Dual role of Rac in the assembly of NADPH oxidase, tethering to the membrane and activation of p67phox: a study based on mutagenesis of p67phox-Rac1 chimeras

NADPH oxidase activation involves the assembly of membrane-localized cytochrome b559 with the cytosolic components p47phox, p67phox, and the small GTPase Rac. Assembly is mimicked by a cell-free system consisting of membranes and cytosolic components, activated by an anionic amphiphile. We reported that a chimeric construct, consisting of residues 1-212 of p67phox and full-length Rac1, activates the oxidase in vitro in an amphiphile-dependent manner, and when prenylated, in the absence of amphiphile and p47phox. We subjected chimera p67phox-(1-212)-Rac1 to mutational analysis and found that: 1) replacement of a single basic residue at the C terminus of the Rac1 moiety by glutamine is sufficient for loss of activity by the non-prenylated chimera; replacement of all six basic residues by glutamines is required for loss of activity by the prenylated chimera. 2) A V204A mutation in the activation domain of the p67phox moiety leads to a reduction in activity. 3) Mutating residues, known to participate in the interaction between free p67phox and Rac1, in the p67phox-(R102E) or Rac1 (A27K, G30S) moieties of the chimera, leads to a marked decrease in activity, indicating a requirement for intrachimeric bonds, in addition to the engineered fusion. 4) Chimeras, inactive because of mutations A27K or G30S in the Rac1 moiety, are reactivated by supplementation with exogenous Rac1-GTP but not with exogenous p67phox. This demonstrates that Rac has a dual role in the assembly of NADPH oxidase. One is to tether p67phox to the membrane; the other is to induce an "activating" conformational change in p67phox.     

Tetratricopeptide repeat (TPR) motifs of p67(phox) participate in interaction with the small GTPase Rac and activation of the phagocyte NADPH oxidase.

The small GTPase Rac functions as a molecular switch in several important cellular events including cytoskeletal reorganization and activation of the phagocyte NADPH oxidase, the latter of which leads to production of superoxide, a precursor of microbicidal oxidants. During formation of the active oxidase complex at the membrane, the GTP-bound Rac appears to interact with the N-terminal region of p67(phox), another indispensable activator that translocates from the cytosol upon phagocyte stimulation. Here we show that the p67(phox) N terminus lacks the CRIB motif, a well known Rac target, but contains four tetratricopeptide repeat (TPR) motifs with highly alpha-helical structure. Disruption of any of the N-terminal three TPRs, but the last one, results in defective interaction with Rac, while all the four are required for the NADPH oxidase activation. We also find that Arg-102 in the third repeat is likely involved in binding to Rac via an ionic interaction, and that replacement of this residue with Glu completely abrogates the capability of activating the oxidase both in vivo and in vitro. Thus the TPR motifs of p67(phox) are packed to function as a Rac target, thereby playing a crucial role in the active oxidase complex formation.     

Rac activation induces NADPH oxidase activity in transgenic COSphox cells, and the level of superoxide production is exchange factor-dependent

Transient expression of constitutively active Rac1 derivatives, (G12V) or (Q61L), was sufficient to induce phagocyte NADPH oxidase activity in a COS-7 cell model in which human cDNAs for essential oxidase components, gp91(phox), p22(phox), p47(phox), and p67(phox), were expressed as stable transgenes. Expression of constitutively active Rac1 in "COS(phox)" cells induced translocation of p47(phox) and p67(phox) to the membrane. Furthermore, translocation of p47(phox) was induced in the absence of p67(phox) expression, even though Rac does not directly bind p47(phox). Rac effector domain point substitutions (A27K, G30S, D38A, Y40C), which can selectively eliminate interaction with different effector proteins, impaired Rac1V12-induced superoxide production. Activation of endogenous Rac1 by expression of constitutively active Rac-guanine nucleotide exchange factor (GEF) derivatives was sufficient to induce high level NADPH oxidase activity in COS(phox) cells. The constitutively active form of the hematopoietic-specific GEF, Vav1, was the most effective at activating superoxide production, despite detection of higher levels of Rac1-GTP upon expression of constitutively active Vav2 or Tiam1 derivatives. These data suggest that Rac can play a dual role in NADPH oxidase activation, both by directly participating in the oxidase complex and by activating signaling events leading to oxidase assembly, and that Vav1 may be the physiologically relevant GEF responsible for activating this Rac-regulated complex.     

