Turning off NADPH oxidase-2 by impeding p67phox activation in infected mouse macrophages reduced viral entry and inflammation.

BACKGROUND: Targeting cells of the host immune system is a promising approach to fight against Influenza A virus (IAV) infection. Macrophage cells use the NADPH oxidase-2 (NOX2) enzymatic complex as a first line of defense against pathogens by generating superoxide ions O2- and releasing H2O2. Herein, we investigated whether targeting membrane -embedded NOX2 decreased IAV entry via raft domains and reduced inflammation in infected macrophages. METHODS: Confocal microscopy and western blots monitored levels of the viral nucleoprotein NP and p67phox, NOX2 activator subunit, Elisa assays quantified TNF-α levels in LPS or IAV-activated mouse or porcine alveolar macrophages pretreated with a fluorescent NOX inhibitor, called nanoshutter NS1. RESULTS: IAV infection in macrophages promoted p67phox translocation to the membrane, rafts clustering and activation of the NOX2 complex at early times. Disrupting rafts reduced intracellular viral NP. NS1 markedly reduced raft clustering and viral entry by binding to the C-terminal of NOX2 also characterized in vitro. NS1 decrease of TNF-α release depended on the cell type. CONCLUSION: NOX2 participated in IAV entry and raft-mediated endocytosis. NOX2 inhibition by NS1 reduced viral entry. NS1 competition with p67phox for NOX2 binding shown by in silico models and cell-free assays was in agreement with NS1 inhibiting p67phox translocation to membrane-embedded NOX2 in mouse and porcine macrophages. GENERAL SIGNIFICANCE: We introduce NS1 as a compound targeting NOX2, a critical enzyme controlling viral levels and inflammation in macrophages and discuss the therapeutic relevance of targeting the C-terminal of NADPH oxidases by probes like NS1 in viral infections.

Strategies for identifying synthetic peptides to act as inhibitors of NADPH oxidases, or "all that you did and did not want to know about Nox inhibitory peptides".

Phagocytes utilize reactive oxygen species (ROS) to kill pathogenic microorganisms. The source of ROS is an enzymatic complex (the NADPH oxidase), comprising a membrane-associated heterodimer (flavocytochrome b (558)), consisting of subunits Nox2 and p22(phox), and four cytosolic components (p47(phox), p67(phox), p40(phox), and Rac). The primordial ROS (superoxide) is generated by the reduction of molecular oxygen by NADPH via redox centers located on Nox2. This process is activated by the translocation of the cytosolic components to the membrane and their assembly with Nox2. Membrane translocation is preceded by interactions among cytosolic components. A number of proteins structurally and functionally related to Nox2 have been discovered in many cells (the Nox family) and these have pleiotropic functions related to the production of ROS. An intense search is underway to design therapeutic means to modulate Nox-dependent overproduction of ROS, associated with diseases. Among drug candidates, a central position is held by synthetic peptides reflecting domains in oxidase components involved in NADPH oxidase assembly. Peptides, corresponding to domains in Nox2, p22(phox), p47(phox), and Rac, found to be oxidase activation inhibitory in vitro, are reviewed. Usually, peptides are inhibitory only when added preceding assembly of the complex. Although competition with intact components seems most likely, less obvious mechanisms are, sometimes, at work. The use of peptides as inhibitory drugs in vivo requires the development of methods to assure cell penetration, resistance to degradation, and avoidance of toxicity, and modest successes have been achieved. The greatest challenge remains the discovery of peptide inhibitors acting specifically on individual Nox isoforms.

A prenylated p47phox-p67phox-Rac1 chimera is a Quintessential NADPH oxidase activator: membrane association and functional capacity.

