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NADPH oxidases: not just for leukocytes anymore!
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NADPH oxidases: not just for leukocytes anymore! Gary M. Bokoch and Ulla G. Knaus Departments of Immunology and Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
In addition to their role in bacterial killing by leukocytes, reactive oxygen species (ROS) have been increasingly recognized as important components of signaling and host defense in other cell types. The formation of ROS in both phagocytic- and non-phagocytic cells involves membrane-localized NADPH oxidases (Noxs). Nox proteins show structural homology to the cytochrome b558 of leukocytes but, until recently, their regulation has been poorly understood. Here, we describe our current understanding of Nox function, and discuss emerging paradigms for regulation of Nox activity by Rac GTPase and/or other cytosolic components. Phagocytic leukocytes play major roles in the innate immune response to pathogens. An important component of this response is the ability of leukocytes to generate reactive oxygen species (ROS) via a membrane-associated NADPH oxidase (Nox) [1,2]. This multi-component enzyme uses electrons derived from intracellular NADPH to generate superoxide anion, which subsequently dismutates to H2O2 and other ROS that are used for host defense against bacterial and fungal pathogens. Genetic defects in Nox result in chronic granulomatous disease, a group of inherited disorders in which innate immunity is impaired [1]. However, the inappropriate or excessive action of Nox has been implicated in the pathogenesis of inflammatory tissue injury and several additional disease states. This highlights the importance of tight regulation of the activity of this system [3]. The phagocyte Nox was the first identified (and remains one of the best-characterized) Rho GTPase-regulated biological systems. It was shown that addition of either of the Rho family members, Rac1 or Rac2, was essential for high-level activity in cell-free Nox systems [4,5], with Rac2 being the predominant active isoform in human neutrophils [6]. Substantial additional evidence has established that Rac2 is an integral and required component of Nox in intact leukocytes, including that rac22/2 murine neutrophils have significantly reduced or absent superoxide production in response to various stimuli [7– 10]. In support of the crucial role of Rac2 in Nox regulation and function, murine neutrophils with a conditional Rac1 deficiency showed no defects in superoxide production in response to the phorbol ester PMA (phorbol myristate acetate) or the chemoattractant fMLF (N-formyl-Met-LeuCorresponding author: Gary M. Bokoch ([email protected]).
Phe) [11]. Rac2 is a member of the Rho family of small GTPases, which control a variety of essential functions in eukaryotes, including those requiring regulated cytoskeletal dynamics. GTPases function as molecular switches that are active when bound to GTP and inactive when bound to GDP. It is only in the active GTP form that GTPases interact with and regulate the function of downstream effector molecules to mediate their cellular effects. Activation of Rho GTPases is thought to occur through the regulated action of guanine nucleotide exchange factors (GEFs) that catalyze the release of bound GDP to enable GTP loading and activation of the GTPase. Conversely, inactivation of the Rho GTPase is mediated via GTPase activating proteins (GAPs), which stimulate their intrinsic ability to hydrolyze GTP to GDP. Components and regulation of the phagocyte NADPH oxidase The Nox system of stimulated phagocytic leukocytes catalyzes the one-electron reduction of oxygen to produce superoxide anion using NADPH as substrate. When the phagocyte is activated through the action of soluble chemoattractants and chemokines, or phagocytic particles, the cytosolic components (Rac2, p47phox and p67phox) of the oxidase are induced to assemble at the level of the membrane-associated flavocytochrome b558 (cyt b) to form the active enzyme. Cyt b has two subunits, gp91phox (Nox2) and p22phox, as well as an NADPH binding site, two heme groups, and bound FAD [1,2]. The formation of this minimal complex enables the flow of electrons via a two-step mechanism, from NADPH to FAD (Step 1), then from FAD to the heme of cyt b, and finally to molecular oxygen (Step 2), whose one-electron reduction leads to the formation of superoxide anion. The cytosolic p47phox regulatory component exists both in a free form and in complexes with the other cytosolic oxidase components, p67phox and p40phox. p67phox appears to serve as the bridging molecule that links p47phox with p40phox [12]. p47phox is phosphorylated on multiple sites through the action of several kinases, resulting in translocation of the p47phox – p67phox (and possible p40phox) complex to the membrane, where it interacts via multiple binding sites with both subunits of the integral cyt b membrane protein to form an active enzyme complex [2,13]. Available biochemical and genetic evidence suggests that phosphorylation of p47phox induces conformational changes that disrupt an inhibitory intramolecular interaction between internal SH3 domains
