The adaptor protein p40(phox) as a positive regulator of the superoxide-producing phagocyte oxidase

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial in host defense, requires the cytosolic proteins p67(phox) and p47(phox). They translocate to the membrane upon cell stimulation and activate flavocytochrome b(558), the membrane-integrated catalytic core of this enzyme system. The activators p67(phox) and p47(phox) form a ternary complex together with p40(phox), an adaptor protein with unknown function, comprising the PX/PB2, SH3 and PC motif- containing domains: p40(phox) associates with p67(phox) via binding of the p40(phox) PC motif to the p67(phox) PB1 domain, while p47(phox) directly interacts with p67(phox) but not with p40(phox). Here we show that p40(phox) enhances membrane translocation of p67(phox) and p47(phox) in stimulated cells, which leads to facilitated production of superoxide. The enhancement cannot be elicited by a mutant p40(phox) carrying the D289A substitution in PC or a p67(phox) with the K355A substitution in PB1, each being defective in binding to its respective partner. Thus p40(phox) participates in activation of the phagocyte oxidase by regulating membrane recruitment of p67(phox) and p47(phox) via the PB1-PC interaction with p67(phox).     

A region C-terminal to the proline-rich core of p47phox regulates activation of the phagocyte NADPH oxidase by interacting with the C-terminal SH3 domain of p67phox

Activation of the phagocyte NADPH oxidase requires the regulatory proteins p47(phox) and p67(phox), each harboring two SH3 domains. p67(phox) interacts with p47(phox) via simultaneous binding of the p67(phox) C-terminal SH3 domain to both the proline-rich region (PRR) of amino acid residues 360-369 and its C-terminally flanking region of p47(phox); the role of the interaction in oxidase regulation has not been fully understood. Here we show that the p47(phox)-p67(phox) interaction is disrupted not only by deletion of the PRR but also by substitution for basic residues in the extra-PRR (K383E/K385E). The substitution impaired oxidase activation partially in vitro and much more profoundly in vivo, indicating the significance of the p47(phox) extra-PRR. Replacement of Ser-379 in the extra-PRR, a residue known to undergo phosphorylation in stimulated cells, by aspartate attenuates the interaction and thus results in a defective superoxide production, suggesting that phosphorylation of Ser-379 is involved in oxidase regulation.     

Conformational changes in p47 upon activation highlighted by mass spectrometry coupled to hydrogen/deuterium exchange and limited proteolysis

The neutrophil NADPH oxidase is an enzymatic complex involved in innate immunity. Phosphorylation of p47^phox promotes its translocation with p67^phox and p40^phox, followed by membrane interaction and assembly with flavocytochrome b₅₅₈ into a functional complex. To characterise p47^phox conformational changes during activation, we used wild-type and the S303/304/328E triple mutant mimicking the phosphorylated state. Hydrogen/deuterium exchange and limited proteolysis coupled to mass spectrometry were used to discriminate between the various structural models. An increase in deuteration confirmed that p47^phox adopts an open and more flexible conformation after activation. Limited proteolysis correlated this change with increased auto-inhibitory region (AIR) accessibility. These results establish a structural link between the AIR release and the exposure of the Phox homology (PX) domain.     

Irreversibly glycated LDL induce oxidative and inflammatory state in human endothelial cells; added effect of high glucose

In diabetes, hyperglycemia and the associated formation of advanced glycation end-products (AGE) and AGE-modified low density lipoproteins (AGE-LDL) can directly affect the cells of the vascular wall. We hypothesize that AGE-LDL may act directly and induce oxidant and inflammatory alterations in human endothelial cells (HEC), this effect being amplified by high glucose. To test this assumption, the activity of NADPH oxidase (NADPHox) was evaluated and the expression of its subunits (p22^phox, NOX4, and p67^phox), of the AGE receptor (RAGE), and of the monocyte chemoattractant protein-1 (MCP-1) were assessed by real-time PCR and Western blot in confluent EA.hy926 cells incubated with AGE-LDL for 24 and 48 h, in normal and high glucose conditions. Exposure of HEC for 48 h to AGE-LDL in 5 mM glucose induced an increase of RAGE expression (50%), NADPHox activity (107%), p22^phox and NOX4 mRNA (50% and 188%, respectively) and MCP-1 expression (80%). AGE-LDL-stimulated p22^phox expression by activating p38 MAP kinase and NF-kB, and MCP-1 expression by activating NF-kB, as demonstrated by the use of specific inhibitors (SB203580 and Bay11-7085). The addition of 25 mM glucose in the culture medium enhanced the effect of AGE-LDL, but also of nLDL, on RAGE, p22^phox, NOX4, p67^phox, and MCP-1 gene expression. In conclusion, AGE-LDL induce an oxidative stress and a pro-inflammatory state in human endothelial cells. Both AGE-LDL and nLDL in the presence of high glucose amplify their effect, revealing a link between hyperlipidemia, diabetes, and endothelial cell dysfunction.     

