their isolated PX domains produced as GFP fusion proteins, as well as their ability to support Nox1 activity in several transfected models. Noxo1alpha, beta, gamma, and delta show different subcellular localization patterns, determined by their PX domains. In HEK293 cells, Noxo1beta exhibits prominent plasma membrane binding, Noxo1gamma shows plasma membrane and nuclear associations, and Noxo1alpha and delta localize primarily on intracellular vesicles or cytoplasmic aggregates, but not the plasma membrane. Nox1 activity correlates with Noxo1 plasma membrane binding in HEK293 cells, since Noxo1beta supports the highest activity and Noxo1gamma and Noxo1alpha support moderate or low activities, respectively. In COS-7 cells, where Noxo1alpha localizes on the plasma membrane, the activities supported by the three isoforms (alpha, beta, and gamma) do not differ significantly. The PX domains of beta and gamma bind the same phospholipids, including phosphatidic acid. These results indicate that the variant PX domains are unique determinants of Noxo1 localization and Nox1 function. Finally, the overexpressed Noxo1 isoforms do not affect p22(phox) localization, although Nox1 is needed to transport p22(phox) to the plasma membrane.     

Expression of isoforms of NADPH oxidase components in rat pancreatic islets

Increased oxidative stress plays a role in the pathogenesis of beta-cell dysfunction and death. We studied isoforms of NADPH oxidase components in islets of Langerhans isolated from rat pancreas and tumoral rat beta-cell line RINm5F cells by RT-PCR and sequencing of its products. RT-PCR revealed that isolated islets constitutively expressed mRNA of NADPH oxidase components, Nox1, Nox2, Nox4 and p22(phox) as membrane-associated components and p47(phox), Noxo1 (homologue of p47(phox)), Noxa1 (homologue of p67(phox)), and p40(phox) as cytosolic components. RINm5F cells showed a similar pattern of expression but Nox2 mRNA was not detected. Expression of Nox1, Nox4, Noxo1 and Noxa1 was confirmed by sequencing the PCR products. Immunohistochemistry revealed the expression of NADPH oxidase component in beta-cells of rat pancreatic islets. Glucose-stimulated insulin secretion from isolated islets was suppressed by diphenyleneiodonium, a flavocytochrome inhibitor, but not by apocynin, an inhibitor of p47(phox) translocation to membranes. Our results suggest that the functional significance of NADPH oxidase in insulin secretion may merit further investigation.     

Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase

Nox1 and Nox4, homologues of the leukocyte NADPH oxidase subunit Nox2 (gp91phox) mediate superoxide anion formation in various cell types. However, their interactions with other components of the NADPH oxidase are poorly defined. We determined whether a direct interaction of Nox1 and Nox4 with the p22phox subunit of the NADPH oxidase occurs. Using confocal microscopy, co-localization of p22phox with Nox1, Nox2, and Nox4 was observed in transiently transfected vascular smooth muscle cells (VSMC) and HEK293 cells. Plasmids coding for fluorescent fusion proteins of p22phox and the Nox proteins with cyan- and yellow-fluorescent protein (cfp and yfp, respectively) were constructed and expressed in VSMC and HEK293 cells. The cfp-tagged p22phox expression level increased upon cotransfection with Nox1 or Nox4. Protein-protein interaction between the fluorescent fusion proteins of p22phox and the Nox partners was observed using the fluorescence resonance energy transfer technique. Immunoprecipitation of native Nox1 from human VSMC revealed co-precipitation of p22phox. Immunoprecipitation from transfected HEK293 cells revealed co-precipitation of native p22phox with yfp-tagged Nox1, Nox2, and Nox4. Following mutation of a histidine (corresponding to the position 115 in human Nox2) to leucine, this interaction was abolished. Transfection of rat p22phox (but not Noxo1 and Noxa1) increased the radical generation in cells expressing Nox4. We provide evidence that p22phox directly interacts with Nox1 and Nox4, to form an superoxide-generating NADPH oxidase and demonstrate that mutation of the potential heme binding site in the Nox proteins disrupts the complex formation of Nox1 and Nox4 with p22phox.     