http://tibs.trends.com 0968-0004/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0968-0004(03)00194-4
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and a C-terminal polyproline domain, thereby exposing the SH3 domains in p47phox for binding to proline-rich regions of other NADPH oxidase components (reviewed in [1,2,13]). This notion is supported by the recently solved X-ray structure of both auto-inhibited and active p47phox by Rittinger and colleagues [14]. They showed that the tandem SH3 domains of p47phox form a ‘super-SH3’ domain in which the ligand-binding surface is formed by a combination of the two individual SH3-ligand-binding domains. The flexible linkage of the two SH3 domains ensures a high local-domain concentration that drives an otherwise low-affinity interaction, while a conserved GlyTrp-Trp motif in each SH3 domain contributes unique interdomain interactions. The super-SH3 domain can then bind to the non-conventional C-terminal polyproline SH3 binding motif. Additional residues outside or at the border of the ligand-binding surface, including Ser303, Ser304 and Ser328, contribute substantially to the auto-inhibited conformation. Substitution of these Ser residues with glutamate residues (to mimic phosphorylation) induces cumulative disruption of the auto-inhibited conformation, enabling p47phox to bind with enhanced affinity to a p22phox peptide containing the p47phox binding site. The ability of multiple Ser phosphorylations within p47phox to destabilize and relieve auto-inhibition by this unique super-SH3 domain provides an important control mechanism to prevent inappropriate activation of Nox activity. An additional level of control of the activation process is achieved through the regulated release of Rac GTPase from cytosolic complexes with Rho GDP dissociation inhibitor (GDI) (see later; Fig. 1), with subsequent activation and translocation to the membrane. Translocation of Rac occurs via an independent mechanism from (but in a coordinated manner with) translocation of the p47phox –p67phox complex [6,15,16]. Price et al. [16] showed that transient expression of constitutively active Rac derivatives was sufficient to induce the membrane translocation of cytosolic oxidase components and assembly of an active oxidase. These data suggest that Rac GTPase must act to coordinate translocation of the p47phox –p67phox complex, consistent with the simultaneous translocation kinetics observed for these three cytosolic factors by Quinn et al. [17]. Addition of activated Rac (added as Rac-GTPgS or constitutively active mutant) is insufficient to induce effective oxidase assembly under standard cell-free assay conditions unless an active conformation of p47phox is induced by the addition of an anionic amphiphile. Therefore, these results suggest that a regulatory action of Rac on p47phox is probable. This might occur through the ability of Rac to modulate the phosphorylation of p47phox through the action of target kinases such as p21-activated kinases (PAKs). p21activated kinases are abundant in neutrophils and their activity is stimulated by chemoattractant [18] and Fc receptors [19]. Moreover, PAK phosphorylates p47phox on several sites of known physiological significance [18,20] and inhibition of PAK activity can impair Nox-mediated ROS generation (K.D. Martyn, U.G. Knaus et al., unpublished). p47phox is now known to be dispensable for Nox activity under cell-free conditions, and appears to serve primarily http://tibs.trends.com
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Fig. 1. Phagocyte NADPH oxidase (Nox) complexes in the context of the intact phagosome. Nox complexes in various stages of assembly are shown on the membrane of the phagosome. Rac2, GDP dissociation inhibitor (GDI), p40phox, p47phox and p67phox components are color coded as indicated; the transmembrane cyt b is depicted as the red gp91phox and the blue p22phox helices. Ingested bacterium (not to scale) is shown in purple.