NOX in liver fibrosis

NADPH oxidase is a multi-protein complex producing reactive oxygen species (ROS) both in phagocytic cells, being essential in host defense, and in non-phagocytic cells, regulating intracellular signalling. In the liver, NADPH oxidase plays a central role in fibrogenesis. A functionally active form of the NADPH oxidase is expressed not only in Kupffer cells (phagocytic cell type) but also in hepatic stellate cells (HSCs) (non-phagocytic cell type), suggesting a role of the non-phagocytic NADPH oxidase in HSC activation. Consistent with this concept, profibrogenic agonists such as Angiotensin II (Ang II) and platelet derived growth factor (PDGF), or apoptotic bodies exert their activity through NADPH oxidase-activation in HSCs. Both pharmacological inhibition with DPI and genetic studies using p47(phox) knockout mice provided evidence for a central role of NADPH oxidase in the regulation of HSC-activity and liver fibrosis. In addition to the p47(phox) component, only Rac1 has been identified as a functional active component of the NADPH oxidase complex in HSCs.     

Protein kinase C-alpha and -delta are required for NADPH oxidase activation in WKYMVm-stimulated IMR90 human fibroblasts

Gene duplications in rodents have given rise to a family of proteases that are expressed exclusively in placenta. To define the biological role of these enzymes specific inhibitors are needed to differentiate their activities from other more ubiquitously expressed proteases, such as cathepsins B and L. Libraries of peptidyl inhibitors based upon a 4-cyclohexanone pharmacophore were screened for inhibition of cathepsins P, L, and B. The tightest binding dipeptidyl inhibitor for cathepsin P contained Tyr in P(2) and Trp in P(2)('), consistent with the specificity of this enzyme for hydrophobic amino acids at these sites in synthetic substrates. An inhibitor containing Trp in both P(2) and P(2)(') provided better discrimination between cathepsin P and cathepsins B and L. Extension of the inhibitors to include P(3), and P(3)(') amino acids identified an inhibitor with Trp in P(2), P(2)('), and P(3), and Phe in P(3)(') that bound to cathepsin P with a K(i) of 32 nM. This specificity for inhibitors with hydrophobic aromatic amino acids in these four positions is unique among the lysosomal cysteine proteases. This inhibitor bound to cathepsin P an order of magnitude tighter than to mouse and human cathepsin L and two orders of magnitude tighter than to human cathepsin B. Cbz-Trp-Trp-4-cyclohexanone-Trp-Phe-OMe can discriminate cathepsin P from cathepsins B and L and consequently can be used to specifically inhibit and identify cathepsin P in cellular systems.     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.     

Bidirectional interactions between NOX2‐type NADPH oxidase and the F‐actin cytoskeleton in neuronal growth cones

NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)‐type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F‐actin content, retrograde F‐actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91^phox localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40^phox. p40^phox itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91^phox and p40^phox with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40^phox with NOX2/gp91^phox at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth.

     

Arachidonic Acid Induces Direct Interaction of the p67phox-Rac Complex with the Phagocyte Oxidase Nox2, Leading to Superoxide Production

The phagocyte NADPH oxidase Nox2, heterodimerized with p22^phox in the membrane, is dormant in resting cells but becomes activated upon cell stimulation to produce superoxide, a precursor of microbicidal oxidants. Nox2 activation requires two switches to be turned on simultaneously: a conformational change of the cytosolic protein p47^phox and GDP/GTP exchange on the small GTPase Rac. These proteins, in an active form, bind to their respective targets, p22^phox and p67^phox, leading to productive oxidase assembly at the membrane. Although arachidonic acid (AA) efficiently activates Nox2 both in vivo and in vitro, the mechanism has not been fully understood, except that AA induces p47^phox conformational change. Here we show that AA elicits GDP-to-GTP exchange on Rac at the cellular level, consistent with its role as a potent Nox2 activator. However, even when constitutively active forms of p47^phox and Rac1 are both expressed in HeLa cells, superoxide production by Nox2 is scarcely induced in the absence of AA. These active proteins also fail to effectively activate Nox2 in a cell-free reconstituted system without AA. Without affecting Rac-GTP binding to p67^phox, AA induces the direct interaction of Rac-GTP-bound p67^phox with the C-terminal cytosolic region of Nox2. p67^phox-Rac-Nox2 assembly and superoxide production are both abrogated by alanine substitution for Tyr-198, Leu-199, and Val-204 in the p67^phox activation domain that localizes the C-terminal to the Rac-binding domain. Thus the “third” switch (AA-inducible interaction of p67^phox·Rac-GTP with Nox2) is required to be turned on at the same time for Nox2 activation.     