Structure of the TPR domain of p67phox in complex with Rac.GTP

p67phox is an essential part of the NADPH oxidase, a multiprotein enzyme complex that produces superoxide ions in response to microbial infection. Binding of the small GTPase Rac to p67phox is a key step in the assembly of the active enzyme complex. The structure of Rac.GTP bound to the N-terminal TPR (tetratricopeptide repeat) domain of p67phox reveals a novel mode of Rho family/effector interaction and explains the basis of GTPase specificity. Complex formation is largely mediated by an insertion between two TPR motifs, suggesting an unsuspected versatility of TPR domains in target recognition and in their more general role as scaffolds for the assembly of multiprotein complexes.     

A prenylated p67phox-Rac1 chimera elicits NADPH-dependent superoxide production by phagocyte membranes in the absence of an activator and of p47phox: conversion of a pagan NADPH oxidase to monotheism

Activation of the superoxide-generating NADPH oxidase of phagocytes is the result of the assembly of a membrane-localized flavocytochrome (cytochrome b(559)) with the cytosolic components p47(phox), p67(phox), and the small GTPase Rac. Activation can be reproduced in an in vitro system in which cytochrome b(559)-containing membranes are mixed with cytosolic components in the presence of an anionic amphiphile. We proposed that the essential event in activation is the interaction between p67(phox) and cytochrome b(559) and that Rac and p47(phox) serve as carriers for p67(phox) to the membrane. When prenylated, Rac can fulfill the carrier function by itself, supporting oxidase activation by p67(phox) in the absence of p47(phox) and amphiphile. We now show that a single chimeric protein, consisting of residues 1-212 of p67(phox) and full-length Rac1 (residues 1-192), prenylated in vitro and exchanged to GTP, becomes a potent oxidase activator in the absence of any other component or stimulus. Oxidase activation by prenylated chimera p67(phox) (1-212)-Rac1 (1-192) is accompanied by its spontaneous association with membranes. Prenylated chimeras p67(phox) (1-212)-Rac1 (178-192) and p67(phox) (1-212)-Rac1 (189-192), containing specific C-terminal regions of Rac1, are inactive; the activity of the first but not of the second chimera can be rescued by supplementation with exogenous nonprenylated Rac1-GTP. An analysis of prenylated p67(phox)-Rac1 chimeras suggests that the basic requirements for oxidase activation are: (i) a "two signals" membrane-localizing motif present in Rac, comprising the prenyl group and a C-terminal polybasic sequence and (ii) an intrachimeric or extrachimeric protein-protein interaction between p67(phox) and Rac1, causing a conformational change in the "activation domain" in p67(phox).     

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.     

Tripartite chimeras comprising functional domains derived from the cytosolic NADPH oxidase components p47phox, p67phox, and Rac1 elicit activator-independent superoxide production by phagocyte membranes: an essential role for anionic membrane phospholipids

The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b(559) with three cytosolic components: p47(phox), p67(phox), and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b(559) within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47(phox), the tetratricopeptide repeat and activation domains of p67(phox), and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC(50)) for and formed a more stable complex (longer half-life) with cytochrome b(559) compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the poly-basic stretch at the C terminus of the Rac1 segment; 2) interaction with p22(phox) via Trp(193) in the N-terminal SH3 domain of the p47(phox) segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67(phox) and Rac1 segments complementary to their physical fusion. The PX domain of the p47(phox) segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.     