Point mutations in the proline-rich region of p22phox are dominant inhibitors of Nox1- and Nox2-dependent reactive oxygen generation

The integral membrane protein p22phox is an indispensable component of the superoxide-generating phagocyte NADPH oxidase, whose catalytic core is the membrane-associated gp91phox (also known as Nox2). p22phox associates with gp91phox and, through its proline-rich C terminus, provides a binding site for the tandem Src homology 3 domains of the activating subunit p47phox. Whereas p22phox is expressed ubiquitously, its participation in regulating the activity of other Nox enzymes is less clear. This study investigates the requirement of p22phox for Nox enzyme activity and explores the role of its proline-rich region (PRR) for regulating activity. Coexpression of specific Nox catalytic subunits (Nox1, Nox2, Nox3, Nox4, or Nox5) along with their corresponding regulatory subunits (NOXO1/NOXA1 for Nox1; p47phox/p67phox/Rac for Nox2; NOXO1 for Nox3; no subunits for Nox4 or Nox5) resulted in marked production of reactive oxygen. Small interfering RNAs decreased endogenous p22phox expression and inhibited reactive oxygen generation from Nox1, Nox2, Nox3, and Nox4 but not Nox5. Truncated forms of p22phox that disrupted the PRR-inhibited reactive oxygen generation from Nox1, Nox2, and Nox3 but not from Nox4 and Nox5. Similarly, p22phox (P156Q), a mutation that disrupts Src homology 3 binding by the PRR, potently inhibited reactive oxygen production from Nox1 and Nox2 but not from Nox4 and Nox5. Expression of p22phox (P156Q) inhibited NOXO1-stimulated Nox3 activity, but co-expression of NOXA1 overcame the inhibitory effect. The P157Q and P160Q mutations of p22phox showed selective inhibition of Nox2/p47phox/p67phox, and selectivity was specific for the organizing subunit (p47phox or NOXO1) rather than the Nox catalytic subunit. These studies stress the importance of p22phox for the function of Nox1, Nox2, Nox3, and Nox4, and emphasize the key role of the PRR for regulating Nox proteins whose activity is dependent upon p47phox or NOXO1.     

Noxa1 is a central component of the smooth muscle NADPH oxidase in mice

NADPH oxidase is the most important source of oxygen-derived radicals (ROS) in the vascular wall. In vascular smooth muscle cells (VSMC), NADPH oxidase is characterized by the expression of the membrane subunit Nox1, which is activated by cytoplasmic proteins binding to its activation domain. We set out to identify the cytoplasmic protein involved in NADPH oxidase activation in mouse VSMC. Western blot analysis revealed that human endothelial cells and leukocytes but not VSMC from the aorta of the rat and the mouse express the classic NADPH oxidase activator p67phox. In mouse VSMC, however, the p67phox homologue Noxa1 was detected. Using antibodies generated against mouse Noxa1, the protein was observed in the cytosolic fraction of mouse VSMC with a molecular weight of about 51 kDa. Immunohistochemistry revealed that Noxa1 is expressed in the smooth muscle layer but not in endothelium or the adventitia of the mouse carotid artery. Fluorescent fusion proteins of Noxa1 were observed to be expressed in the cytoplasm of VSMC and coexpression of the NADPH oxidase organizer Noxo1 targeted the complex to membrane. An antisense plasmid of Noxa1 attenuated the endogenous Noxa1 protein expression in VSMC. This plasmid attenuated the ROS formation in mouse VSMC as detected using L012 chemiluminescence and prevented the agonist-induced ROS production in response to basic fibroblast growth factor and epidermal growth factor. In conclusion, these data indicate that Noxa1 replaces p67phox in VSMC and plays a central role in the activation of the NADPH oxidase in the vascular wall.     

C-terminal truncation of Noxa1 greatly enhances its ability to activate Nox2 in a pure reconstitution system

Noxa1 activates Nox2 together with Noxo1 and Rac in a pure reconstitution system, but the resulting activity is considerably lower than that induced by p67^phox and p47^phox. In this study, we found that C-terminal-truncated forms of Noxa1 exhibited higher activities than full-length Noxa1. Of the truncations examined, Noxa1(1-225) showed the highest ability for activation. Kinetic studies revealed that Noxa1(1-225) had a threefold higher V_max value than full-length Noxa1 with a similar EC₅₀ value. The affinities of Noxo1 and RacQ61L were not much altered by the truncation. Conversely, the affinity of FAD for the Nox2 complex was enhanced after the truncation. In the absence of Noxo1, Noxa1(1-225) showed much higher activity with a lower EC₅₀ than full-length Noxa1. Noxa1(1-225) showed comparable activity to that of p67^phox with either Noxo1 or p47^phox, although the stability was lower than that with p67^phox and p47^phox. These findings indicate that the role of the C-terminal half of Noxa1 is autoinhibition. The data suggest a two-step autoinhibition mechanism, comprising self-masking to interrupt the binding to the oxidase, and holding of the activation domain in a suboptimal position to the oxidase. This study reveals that when both types of inhibition are released, Noxa1 achieves high-level superoxide production.     