in the intact cell to: (1) provide a regulatory response element for Nox assembly induced by extracellular activators, and (2) act as an adaptor to facilitate binding of p67phox with cyt b [21,22]. It should be emphasized that this adaptor function of p47phox is crucial for normal Nox activation in response to stimuli in intact leukocytes, as shown by the marked impairment of superoxide formation in chronic granulomatous disease patients lacking p47phox [1,2]. Similar to p47phox, the cytosolic component p40phox might facilitate complex assembly under certain circumstances [23], but is not required for Nox activity in the cellfree system. Both p47phox and p40phox contain conserved ‘phox homology’ or PX domains that preferentially bind PtdIns(3,4)P2 and PtdIns(3)P, respectively [24,25]. It has been suggested that these domains facilitate the translocation of p47phox and p40phox to both plasma membrane and internal membranes to help assemble a functional enzyme complex [24 – 27]. Although this remains a tempting speculation, the physiological significance of these domains in oxidase assembly and regulation remains to be elucidated. In contrast to p47phox and p40phox, p67phox is an essential regulatory component of the enzyme. Deletional and mutational analysis was used to identify a putative activation domain in p67phox (aa 199– 210 and perhaps aa 187– 193) that is required for stimulation of oxidant
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formation by cyt b in cell-free systems [28,29]. This domain appears to interact directly with cyt b to regulate the transfer of electrons from NADPH to cyt-b-bound flavin (step 1) through as-yet undefined mechanisms [30]. Considering its crucial importance to the regulation of electron flow in Nox, little is known about the mechanism of action of p67phox. Rac GTPase function in Nox regulation As indicated earlier, Rac2 GTPase is also a required component of the active phagocyte Nox. Upon cell activation, Rac2 dissociates from a pre-existing cytosolic complex with GDI by an as-yet undetermined mechanism. GDP is exchanged for GTP through the action of membrane-localized GEFs [31] possibly including P-Rex1, a leukocyte-specific PtdIns(3,4,5)P3- and G-protein bg subunit-regulated Rac GEF [32] and Vav1 [16]. Rac – now in its GTP-bound active form – becomes membraneassociated. Rac supports Nox activity only in its GTPbound active form. (There might be an interesting exception to this – it has been proposed that Rac-GDP bound in the complex with RhoGDI might exhibit a conformational state similar to that of the active GTPbound state; see [33].) The GTP-dependence for oxidase activation by Rac indicates that the Rac switch I domain is involved (the switch II domain only undergoes minimal conformational changes upon GTP binding). Consistent with this, point mutations within the switch I or effector domain of Rac were unable to support oxidase activity [34,35]. The finding that Rac binds directly to p67phox (but not to p47phox) via the switch I domain provided important insights into Rac function in the oxidase [36]. It was shown that the tetratricopeptide repeat (TPR) in the N-terminus of p67phox was the site of Rac binding [37], and this was confirmed upon determination of the crystal structure of the Rac– p67(TPR) complex [38]. The structure revealed specific stabilizing interactions between p67phox and Rac, including amino acids A27 and G30 of Rac, and the TPR domain of p67phox, and suggested the availability of the insert domain present in members of the Rho GTPase subfamily for possible additional protein interactions. Peptide-walking experiments indicated that blocking the insert domain of Rac abrogated Nox activation [39]. However, studies using insert-domain deletion mutants of Rac gave conflicting results, suggesting that the insert domain was either essential [40,41] or unnecessary [42,43] for Rac oxidase activity. Interestingly, it has been shown that the insert region of Rac1 is essential for ROS formation and mitogenesis in HeLa cells [44,45]. Mechanisms of Rac action in Nox regulation Currently, there are three major molecular models describing how Rac GTPase regulates Nox activity (see [46] for a more complete discussion of the data for each). Both Lambeth et al. and Pick et al. (Fig. 2a,b) suggest that p67phox, which contains the defined activation domain for cyt b, is the only protein influencing the rate-limiting electron-transfer step (step1) of Nox. Rac is considered to act solely as an adaptor molecule that binds to p67phox through the switch I region and aids in the correct http://tibs.trends.com