Small angle neutron scattering and gel filtration analyses of neutrophil NADPH oxidase cytosolic factors highlight the role of the C-terminal end of p47phox in the association with p40phox

The NADPH oxidase of phagocytic cells is regulated by the cytosolic factors p47(phox), p67(phox), and p40(phox) as well as by the Rac1-Rho-GDI heterodimer. The regulation is a consequence of protein-protein interactions involving a variety of protein domains that are well characterized in signal transduction. We have studied the behavior of the NADPH oxidase cytosolic factors in solution using small angle neutron scattering and gel filtration. p47(phox), two truncated forms of p47(phox), namely, p47(phox) without its C-terminal end (residues 1-358) and p47(phox) without its N-terminal end (residues 147-390), and p40(phox) were found to be monomeric in solution. The dimeric form of p67(phox) previously observed by gel filtration experiments was confirmed. Our small angle neutron scattering experiments show that p40(phox) binds to the full-length p47(phox) in solution in the absence of phosphorylation. We demonstrated that the C-terminal end of p47(phox) is essential in this interaction. From the comparison of the presence or absence of interaction with various truncated forms of the proteins, we confirmed that the SH3 domain of p40(phox) interacts with the C-terminal proline rich region of p47(phox). The radii of gyration observed for p47(phox) and the truncated forms of p47(phox) (without the C-terminal end or without the N-terminal end) show that all these molecules are elongated and that the N-terminal end of p47(phox) is globular. These results suggest that the role of amphiphiles such as SDS or arachidonic acid or of p47(phox) phosphorylation in the elicitation of NADPH oxidase activation could be to disrupt the p40(phox)-p47(phox) complex rather than to break an intramolecular interaction in p47(phox).     

Mapping of functional domains in the p22(phox) subunit of flavocytochrome b(559) participating in the assembly of the NADPH oxidase complex by "peptide walking".

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membranal heterodimeric flavocytochrome (cytochrome b(559)), composed of gp91(phox) and p22(phox) subunits, and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (1 or 2). All redox stations involved in electron transport from NADPH to oxygen are located in gp91(phox). NADPH oxidase activation is the consequence of assembly of cytochrome b(559) with cytosolic proteins, a process reproducible in a cell-free system, consisting of phagocyte membranes, and recombinant cytosolic components, activated by an anionic amphiphile. p22(phox) is believed to act as a linker between the cytosolic components and gp91(phox). We applied "peptide walking" to mapping of domains in p22(phox) participating in NADPH oxidase assembly. Ninety one synthetic overlapping pentadecapeptides, spanning the p22(phox) sequence, were tested for the ability to inhibit NADPH oxidase activation in the cell-free system and to bind individual cytosolic NADPH oxidase components. We conclude the following. 1) The p22(phox) subunit of cytochrome b(559) serves as an anchor for both p47(phox) and p67(phox). 2) p47(phox) binds not only to the proline-rich region, located at residues 151-160 in the cytosolic C terminus of p22(phox), but also to a domain (residues 51-63) located on a loop exposed to the cytosol. 3) p67(phox) shares with p47(phox) the ability to bind to the proline-rich region (residues 151-160) and also binds to two additional domains, in the cytosolic loop (residues 81-91) and at the start of the cytosolic tail (residues 111-115). 4) The binding affinity of p67(phox) for p22(phox) peptides is lower than that of p47(phox). 5) Binding of both p47(phox) and p67(phox) to proline-rich p22(phox) peptides occurs in the absence of an anionic amphiphile. A revised membrane topology model of p22(phox) is proposed, the core of which is the presence of a functionally important cytosolic loop (residues 51-91).     