Nox1 activation by βPix and the role of Ser-340 phosphorylation

Rac is an activating factor for Nox1, an O₂⁻-generating NADPH oxidase, expressed in the colon and other tissues. Rac requires a GDP-GTP exchange factor for activation. Nox1 activation by βPix has been demonstrated in cell lines. We examined the effects of βPix and its phosphomimetic mutant on endogenous Nox1 in Caco-2 cells transfected with Noxo1 and Noxa1. βPix expression enhanced O₂⁻ production in resting cells and cells stimulated with EGF or phorbol ester. βPix(S340E) further enhanced O₂⁻ production, while βPix(S340A) eliminated the βPix effect. βPix(S340E), but not βPix(S340A), had higher affinity and GEF activity for Rac than wild-type βPix. These results suggest that βPix phosphorylation at Ser-340 upregulates Nox1 through Rac activation, confirming Rac as a trigger for acute Nox1-dependent ROS production.     

Activation of NADPH oxidase 1 in tumour colon epithelial cells

In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP(+). The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O(2)(-)-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N-Rac1(Q61L)] between truncated NOXA1(1-211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1-292) [C-terminal truncated NOXO1(1-292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O(2)(-)-producing activity in Caco-2 cells.     

Nox1-dependent reactive oxygen generation is regulated by Rac1

Rac1 has been implicated in the generation of reactive oxygen species (ROS) in several cell types, but the enzymatic origin of the ROS has not been proven. The present studies demonstrate that Nox1, a homolog of the phagocyte NADPH-oxidase component gp91(phox), is activated by Rac1. When Nox1 is co-expressed along with its regulatory subunits NOXO1 and NOXA1, significant ROS generation is seen. Herein, co-expression of constitutively active Rac1(G12V), but not wild-type Rac1, resulted in marked further stimulation of activity. Decreased Rac1 expression using small interfering RNA reduced Nox1-dependent ROS. CDC42(G12V) failed to increase activity, and small interfering RNA directed against CDC42 failed to decrease activity, pointing to specificity for Rac. TPR domain mutants of NOXA1 that interfere with Rac1 binding were ineffective in supporting Nox1-dependent ROS generation. Immunoprecipitation experiments demonstrated a complex containing Rac1(G12V), NOXO1, NOXA1, and Nox1. CDC42(G12V) could not substitute for Rac1(G12V) in such a complex. Nox1 formed a complex with Rac1(G12V) that was independent of NOXA1 and NOXO1, consistent with direct binding of Rac1(G12V) to Nox1. Rac1(G12V) interaction with NOXA1 was enhanced by Nox1 and NOXO1, suggesting cooperative binding. A model is presented comparing activation by regulatory subunits of Nox1 versus gp91(phox) (Nox2) in which Rac1 activation provides a major trigger that acutely activates Nox1-dependent ROS generation.     

Crucial role of two potential cytosolic regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidase activation

Assembly of cytosolic factors p67(phox) and p47(phox) with cytochrome b(558) is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop (191)TSSTKTIRRS(200) and the C-terminal (484)DESQANHFAVHHDEEKD(500) of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47(phox) translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop(Nox4)-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the alpha-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47(phox) membrane translocation. This suggests that the alpha-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.     

Death receptors 4 and 5 activate Nox1 NADPH oxidase through riboflavin kinase to induce reactive oxygen species-mediated apoptotic cell death

Stimulation of the proapoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, death receptors 4 (DR4) and 5 (DR5), conventionally induces caspase-dependent apoptosis in tumor cells. Here we report that stimulation of DR4 and/or DR5 by the agonistic protein KD548-Fc, an Fc-fused DR4/DR5 dual-specific Kringle domain variant, activates plasma membrane-associated Nox1 NADPH oxidase to generate superoxide anion and subsequently accumulates intracellular reactive oxygen species (ROS), leading to sustained c-Jun N-terminal kinase activation and eventual apoptotic cell death in human HeLa and Jurkat tumor cells. KD548-Fc treatment induces the formation of a DR4/DR5 signaling complex containing riboflavin kinase (RFK), Nox1, the Nox1 subunits (Rac1, Noxo1, and Noxa1), TNF receptor-associated death domain (TRADD), and TNF receptor-associated factor 2 (TRAF2). Depletion of RFK, but not the Nox1 subunits, TRADD and TRAF2, failed to recruit Nox1 and Rac1 to DR4 and DR5, demonstrating that RFK plays an essential role in linking DR4/DR5 with Nox1. Knockdown studies also reveal that RFK, TRADD, and TRAF2 play critical, intermediate, and negligible roles, respectively, in the KD548-Fc-mediated ROS accumulation and downstream signaling. Binding assays using recombinantly expressed proteins suggest that DR4/DR5 directly interact with cytosolic RFK through RFK-binding regions within the intracellular death domains, and TRADD stabilizes the DR4/DR5-RFK complex. Our results suggest that DR4 and DR5 have a capability to activate Nox1 by recruiting RFK, resulting in ROS-mediated apoptotic cell death in tumor cells.     