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orientation of this active regulator of the system to cyt b. The two models differ in that Lambeth et al. consider that the Rac insert domain might bind to cyt b but – as with p47phox – this interaction serves only to position and facilitate binding of p67phox to cyt b. The model favored by Pick et al. proposes that Rac does not interact directly with cyt b, but rather interacts solely with membrane phospholipids via the prenylated C-terminus to carry p67phox into its correct position with cyt b. The regulatory model proposed by Diebold and Bokoch [40] differs from the previous two models (Fig. 2c). They propose that Rac differentially regulates the two steps involved in electron transfer from NADPH to molecular oxygen. Rac is required to act separately from, and in addition to, p67phox to regulate the Step 1 reaction. However, Rac must subsequently interact with p67phox for Step 2 of the reaction to occur. Fluorescent binding data indicate that Rac interacts directly with cyt b to support the Step 1 reaction. Consistent with this model, we have recently demonstrated that recombinant Rac2 can specifically bind cyt b purified from neutrophils in pull-down assays (B.A. Diebold and G.M. Bokoch, unpublished). It is clear that the three models differ in how they view oxidase regulation by Rac GTPase: adaptor versus active participant. At least part of the discrepancy between these models might be related to the use of prenylated Rac (Model C) versus non-prenylated Rac (Models A and B), or chimeric proteins (Model B) in the cell-free system. The concentration of prenylated Rac required in the cell-free assay is at least 100-fold lower than non-prenylated Rac. Using high concentrations of unprocessed GTPase probably obscures relevant high-affinity protein–protein interactions that normally occur at physiological concentrations of reactants within the plane of the membrane. Identification of non-phagocyte Nox homologs Earlier observations describing stimulus-dependent ROS formation at modest levels in non-phagocytic cells, as well as the presence of the known Nox component p22phox in almost all cell types examined, prompted a search for gp91phox (also known as Nox2) homologs. Within the past three years, multiple mammalian Nox2 homologs have been identified in various tissues and can be classified into three groups. (1) Nox1, Nox3, Nox4, and a short form of Nox5 resemble Nox2 in that they consist of six transmembrane a-helices containing conserved histidines implicated in heme ligation in Nox2, and also exhibit consensus sequences comprising putative flavin- and NAD(P)H-binding sites in the carboxyl termini [47,48]. (2) The longer forms of Nox5 have additional similarities with plant oxidases, such as the Atrbohs gene identified in Arabidopsis [49], with two calcium-binding EF hand motifs in their N-terminal elongation [50]. (3) The third class, consisting of Duox1 and Duox2 (also known as Thox and Lnox), features an N-terminal peroxidase domain that is separated from dual EF hands by an additional transmembrane segment [51 – 53]. Duox homologs have been identified in Caenorhabditis elegans and Drosophila [48].
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Fig. 2. Comparison of proposed models of NADPH oxidase (Nox) regulation by Rac GTPase. Shown are the three major models proposed for phagocyte Nox regulation by Rac GTPase (see main text). Protein components are as indicated. The p67phox activation domain and the Rac insert domain are dark green and turquoise, respectively. The wavy line indicates the Rac isoprenyl group. Reproduced, with permission, from Ref. [46].
Biological functions of Nox proteins The biological functions of ROS generated by Nox/Duox proteins are currently based on many hypotheses and little solid data. Their roles might vary depending on the Nox/Duox isoform and the cell type involved. The tissue distribution of the mammalian Nox homologs appears to be restricted, perhaps reflecting specific biological roles of these enzymes. The expression of Nox3 is limited to fetal tissues, where it might play a role in developmental signaling [54]. Nox4 (also known as Renox) has been found primarily in kidney, uterus and testis, whereas the EFhand-containing Nox5 protein is restricted to B lymphocytes and mantel cells in lymph nodes [54 –56]. Although Duox proteins have been cloned from their predominant tissue source (the thyroid), they have also been detected in lung, placenta and testis [52]. Presumably, the presence of the additional peroxidase domain in the Duox enzymes reflects some specific contributions of the coupled Nox– peroxidase tandem to the physiological roles of these enzymes in these tissues. In this respect, a role for Duox in stabilization of the cuticle in C. elegans through the generation of stabilizing oxidized tyrosine cross-links has been described [52]. Recently, Geiszt et al. postulated a Duox – lactoperoxidase system, present in mucosal surfaces and lung tissues, as a microbicidal host defense mechanism [57]. Nox1, which is structurally most closely related to Nox2 [58,59], is ideally located in the colon and intestinal barrier tissues to come into close contact with both normal and pathogenic bacteria, where it could potentially serve as a component of innate immune defense. It is unclear, however, if mechanisms based upon the specific patternrecognition of pathogens exist that could trigger a Nox1dependent ROS defense system. Kawahara and colleagues [60] observed a Toll-like receptor-mediated pathway triggered by Helicobacter pylori lipopolysaccharide that initiated transcriptional upregulation of oxidant production in Nox1-expressing gastric pit cells, suggesting Nox1 activities might be transcriptionally regulated rather than acutely activated. In the near future, the development of Nox1 knockout mice will certainly shed more light on the potential roles of Nox1 function in innate immunity. http://tibs.trends.com