Protein delivery by Pseudomonas type III secretion system: Ex vivo complementation of p67(phox)-deficient chronic granulomatous disease

Bacterial type III secretion system drives the translocation of virulence factors into the cystosol of host target cells. In phagocytes and in Epstein-Barr virus immortalized B lymphocytes, NADPH oxidase generates O(-2) through an electron transfer chain the activity of which depends on the assembly of three, p67(phox), p47(phox) and p40(phox) cytosolic activating factors with Rac 1/2 and a membrane redox component, cytochrome b(558). In p67(phox) deficient chronic granulomatous disease (CGD) patients, p67-phox is missing and NADPH oxidase activity is abolished. ExoS is a virulence factor of Pseudomonas aeruginosa which is secreted via the type III secretion system: it was fused with p67(phox). Pseudomonas aeruginosa synthesized and translocated the hybrid ExoS-p67(phox) fusion protein into the cytosol of B lymphocytes via the type III secretion system. Purified ExoS-p67(phox) hybrid protein was as efficient as normal recombinant p67(phox) in cell-free reconstitution of NADPH oxidase activity. Therefore, ExoS-p67(phox) was transferred via the type III secretion system of Pseudomonas aeruginosa into the cytosol of B lymphocytes from a p67(phox)-deficient CGD patient and functionally reconstituted NADPH oxidase activity. In the complementation process, ExoS acted as a molecular courier for protein delivery: the reconstitution of an active NADPH oxidase complex suggests type III secretion system to be a new approach for cellular therapy.     

Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly

The superoxide (O(2))-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome (cytochrome b(559)) and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O(2) production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome b(559). This process can be reproduced in an in vitro system consisting of phagocyte membranes, p47(phox), p67(phox), and Rac, activated by an anionic amphiphile. We now show that post-translationally processed (prenylated) Rac1 initiates NADPH oxidase assembly, expressed in O(2) production, in a cell-free system containing phagocyte membrane vesicles and p67(phox), in the absence of an activating amphiphile and of p47(phox). Prenylated Cdc42Hs, a GTPase closely related to Rac, is inactive under the same conditions. Results obtained with phagocyte membrane vesicles can be reproduced fully by replacing these with partially purified cytochrome b(559), incorporated in phosphatidylcholine vesicles. Prenylated, but not nonprenylated, Rac1 binds spontaneously to phagocyte membrane vesicles and also to artificial, protein-free, phosphatidylcholine vesicles, a process counteracted by GDP dissociation inhibitor for Rho. Binding of prenylated Rac1 to membrane vesicles is accompanied by the recruitment of p67(phox) to the same location and the formation of an assembled NADPH oxidase complex, producing O(2) upon the addition of NADPH. Amphiphile and p47(phox)-independent NADPH oxidase activation by prenylated Rac1 is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67(phox) to the membrane environment, to be followed by the interaction of p67(phox) with cytochrome b(559).     

Mutagenesis of an arginine- and lysine-rich domain in the gp91(phox) subunit of the phagocyte NADPH-oxidase flavocytochrome b558

Site-directed mutagenesis was used to generate a series of mutants harboring point or multiple substitutions within the hydrophilic, polybasic domain of gp91(phox) encompassed by residues 86-102, which was previously identified as a site of interaction with p47(phox) during phagocyte NADPH oxidase assembly. Recombinant wild-type or mutant gp91(phox) was expressed in a human myeloid leukemia cell line in which the endogenous gp91(phox) gene was disrupted by gene targeting. NADPH oxidase activity was measured in a cytochrome c reduction assay following granulocytic differentiation of cells that expressed recombinant gp91(phox). Expression of a gp91(phox) mutant in which amino acids 89-97 were replaced with nine alternate amino acids abolished NADPH oxidase activity. Expression of gp91(phox) mutants R89T, D95A, D95R, R96A, R96E, or K102T did not significantly affect NADPH oxidase activity. However, mutations of individual or paired arginine residues at positions 91 and 92 had substantial effects on superoxide generation. The R91E/R92E mutation completely abolished both NADPH oxidase activity and membrane-translocation of the cytosolic oxidase proteins p47(phox), p67(phox), Rac1, and Rac2. The phorbol 12-myristate 13-acetate-induced rate of superoxide production was reduced by approximately 75% in cells expressing R91T/R92A, R91E, or R92E gp91(phox) along with an increased lag time to the maximal rates of superoxide production relative to cells expressing wild-type gp91(phox). Taken together, these results demonstrate that Arg91 and Arg92 of gp91(phox) are essential for flavocytochrome b558 function in granulocytes and suggest that these residues participate in the interaction of gp91(phox) with the cytosolic oxidase proteins.     