NADPH oxidase in human lung fibroblasts

Reactive oxygen species produced by NADPH oxidase appear to play a role in the response of human lung fibroblast cells to rhinovirus infection. The purpose of the following studies was to characterize the NADPH oxidase components in these cells, to examine the effect of rhinovirus challenge on the expression of these proteins, and to confirm previous studies suggesting a role for p47-phox in the oxidant response to rhinovirus challenge. The results revealed that the NADPH oxidase components p47-phox, p67-phox, p22-phox, and NOX4 were expressed in lung fibroblast cells. In contrast, gp91-phox was not expressed in this cell line. Expression of p67-phox was upregulated by rhinovirus challenge. The functional role of NADPH oxidase in the rhinovirus-induced oxidant stress and elaboration of IL-8 was confirmed by detection of significant reductions in oxidant stress and IL-8 elaboration following transfection of the cells with antisense nucleotides to p47-phox. The lack of gp91-phox in cultured lung fibroblast cells, the induction of p67-phox by rhinovirus, and the confirmation of participation of p47-phox in rhinovirus-induced oxidant stress are significant findings of this study and form a basis for future investigations into understanding the mechanisms of the NADPH oxidase response to rhinovirus infection.     

Evidence that the product of the human X-linked CGD gene, gp91-phox, is a voltage-gated H(+) pathway

Expression of gp91-phox in Chinese hamster ovary (CHO91) cells is correlated with the presence of a voltage-gated H(+) conductance. As one component of NADPH oxidase in neutrophils, gp91-phox is responsible for catalyzing the production of superoxide (O(2).(2)). Suspensions of CHO91 cells exhibit arachidonate-activatable H(+) fluxes (Henderson, L.M., G. Banting, and J.B. Chappell. 1995. J. Biol. Chem. 270:5909-5916) and we now characterize the electrical properties of the pathway. Voltage-gated currents were recorded from CHO91 cells using the whole-cell configuration of the patch-clamp technique under conditions designed to exclude a contribution from ions other than H(+). As in other voltage-gated proton currents (Byerly, L., R. Meech, and W. Moody. 1984. J. Physiol. 351:199-216; DeCoursey, T.E., and V.V. Cherny. 1993. Biophys. J. 65:1590-1598), a lowered external pH (pH(o)) shifted activation to more positive voltages and caused the tail current reversal potential to shift in the manner predicted by the Nernst equation. The outward currents were also reversibly inhibited by 200 microM zinc. Voltage-gated currents were not present immediately upon perforating the cell membrane, but showed a progressive increase over the first 10-20 min of the recording period. This time course was consistent with a gradual shift in activation to more negative potentials as the pipette solution, pH 6.5, equilibrated with the cell contents (reported by Lucifer yellow included in the patch pipette). Use of the pH-sensitive dye 2'7' bis-(2-carboxyethyl)-5(and 6) carboxyfluorescein (BCECF) suggested that the final intracellular pH (pH(i)) was approximately 6.9, as though pH(i) was largely determined by endogenous cellular regulation. Arachidonate (20 microM) increased the amplitude of the currents by shifting activation to more negative voltages and by increasing the maximally available conductance. Changes in external Cl(-) concentration had no effect on either the time scale or the appearance of the currents. Examination of whole cell currents from cells expressing mutated versions of gp91-phox suggest that: (a) voltage as well as arachidonate sensitivity was retained by cells with only the NH(2)-terminal 230 amino acids, (b) histidine residues at positions 111, 115, and 119 on a putative membrane-spanning helical region of the protein contribute to H(+) permeation, (c) histidine residues at positions 111 and 119 may contribute to voltage gating, (d) the histidine residue at position 115 is functionally important for H(+) selectivity. Mechanisms of H(+) permeation through gp91-phox include the possible protonation/deprotonation of His-115 as it is exposed alternatively to the interior and exterior faces of the cell membrane (see Starace, D.M., E. Stefani, and F. Bezanilla. 1997. Neuron. 19:1319-1327) and the transfer of protons across an "H-X-X-X-H-X-X-X-H" motif lining a conducting pore.     