Conversely, Nox1 had been initially implicated in tumorigenesis by virtue of its growth-stimulating and angiogenic properties [58,61,62]. Overexpression of Nox1 in NIH3T3 and DU435 cells induced the formation of highly vascularized tumors when introduced into nude mice [61,62]. These were abolished by co-expression of the ROS-degrading enzymes catalase and superoxide dismutase. Interestingly, it has been observed that multiple cancer cell lines are capable of constitutively releasing large amounts of ROS, whose enzymatic source(s) are unknown. A role for Nox proteins in these processes is reasonable to suggest, because nearly all of these cancer cell lines express several Nox isoforms, and their oxidant production is sensitive to the flavoenzyme inhibitor diphenylene iodonium (DPI). Consistent with a potential growth-stimulating role of Nox proteins, a recent report suggested that the localized generation of ROS by the calcium-dependent plant Nox, RHD2/AtrbohC, is required for root-cell expansion in plants [63]. Conversely, Geiszt and colleagues used human tumor tissue array analysis to show that Nox1 expression was enhanced in moredifferentiated colon cancer samples [64]. Modification of Nox1 mRNA levels by antisense treatment or induced differentiation did not affect proliferation of colon cancer cell lines. A more general effect of Nox-generated oxidants on intracellular signaling pathways is also probable. Several important intracellular signaling proteins, for example, tyrosine phosphatases and transcription factors, are redox-sensitive, and their inhibition and/or activation in response to changes in oxidation state has the potential to affect cellular function in many ways. Reactive oxygen species generated by Nox proteins might also play a role in the ability of cells to sense and respond to changes in oxygen levels by modulating the oxidation state of the transcription factor HIF-1, thereby inducing hypoxiaregulated gene expression. Regulation of non-phagocyte Nox isoforms Although the regulation of the phagocyte Nox2 is welldocumented, very little is known about the molecular mechanisms involved in Nox isoform regulation. Although the Nox homologs resemble gp91phox (Nox2) in their basic
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overall structure, the formation of physical and/or functional complexes with p22phox for ROS production has not yet been established. Indirect evidence indicates that for Nox4 – the only Nox protein constitutively active in various epithelial cell types – p22phox is essential for ROS generation and association on internal cell membranes (L.M. Frederick, U.G. Knaus et al., unpublished). Takeya and colleagues reported enhanced Nox1-mediated superoxide production by elevating p22phox levels in CHO cells [65]. In general, regulation of Nox homologs, including Nox1, appears not to follow the existing paradigm established for the phagocyte Nox. For example, treatment of Nox1-expressing epithelial cell lines with phorbol esters and/or arachidonic acid – both potent activators of Nox2 in phagocytes – elicited no elevation of intracellular ROS levels or ROS release. Recently, some light has been shed on this issue by the identification of two novel Nox regulatory proteins, p41 [Nox Organizer 1 (NOXO1)] and p51 [Nox activator 1 (NOXA1)], with significant homology to the Nox regulatory components p47phox and p67phox, respectively. The p47phox homolog, p41, and its splice variants in the N-terminal PX domain, were initially identified through sequence homology searches of existing databases by Geiszt and Leto, and subsequently characterized by several groups [65,66,67]. They conserve several p47phox features, such as two SH3 domains and a proline-rich protein interaction motif. Absent, however, is the polybasic domain at the carboxyl terminus of p47phox, which is surrounded by several serine-containing regulatory phosphorylation sites. As discussed earlier, this polybasic domain mediates the basal auto-inhibited conformation of p47phox, which is relieved through the stimulusdependent phosphorylation of the adjacent regulatory serine residues. As the p41 protein lacks most of this autoinhibitory region, it spontaneously binds to p22phox in vitro [65] and forms a stimulus-independent regulatory complex (Fig. 3). Consistent with this, co-expression of Nox1 with p41 and p51 leads to stimulus-independent superoxide formation