Phosphatidylinositol 3-phosphate-dependent and -independent functions of p40phox in activation of the neutrophil NADPH oxidase

In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40(phox) remains less well defined than those of p47(phox) and p67(phox). We investigated the biological role of p40(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40(phox) reduces both the maximal rate and total amount of ROS produced without altering the K(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40(phox) chimeras, and deletion constructs, we found that the p40(phox) PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67(phox) requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40(phox), however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40(phox) can increase oxidase activity approximately 2.5-fold above that of wild-type p40(phox) but maintain the requirement for PX and SH3 domain function. We present a model where p40(phox) translocates p67(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.     

The Rac effector p67phox regulates phagocyte NADPH oxidase by stimulating Vav1 guanine nucleotide exchange activity

The phagocyte NADPH oxidase catalyzes the reduction of molecular oxygen to superoxide and is essential for microbial defense. Electron transport through the oxidase flavocytochrome is activated by the Rac effector p67(phox). Previous studies suggest that Vav1 regulates NADPH oxidase activity elicited by the chemoattractant formyl-Met-Leu-Phe (fMLP). We show that Vav1 associates with p67(phox) and Rac2, but not Rac1, in fMLP-stimulated human neutrophils, correlating with superoxide production. The interaction of p67(phox) with Vav1 is direct and activates nucleotide exchange on Rac, which enhances the interaction between p67(phox) and Vav1. This provides new molecular insights into regulation of the neutrophil NADPH oxidase, suggesting that chemoattractant-stimulated superoxide production can be amplified by a positive feedback loop in which p67(phox) targets Vav1-mediated Rac activation.     

The guanine nucleotide exchange factor trio activates the phagocyte NADPH oxidase in the absence of GDP to GTP exchange on Rac. "The emperor's nw clothes"

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome b(559) and four cytosolic components as follows: p47(phox), p67(phox), p40(phox), and the small GTPase Rac (1 or 2). Activation of the oxidase is the result of assembly of the cytosolic components with cytochrome b(559) and can be mimicked in vitro by mixtures of membrane and cytosolic components exposed to an anionic amphiphile, serving as activator. We reported that prenylation of Rac1 endows it with the ability to support oxidase activation in conjunction with p67(phox) but in the absence of amphiphile and p47(phox). We now show the following 6 points. 1) The Rac guanine nucleotide exchange factor Trio markedly potentiates oxidase activation by prenylated Rac1-GDP. 2) This occurs in the absence of exogenous GTP or any other source of GTP generation, demonstrating that the effect of Trio does not involve GDP to GTP exchange on Rac1. 3) Trio does not potentiate oxidase activation by prenylated Rac1-GTP, by nonprenylated Rac1-GDP in the presence or absence of amphiphile, and by a prenylated [p67(phox)-Rac1] chimera in GDP-bound form. 4) Rac1 mutants defective in the ability to bind Trio or to respond to Trio by nucleotide exchange fail to respond to Trio by enhanced oxidase activation. 5) A Trio mutant with conserved Rac1-binding ability but lacking nucleotide exchange activity fails to enhance oxidase activation. 6) The effect of Trio is mimicked by displacement of Mg(2+) from Rac1-GDP. These results reveal the existence of a novel mechanism of Rac activation by a guanine nucleotide exchange factor and suggest that the induction by Trio of a conformational change in Rac1, in the absence of nucleotide exchange, is sufficient for enhancing its effector function.     