N-linked glycosylation of the superoxide-producing NADPH oxidase Nox1

Nox1 is a membrane-integrated protein that belongs to the Nox family of superoxide-producing NADPH oxidases. Here we show that human Nox1 undergoes glycosylation at Asn-162 and Asn-236 in the second and third extracellular loops, respectively. Simultaneous threonine substitution for these residues completely abrogates the glycosylation, but does not prevent Nox1 from forming a heterodimer with p22^phox, trafficking to the cell surface, or producing superoxide. In the absence of p22^phox, Nox1 is transported to the plasma membrane mainly as a form with high mannose N-glycans, although their conversion into complex N-glycans is induced by expression of p22^phox. These findings indicate that glycosylation and subsequent N-glycan maturation of Nox1 are both dispensable for its cell surface recruitment. Superoxide production by unglycosylated Nox1 is largely dependent on p22^phox, which is abrogated by glutamine substitution for Pro-156 in p22^phox, a mutation leading to a defective interaction with the Nox1-activating protein Noxo1. Thus p22^phox directly contributes to Nox1 activation in a glycosylation-independent manner, besides its significant role in Nox1 glycan maturation.     

Identification of a conserved Rac-binding site on NADPH oxidases supports a direct GTPase regulatory mechanism

The NADPH oxidases (Noxs) are a family of superoxide-generating enzymes implicated in a variety of biological processes. Full activity of Nox1, -2, and -3 requires the action of a Rac GTPase. A direct regulatory interaction of Rac with Nox2 has been proposed as part of a two-step mechanism for regulating electron transfer during superoxide formation. Using truncation analysis of Rac binding to the cytoplasmic tail of Nox2, along with peptides derived from this region in cell-free assays, we identify a Rac interaction site within amino acids 419-430 of Nox2. This region is required for binding Rac2 but not p47(phox) or p67(phox) cytosolic regulatory factors. A cell-permeant version of the peptide encompassing amino acids 419-430 specifically inhibits NADPH oxidase activation in intact human neutrophils. Mutational analysis of the putative Rac-binding site revealed specific residues, particularly Lys-421, Tyr-425, and Lys-426, individually required for Rac-dependent NADPH oxidase activity that are conserved in the Rac-regulated Nox1, Nox2, and Nox3 enzymes but not in Nox4 or Nox5. Mutation of the conserved residues in the Rac-binding site of Nox1 also result in the loss of Rac-dependent activity. Our data identify a functional Rac interaction site conserved in Rac-dependent Noxs and support a direct regulatory interaction of Rac GTPases to promote activation of these NADPH oxidases.     

Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species

NADPH oxidases of the Nox family exist in various supergroups of eukaryotes but not in prokaryotes, and play crucial roles in a variety of biological processes, such as host defense, signal transduction, and hormone synthesis. In conjunction with NADPH oxidation, Nox enzymes reduce molecular oxygen to superoxide as a primary product, and this is further converted to various reactive oxygen species. The electron-transferring system in Nox is composed of the C-terminal cytoplasmic region homologous to the prokaryotic (and organelle) enzyme ferredoxin reductase and the N-terminal six transmembrane segments containing two hemes, a structure similar to that of cytochrome b of the mitochondrial bc(1) complex. During the course of eukaryote evolution, Nox enzymes have developed regulatory mechanisms, depending on their functions, by inserting a regulatory domain (or motif) into their own sequences or by obtaining a tightly associated protein as a regulatory subunit. For example, one to four Ca(2+)-binding EF-hand motifs are present at the N-termini in several subfamilies, such as the respiratory burst oxidase homolog (Rboh) subfamily in land plants (the supergroup Plantae), the NoxC subfamily in social amoebae (the Amoebozoa), and the Nox5 and dual oxidase (Duox) subfamilies in animals (the Opisthokonta), whereas an SH3 domain is inserted into the ferredoxin-NADP(+) reductase region of two Nox enzymes in Naegleria gruberi, a unicellular organism that belongs to the supergroup Excavata. Members of the Nox1-4 subfamily in animals form a stable heterodimer with the membrane protein p22(phox), which functions as a docking site for the SH3 domain-containing regulatory proteins p47(phox), p67(phox), and p40(phox); the small GTPase Rac binds to p67(phox) (or its homologous protein), which serves as a switch for Nox activation. Similarly, Rac activates the fungal NoxA via binding to the p67(phox)-like protein Nox regulator (NoxR). In plants, on the other hand, this GTPase directly interacts with the N-terminus of Rboh, leading to superoxide production. Here I describe the regulation of Nox-family oxidases on the basis of three-dimensional structures and evolutionary conservation.