The superoxide-generating NADPH oxidase complex of resting phagocytes includes cytochrome b(559), a membrane-associated heterodimer composed of two subunits (Nox2 and p22(phox)), and four cytosolic proteins (p47(phox), p67(phox), Rac, and p40(phox)). Upon stimulation, the cytosolic components translocate to the membrane, as the result of a series of interactions among the cytosolic components and among the cytosolic components and cytochrome b(559) and its phospholipid environment. We described the construction of a tripartite chimera (trimera) consisting of strategic domains of p47(phox), p67(phox), and Rac1, in which interactions among cytosolic components were replaced by fusion (Berdichevsky, Y., Mizrahi, A., Ugolev, Y., Molshanski-Mor, S., and Pick, E. (2007) J. Biol. Chem. 282, 22122-22139). We now fused green fluorescent protein (GFP) to the N terminus of the trimera and found the following. 1) The GFP-p47(phox)-p67(phox)-Rac1 trimera activates the oxidase in amphiphile-dependent and -independent (anionic phospholipid-enriched membrane) cell-free systems. 2) Geranylgeranylation of the GFP-trimera makes it a potent oxidase activator in unmodified (native) membranes and in the absence of amphiphile. 3) Prenylated GFP-trimera binds spontaneously to native membranes (as assessed by gel filtration and in-line fluorometry), forming a tight complex capable of NADPH-dependent, activator-independent superoxide production at rates similar to those measured in canonical cell-free systems. 4) Prenylation of the GFP-trimera supersedes completely the dependence of oxidase activation on the p47(phox) phox homology domain and, partially, on the Rac1 polybasic domain, but the requirement for Trp(193) in p47(phox) persists. Prenylated GFP-p47(phox)-p67(phox)-Rac1 trimera acts as a quintessential single molecule oxidase activator of potential use in high throughput screening of inhibitors.

G6PC3 mutations are associated with a major defect of glycosylation: a novel mechanism for neutrophil dysfunction.

Glucose-6-phosphatase, an enzyme localized in the endoplasmic reticulum (ER), catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. In humans, there are three differentially expressed glucose-6-phosphatase catabolic genes (G6PC1-3). Recently, it has been shown that mutations in the G6PC3 gene result in a syndrome associating congenital neutropenia and various organ malformations. The enzymatic function of G6PC3 is dependent on G6P transport into the ER, mediated by G6P translocase (G6PT). Mutations in the gene encoding G6PT result in glycogen storage disease type-1b (GSD-1b). Interestingly, GSD-1b patients exhibit a similar neutrophil dysfunction to that observed in G6PC3-deficient patients. To better understand the causes of neutrophil dysfunction in both diseases, we have studied the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of patients with G6PC3 and G6PT syndromes. Unexpectedly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments indicated hypo-glycosylation of gp91(phox), the electron-transporting component of the NADPH oxidase, in all of these patients. Rigorous mass spectrometric glycomic profiling showed that most of the complex-type antennae which characterize the neutrophil N-glycome of healthy individuals were severely truncated in the patients' neutrophils. A comparable truncation of the core 2 antenna of the O-glycans was also observed. This aberrant neutrophil glycosylation is predicted to have profound effects on the neutrophil function and merit designation of both syndromes as a new class of congenital disorders of glycosylation.

p67phox -derived self-assembled peptides prevent Nox2 NADPH oxidase activation by an auto-inhibitory mechanism.

Activation of the Nox2-dependent NADPH oxidase is the result of a conformational change in Nox2 induced by interaction with the cytosolic component p67phox . In preliminary work we identified a cluster of overlapping 15-mer synthetic peptides, corresponding to p67phox residues 259-279, which inhibited oxidase activity in an in vitro, cell-free assay, but the results did not point to a competitive mechanism. We recently identified an auto-inhibitory intramolecular bond in p67phox , one extremity of which was located within the 259-279 sequence, and we hypothesized that inhibition by exogenous peptides might mimic intrinsic auto-inhibition. In this study, we found that: (i) progressive N- and C-terminal truncation of inhibitory p67phox peptides, corresponding to residues 259-273 and 265-279, revealed that inhibitory ability correlated with the presence of residues 265 NIVFVL270 , exposed at either the N- or C-termini of the peptides; (ii) inhibition of oxidase activity was associated exclusively with self-assembled peptides, which pelleted upon centrifugation at 12,000 ×g; (iii) self-assembled p67phox peptides inhibited oxidase activity by specific binding of p67phox and the ensuing depletion of this component, essential for interaction with Nox2; and (iv) peptides subjected to scrambling or reversing the order of residues in NIVFVL retained the propensity for self-assembly, oxidase inhibitory ability, and specific binding of p67phox , indicating that the dominant parameter was the hydrophobic character of five of the six residues. This appears to be the first description of inhibition of oxidase activity by self-assembled peptides derived from an oxidase component, acting by an auto-inhibitory mechanism.