Characterization of the flavoprotein domain of gp91phox which has NADPH diaphorase activity

A series of truncated forms of gp91phox were expressed in Escherichia coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin. TRX-gp91phox (306-569), which contains the putative FAD and NADPH binding sites, showed weak NADPH-dependent NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91phox (304-423) and TRX-gp91phox (424-569) were inactive. Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91phox (306-569), and showed the same K(m) for NADPH as that for superoxide generating activity by the intact enzyme. Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium. In the presence of Rac1, the cytosolic regulatory protein p67phox stimulated the NBT reductase activity, but p47phox had no effect. Truncated p67phox containing the activation domain (residues 199-210) [C.-H. Han, J.R. Freeman, T. Lee, S.A. Motalebi, and J.D. Lambeth (1998) J. Biol. Chem. 273, 16663-16668] stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity. Our data indicate that: (i) TRX-gp91phox (306-569) contains binding sites for both pyridine and flavin nucleotides; (ii) this flavoprotein domain shows weak diaphorase activity; and (iii) the flavin-binding domain of gp91phox is the target of regulation by the activation domain of p67phox.     

Specificity of p47phox SH3 domain interactions in NADPH oxidase assembly and activation.

The delineation of molecular structures that dictate Src homology 3 (SH3) domain recognition of specific proline-rich ligands is key to understanding unique functions of diverse SH3 domain-containing signalling molecules. We recently established that assembly of the phagocyte NADPH oxidase involves multiple SH3 domain interactions between several oxidase components (p47phox, p67phox, and p22phox). p47phox was shown to play a central role in oxidase activation in whole cells by mediating interactions with both the transmembrane component p22phox and cytosolic p67phox. To understand the specific roles of each SH3 domain of p47phox in oxidase assembly and activation, we mutated critical consensus residues (Tyr167 or Tyr237-->Leu [Y167L or Y237L], W193R or W263R, and P206L or P276L) on each of their binding surfaces. The differential effects of these mutations indicated that the first SH3 domain is responsible for the p47phox-p22phox interaction and plays a predominant role in oxidase activity and p47phox membrane assembly, while the second p47phox SH3 domain interacts with the NH2-terminal domain of p67phox. Binding experiments using the isolated first SH3 domain also demonstrated its involvement in intramolecular interactions within p47phox and showed a requirement for five residues (residues 151 to 155) on its N-terminal boundary for binding to p22phox. The differential effects of nonconserved-site mutations (W204A or Y274A and E174Q or E244Q) on whole-cell oxidase activity suggested that unique contact residues within the third binding pocket of each SH3 domain influence their ligand-binding specificities.     

Liposomes comprising anionic but not neutral phospholipids cause dissociation of Rac(1 or 2) x RhoGDI complexes and support amphiphile-independent NADPH oxidase activation by such complexes

Activation of the phagocyte NADPH oxidase involves the assembly of a membrane-localized cytochrome b559 with the cytosolic components p47(phox), p67(phox), p40(phox), and the GTPase Rac (1 or 2). In resting phagocytes, Rac is found in the cytosol as a prenylated protein in the GDP-bound form, associated with the Rho GDP dissociation inhibitor (RhoGDI). In the process of NADPH oxidase activation, Rac is dissociated from RhoGDI and translocates to the membrane, in concert with the other cytosolic components. The mechanism responsible for dissociation of Rac from RhoGDI is poorly understood. We generated Rac(1 or 2) x RhoGDI complexes in vitro from recombinant Rac(1 or 2), prenylated enzymatically, and recombinant RhoGDI, and purified these by anion exchange chromatography. Exposing Rac(1 or 2)(GDP) x RhoGDI complexes to liposomes containing four different anionic phospholipids caused the dissociation of Rac(1 or 2)(GDP) from RhoGDI and its binding to the anionic liposomes. Rac2(GDP) x RhoGDI complexes were more resistant to dissociation, reflecting the lesser positive charge of Rac2. Liposomes consisting of neutral phospholipid did not cause dissociation of Rac(1 or 2) x RhoGDI complexes. Rac1 exchanged to the hydrolysis-resistant GTP analogue, GMPPNP, associated with RhoGDI with lower affinity than Rac1(GDP) and Rac1(GMPPNP) x RhoGDI complexes were more readily dissociated by anionic liposomes. Rac1(GMPPNP) x RhoGDI complexes elicited NADPH oxidase activation in native phagocyte membrane liposomes in the presence of p67(phox), without the need for an anionic amphiphile, as activator. Both Rac1(GDP) x RhoGDI and Rac1(GMPPNP) x RhoGDI complexes elicited amphiphile-independent, p67(phox)-dependent NADPH oxidase activation in phagocyte membrane liposomes enriched in anionic phospholipids but not in membrane liposomes enriched in neutral phospholipids.     