in HEK293, and Cos7 or CaCo2 cells, respectively [65– 67] (Fig. 3d). Substitution of p47phox for p41 led to stimulus (PMA)-dependent activity in HEK293 cells [66] (Fig. 3c). Conversely, two groups reported Nox1dependent ROS production in the presence of p41, and p51 was enhanced fourfold by PMA stimulation [65,67]. Substitution of p41 or p51 with p47phox or p67phox, respectively, abolished both constitutive and PMA-stimulated oxidant generation in NIH3T3 cells. The Nox component p67phox was partially able to substitute for p51 in Nox1-transfected Cos7 cells (Fig. 3b). Overall, these data suggest that the activity of Nox1 when associated with p41 and p51 might be regulated by mechanisms other than stimulus-dependent phosphorylation, for example, through a change in regulatory protein levels via a transcriptional process and/or protein degradation. Indeed, the expression profile of p41 closely reflects that of Nox1, being expressed mainly in colon, testis, pancreas, small intestine and liver, and both proteins might colocalize on endosomes [65– 67]. One can envision mixed modes of Nox regulation depending upon the cell type and http://tibs.trends.com
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the relative presence of the various cytosolic regulatory proteins (Fig. 3). The p51 Nox regulatory component is more ubiquitously expressed, albeit at fairly low levels, in pancreas, colon, testis, kidney, small intestine, muscle, heart, prostate, thyroid and lung [65– 67]. Comparison with the p67phox sequence reveals that this protein contains most of the p67phox domains known to be of functional importance, except for the N-terminal SH3 domain and the p40phoxbinding site. The presence of the cyt b activation domain and the four N-terminal TPRs that mediate interaction with Rac GTPase suggests that p51 – as with p67phox – can directly interact with Nox homologs and activated Rac GTPase. These structural features strongly suggest that Rac GTPase might constitute an essential element for Nox regulation. In fact, constitutively active Rac1 and Rac2 mutants associate with p51 in vitro [65]. A role for Rac in the regulation of Nox function is also supported by the observation of direct regulatory interactions of Rac with Nox2 [40]. GTPase-mediated regulation of Nox isoforms It has been known for some time that Ras and Rac GTPases regulate oxidant-signaling pathways that are crucial for mitogenesis and oncogenesis. Transient expression of a constitutively activated form of Ras in NIH3T3 cells induced a significant increase in intracellular ROS that could be inhibited by expression of a dominant negative allele of Rac1 [68,69]. Reactive oxygen species production was suppressed by treatment with the flavoprotein inhibitor diphenylene iodonium, suggesting that a Nox protein might be involved. Rac1 also regulates ROS production leading to reperfusion injury during reoxygenation of vascular smooth muscle [70,71]. Recombinant adenoviral expression of a dominant-negative Rac1 suppressed tissue damage in an in vivo model of mouse hepatic ischemia/reperfusion injury. This was also observed in mice deficient for the gp91phox of phagocytic Nox2, suggesting that the Rac mutant inhibited ROS production controlled by a Nox family member other than Nox2 [71]. Thus, it appears that ROS production in nonphagocytic cells involves the regulation of a Nox-like enzyme by Rac GTPase. Concluding remarks Although the observations outlined here strongly indicate a regulatory role for Rac GTPase in Nox function, unfortunately there are little data at this time that directly link Rac to regulation of the activity of the nonphagocytic Nox proteins. Observations from several laboratories, mainly based upon yeast two-hybrid studies and in vitro binding assays using recombinant proteins, point to the potential association of active Rac with Nox carboxyl-terminal residues and with p51, presumably through the TPR motifs. Conversely, a requirement for Rac in ROS generation by any Nox homolog apart from the phagocyte Nox2 has yet to be demonstrated. Clearly, the mechanisms of cellular regulation of Nox proteinmediated ROS formation and the role(s) of Rac GTPase in this process remain an area for further investigation.
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Fig. 3. Potential modes of NADPH oxidase (Nox) isoform regulation. Shown are potential models for interaction and regulation of Nox isoforms in comparison with the phagocyte NADPH oxidase component Nox2. Reactive oxygen species generation via Nox isoforms can be either stimulus-dependent (a,b,c,f) or constitutive (d,e). Models b– f are based on results from experiments using Nox-transfected epithelial cells (HEK293, Cos7 and CHO) and are described in detail in the text. Color coding indicates a particular class of Nox-associated regulatory proteins, and variations in color shades represent homology between components. Question marks represent hypothetical interactions that have not been demonstrated experimentally.