The molecular basis of Rac-GTP action-promoting binding of p67phox to Nox2 by disengaging the ß hairpin from downstream residues.

p67phox fulfils a key role in the assembly/activation of the NADPH oxidase by direct interaction with Nox2. We proposed that Rac-GTP serves both as a carrier of p67phox to the membrane and an inducer of a conformational change enhancing its affinity for Nox2. This study provides evidence for the latter function: (i) oxidase activation was inhibited by p67phox peptides (106-120) and (181-195), corresponding to the ß hairpin and to a downstream region engaged in intramolecular bonds with the ß hairpin, respectively; (ii) deletion of residues 181-193 and point mutations Q115R or K181E resulted in selective binding of p67phox to Nox2 peptide (369-383); (iii) both deletion and point mutations led to a change in p67phox , expressed in increased apparent molecular weights; (iv) p67phox was bound to p67phox peptide (181-195) and to a cluster of peptides (residues 97-117), supporting the participation of selected residues within these sequences in intramolecular bonds; (v) p67phox failed to bind to Nox2 peptide (369-383), following interaction with Rac1-GTP, but a (p67phox -Rac1-GTP) chimera exhibited marked binding to the peptide, similar to that of p67phox deletion and point mutants; and (vi) size exclusion chromatography of the chimera revealed its partition in monomeric and polymeric forms, with binding to Nox2 peptide (369-383) restricted to polymers. The molecular basis of Rac-GTP action entails unmasking of a previously hidden Nox2-binding site in p67phox , following disengagement of the ß hairpin from more C-terminal residues. The domain in Nox2 binding the "modified" p67phox comprises residues within the 369-383 sequence in the cytosolic dehydrogenase region.

Cell-Free NADPH Oxidase Activation Assays: A Triumph of Reductionism.

The superoxide (O2·-)-generating NADPH oxidase complex of phagocytes comprises a membrane-associated heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of NOX2 and p22phox) and four cytosolic regulatory proteins, p47phox, p67phox, p40phox, and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2·- generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome, a process known as NADPH oxidase assembly. A consequent conformational change in NOX2 initiates the electron flow along a redox gradient, from NADPH to molecular oxygen (O2), leading to the one-electron reduction of O2 to O2·-. Historically, methodological difficulties in the study of the assembled complex derived from stimulated cells, due to its lack of stability, led to the design of "cell-free" systems (also known as "broken cells" or in vitro systems). In a major paradigm shift, the cell-free systems have as their starting point NADPH oxidase components derived from resting (unstimulated) phagocytes, or as in the predominant method at present, recombinant proteins representing the components of the NADPH oxidase complex. In cell-free systems, membrane receptor stimulation and the signal transduction sequence are absent, the accent being placed on the actual process of assembly, all of which takes place in vitro. Thus, a mixture of the individual components of the NADPH oxidase is exposed in vitro to an activating agent, the most common being anionic amphiphiles, resulting in the formation of a complex between cytochrome b 558 and the cytosolic components and O2·- generation in the presence of NADPH. Alternative activating pathways require posttranslational modification of oxidase components or modifying the phospholipid milieu surrounding cytochrome b 558. Activation is commonly quantified by measuring the primary product of the reaction, O2·-, trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of rates of O2·- production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount role in the identification and characterization of the components of the NADPH oxidase complex, the performance of structure-function studies, the deciphering of the mechanisms of assembly, the search for inhibitory drugs, and the diagnosis of various forms of chronic granulomatous disease (CGD).

p67phox binds to a newly identified site in Nox2 following the disengagement of an intramolecular bond-Canaan sighted?

Activation of the phagocyte NADPH oxidase involves a conformational change in Nox2. The effector in this process is p67phox and there is evidence for a change in the configuration of p67phox being required for binding to Nox2. To study this, we measured binding of p67phox to a library of Nox2 peptides and binding of NusA-Nox2 fusion proteins to p67phox . We found, serendipitously, that deletion of residues 259-279 in p67phox (p67phox Δ(259-279)), endowed it with the ability to bind selectively to Nox2 peptide 369-383 (peptide 28). There was no binding to scrambled Nox2 peptide 28 and to Nox4 peptide 28. Binding was cysteine independent and resistant to reducing and alkylating agents. Truncations of peptide 28 revealed that the actual binding site consisted of residues 375-383. Binding of p67phox Δ(259-279) to peptide 28 was mimicked by that of a (p67phox -RacGTP) chimera. Both p67phox Δ(259-279) and the (p67pho -RacGTP) chimera bound a NusA-Nox2 fusion protein, comprising residues 375-383. Specific single residue deletion mutants, within the p67phox sequence 259-279, were also bound to Nox2 peptide 28. Peptides synthesized to correspond to the 259-279 sequence in p67phox , were found to autobind p67phox , suggesting that an intramolecular bond exists in p67phox , one pole of which was located within residues 259-279. We conclude that "resting" p67phox exists in a "closed" conformation, generated by an intramolecular bond. Deletion of specific residues within the 259-279 sequence, in vitro, or interaction with RacGTP, in vivo, causes "opening" of the bond and results in binding of p67phox to a specific, previously unknown, site in Nox2.