Leishmania donovani lipophosphoglycan blocks NADPH oxidase assembly at the phagosome membrane

Phagocytosis of Leishmania donovani promastigotes is characterized by an inhibition of phagolysosome biogenesis mediated by the surface glycolipid lipophosphoglycan (LPG). However, the consequences of this inhibition on macrophage function remain to be determined. In this study, we investigated the impact of LPG-mediated phagosome remodelling on the assembly and function of the NADPH oxidase complex. Phagocytosis of both wild-type and LPG-defective L. donovani promastigotes triggered the release of similar levels of superoxide. However, wild-type promastigotes, but not LPG-defective mutants, inhibited generation of superoxide at the phagosome. Confocal microscopy imaging revealed that the membrane component gp91(phox) and the Rho-family GTPase Rac1 were present on phagosomes containing either wild-type or LPG-defective promastigotes. In contrast, the NADPH oxidase cytosolic components p47(phox) and p67(phox) were excluded from phagosomes in a LPG-dependent fashion. This inhibition is not the consequence of a general defect in the initiation of the NADPH oxidase activation process because both wild-type and LPG-defective promastigotes induced p47(phox) phosphorylation and the formation of complexes containing p47(phox) and p67(phox). Thus, by remodelling their intracellular habitat, L. donovani promastigotes prevent the assembly of a functional phagosomal NADPH oxidase complex, thereby evading an important host innate defence mechanism.     

Activation of the superoxide-generating NADPH oxidase by chimeric proteins consisting of segments of the cytosolic component p67(phox) and the small GTPase Rac1.

Activation of the superoxide (O2(-))-generating NADPH oxidase of phagocytes is the consequence of the assembly of a membrane-associated flavocytochrome b(559) with the cytosolic proteins p47(phox) and p67(phox) and the small GTPase Rac (1 or 2). We proposed that Rac1 serves as a membrane-targeting molecule for p67(phox). This hypothesis was tested by constructing recombinant chimeric proteins, joining various functional domains of p67(phox) and Rac1, and expressing these in Escherichia coli. Chimeras were assayed for the ability to support O2(-) production by phagocyte membranes in an amphiphile-activated cell-free system in the presence or absence of p47(phox). A chimera consisting of p67(phox) truncated at residue 212 and fused to a full-length Rac1 [p67(phox)(1-212)-Rac1(1-192)] was a potent NADPH oxidase activator. A p67(phox)(1-212)-Rac1(178-192) chimera, to which Rac1 contributed only the C-terminal polybasic domain, was a weaker but consistent activator. Chimeras comprising the full length of Rac1 bound GTP/GDP, like bona fide GTPases. The activity of p67(phox)-Rac1 chimeras was dependent on the presence of the tetratricopeptide repeat and activation domains, in the p67(phox) segment, and on an intact polybasic region, at the C terminus of the Rac1 segment, but not on the insert region of Rac1. Partial activation by chimeras, in the GTP-bound form, was also possible in the absence of p47(phox). Evidence is offered in support of the proposal that the GTP- and GDP-bound forms of chimera p67(phox)(1-212)-Rac1(1-192) have distinct conformations, corresponding to the presence and absence of intrachimeric bonds, respectively.