Molecular composition and regulation of the Nox family NAD(P)H oxidases

Reactive oxygen species (ROS) are conventionally regarded as inevitable deleterious by-products in aerobic metabolism with a few exceptions such as their significant role in host defense. The phagocyte NADPH oxidase, dormant in resting cells, becomes activated during phagocytosis to deliberately produce superoxide, a precursor of other microbicidal ROS, thereby playing a crucial role in killing pathogens. The catalytic center of this oxidase is the membrane-integrated protein gp91(phox), tightly complexed with p22(phox), and its activation requires the association with p47(phox), p67(phox), and the small GTPase Rac, which normally reside in the cytoplasm. Since recent discovery of non-phagocytic gp91(phox)-related enzymes of the NAD(P)H oxidase (Nox) family--seven homologues identified in humans--deliberate ROS production has been increasingly recognized as important components of various cellular events. Here, we describe a current view on the molecular composition and post-translational regulation of Nox-family oxidases in animals.     

The neutrophil NADPH oxidase.

The NADPH oxidase of phagocytes catalyzes the conversion of oxygen to O2(-). This multicomponent enzyme complex contains five essential protein components, two in the membrane and three in the cytosol. Unassembled and inactive in resting phagocytes, the oxidase becomes active after translocation of cytosolic components to the membrane to assemble a functional oxidase. Multiple factors regulate its assembly and activity, thus serving to maintain this highly reactive system under spatial and temporal control until recruited for antimicrobial or proinflammatory events. The recent identification of homologs of one of the membrane components in nonphagocytic cells will expand understanding of the biological contexts in which this system may function.     

FPRL1-mediated induction of superoxide in LL-37-stimulated IMR90 human fibroblast

Molecular mechanisms underlying the generation of reactive oxygen species in LL-37-stimulated cells are poorly understood. Previously, we demonstrated that in human fibroblasts the exposure to WKYMVm induced p47^phox phosphorylation and translocation and, in turn, NADPH oxidase activation. These effects were mediated by the activation of the Formyl-peptide receptor-like 1 (FPRL1) and the downstream signaling involved ERKs phosphorylation and PKCα- and PKCδ-activation. Since LL-37 uses FPRL1 as a receptor to mediate its action on several cell types, we investigated in LL-37-stimulated IMR90 cells molecular mechanisms involved in NADPH-dependent superoxide generation. The exposure to LL-37, which is expressed in fibroblasts, induced ERKs activation, p47^phox phosphorylation and translocation as well as NADPH oxidase activation. These effects were prevented by pertussis toxin, PD098059 and WRWWWW, a FPRL1-selective antagonist. Furthermore, the stimulation with LL-37 of HEK293 cells, transfected to stably express FPRL1, induced a rapid activation of ERKs and p47^phox phosphorylation.     

Cooperation of p40(phox) with p47(phox) for Nox2-based NADPH oxidase activation during Fcγ receptor (FcγR)-mediated phagocytosis: mechanism for acquisition of p40(phox) phosphatidylinositol 3-phosphate (PI(3)P) binding

During activation of the phagocyte (Nox2-based) NADPH oxidase, the cytoplasmic Phox complex (p47(phox)-p67(phox)-p40(phox)) translocates and associates with the membrane-spanning flavocytochrome b(558). It is unclear where (in cytoplasm or on membranes), when (before or after assembly), and how p40(phox) acquires its PI(3)P-binding capabilities. We demonstrated that in addition to conformational changes induced by H(2)O(2) in the cytoplasm, p40(phox) acquires PI(3)P-binding through direct or indirect membrane targeting. We also found that p40(phox) is essential when p47(phox) is partially phosphorylated during FcγR-mediated oxidase activation; however, p40(phox) is less critical when p47(phox) is adequately phosphorylated, using phosphorylation-mimicking mutants in HEK293(Nox2/FcγRIIa) and RAW264.7(p40/p47KD) cells. Moreover, PI binding to p47(phox) is less important when the autoinhibitory PX-PB1 domain interaction in p40(phox) is disrupted or when p40(phox) is targeted to membranes. Furthermore, we suggest that high affinity PI(3)P binding of the p40(phox) PX domain is critical during its accumulation on phagosomes, even when masked by the PB1 domain in the resting state. Thus, in addition to mechanisms for directly acquiring PI(3)P binding in the cytoplasm by H(2)O(2), p40(phox) can acquire PI(3)P binding on targeted membranes in a p47(phox)-dependent manner and functions both as a "carrier" of the cytoplasmic Phox complex to phagosomes and an "adaptor" of oxidase assembly on phagosomes in cooperation with p47(phox), using positive feedback mechanisms.     

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.