Using Synthetic Peptides for Exploring Protein-Protein Interactions in the Assembly of the NADPH Oxidase Complex.

The NADPH oxidase complex, responsible for reactive oxygen species (ROS) generation by phagocytes, consists of a membrane-associated flavocytochrome b 558 (a heterodimer of NOX2 and p22phox) and the cytosolic components p47phox, p67phox, Rac(1 or 2), and p40phox. NOX2 carries all redox stations through which electrons flow from NADPH to molecular oxygen, to generate the primary ROS, superoxide. For the electron flow to start, a conformational change in NOX2 is required. The dominant hypothesis is that this change is the result of the interaction of NOX2 with one or more of the cytosolic components (NADPH oxidase assembly). At the most basic level, assembly is the sum of several protein-protein interactions among oxidase components. This chapter describes a reductionist approach to the identification of regions in oxidase components involved in assembly. This approach consists of "transforming" one component in an array of overlapping synthetic peptides and assessing binding to the peptides of another component, represented by a recombinant protein. The peptides are tagged with biotin, at the N- or C-terminus, and immobilized on streptavidin-coated 96-well plates. The protein partners are expressed with a 6His tag and added to the plates in the fluid phase. Binding of the protein to the peptides is quantified by a kinetic ELISA , using a peroxidase-conjugated anti-polyhistidine antibody. Protein-peptide binding assays were applied successfully to (a) identifying the binding site on one component (represented by peptides) for another component (proteins), (b) precisely defining the "binding sequence," (c) acquiring information on the binding site in the partner protein, (d) investigating the effect of conformational changes in proteins on binding to peptides, (e) determining the effect of physicochemical modification of peptides on binding of proteins, and (f) identifying epitopes recognized by anti-oxidase component antibodies by binding of antibody to peptide arrays derived from the component.

Binding of p67phox to Nox2 is stabilized by disulfide bonds between cysteines in the 369 Cys-Gly-Cys371 triad in Nox2 and in p67phox.

A central event in the activation of the phagocyte NADPH oxidase involves binding of p67phox to the dehydrogenase region of Nox2. The identity of the binding site in Nox2 is unknown. By measuring binding of p67phox to synthetic Nox2 peptides, we previously identified a sequence corresponding to Nox2 residues 357-383, as a potential binding site. A key role was attributed to a 369 Cys-Gly-Cys371 triad, shared by peptides 357-371 (peptide 24) and 369-383 (peptide 28). In this study, we show that (1) oxidation of cysteines in peptides 24 and 28 by a variety of oxidants markedly enhances the binding of p67phox ; (2) replacing cysteines by arginine abolishes the response to oxidants and the enhanced binding of p67phox ; (3) oxidants act by generating an intramolecular disulfide bond linking cysteines 369 and 371, generating such bond during peptide synthesis reproduces the effect of oxidants; (4) for the disulfide bond to lead to enhanced binding, cysteines must be separated by an intervening residue; bonds joining adjacent cysteines, or cysteines located on two peptides, do not enhance binding; (5) dissociating disulfide bonds by reducing agents abolishes enhanced binding; (6) treating p67phox with the alkylating agent N-ethylmaleimide suppresses binding; and (7) mutating all nine cysteines in p67phox to serines abolishes binding and diminishes the ability of p67phox to support NADPH oxidase activation in vitro. Results show that the primary interaction of p67phox with Nox2 is followed by a stabilizing step, based on the establishment of disulfide bonds between cysteine(s) in the 369 Cys-Gly-Cys371 triad and cysteine(s) in p67phox .