References 1 Babior, B.M. (1999) NADPH oxidase: an update. Blood 93, 1464– 1476 2 Lambeth, J.D. (2000) Regulation of the phagocyte respiratory burst oxidase by protein interactions. Biochem. Mol. Biol. Int. 33, 427– 439 3 Benard, V. et al. (1999) Potential drug targets: small GTPases that regulate leukocyte function. Trends Pharmacol. Sci. 20, 365 – 370 4 Abo, A. et al. (1991) Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature 353, 668– 670 5 Knaus, U.G. et al. (1991) Regulation of phagocytic oxygen radical production by the GTP-binding protein Rac 2. Science 254, 1512– 1515 6 Heyworth, P.G. et al. (1994) Rac translocates independently of the neutrophil NADPH oxidase components p47phox and p67phox. J. Biol. Chem. 269, 30749 – 30752 7 Dorseuil, O. et al. (1992) Inhibition of superoxide in B lymphocytes by Rac antisense oligonucleotides. J. Biol. Chem. 267, 20540 – 20542 8 Roberts, A.W. et al. (1999) Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense. Immunity 10, 183 – 196 9 Kim, C. and Dinauer, M.C. (2001) Rac2 is an essential regulator of neutrophil nicotinamide adenine dinucleotide phosphate oxidase activation in response to specific signaling pathways. J. Immunol. 166, 1223 – 1232 10 Dinauer, M.C. (2003) Regulation of neutrophil function by Rac GTPases. Curr. Opin. Hematol. 10, 8 – 15 11 Glogauer, M. et al. (2003) Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions. J. Immunol. 170, 5652– 5657 12 Lapouge, K. et al. (2002) Architecture of the p40– p47– p67phox complex in the resting state of the NADPH oxidase. A central role for p67phox. J. Biol. Chem. 277, 10121 – 10128 13 DeLeo, F.R. and Quinn, M.T. (1996) Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins. J. Leukoc. Biol. 60, 677– 691 14 Groemping, Y. et al. (2003) Molecular basis of phosphorylation-induced activation of the NADPH oxidase. Cell 113, 343 – 355 15 Dorseuil, O. et al. (1995) Dissociation of Rac translocation from p47phox/ p67phox movements in human neutrophils by tyrosine kinase inhibitors. J. Leukoc. Biol. 58, 108 – 113 16 Price, M.O. et al. (2002) Rac activation induces NADPH oxidase activity in transgenic COSphox cells, and the level of superoxide production is exchange factor-dependent. J. Biol. Chem. 277, 19220 – 19228 17 Quinn, M.T. et al. (1993) Translocation of Rac correlates with NADPH oxidase activation. J. Biol. Chem. 268, 20983 – 20987 http://tibs.trends.com
18 Knaus, U.G. et al. (1995) Regulation of human leukocyte p21-activated kinases through G protein-coupled receptors. Science 269, 221 – 223 19 Jones, S.L. et al. (1998) Two signaling mechanisms for activation of aM b2 avidity in polymorphonuclear neutrophils. J. Biol. Chem. 273, 10556 – 10566 20 Ago, T. et al. (1999) Mechanism for phosphorylation-induced activation of the phagocyte NADPH oxidase protein p47(phox). Triple replacement of serines 303, 304, and 328 with aspartates disrupts the SH3 domain-mediated intramolecular interaction in p47(phox), thereby activating the oxidase. J. Biol. Chem. 274, 33644 – 33653 21 Freeman, J.L. and Lambeth, J.D. (1996) NADPH oxidase activity is independent of p47-phox in vitro. J. Biol. Chem. 271, 22578 – 22582 22 Koshkin, V. et al. (1996) The cytosolic component p47phox is not a sine qua non participant in the activation of NADPH oxidase, but is required for optimal superoxide production. J. Biol. Chem. 271, 30326 – 30329 23 Kuribayashi, F. et al. (2002) The adapter protein p40(phox) as a positive regulator of the superoxide-producing phagocyte oxidase. EMBO J. 21, 6312 – 6320 24 Ellson, C.D. et al. (2001) PtDIns(3) P regulates the neutrophil oxidase complex by binding to the PX domain of p40phox. Nat. Cell Biol. 3, 679– 682 25 Kanai, F. et al. (2001) The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nat. Cell Biol. 3, 675 – 678 26 Zhan, Y. et al. (2002) The p40phox and p47phox PX domains of NADPH oxidase target cell membranes via direct and indirect recruitment by phosphoinositides. J. Biol. Chem. 277, 4512– 4518 27 Simonsen, A. and Stenmark, H. (2001) PX domains: attracted by phosphoinositides. Nat. Cell Biol. 3, E179– E182 28 Han, C-H. et al. (1998) Regulation of the neutrophil respiratory burst oxidase: identification of an activation domain in p67phox. J. Biol. Chem. 273, 16663 – 16668 29 Grizot, S. et al. (2001) The active N terminal region of p67phox. J. Biol. Chem. 276, 21627 – 21631 30 Nishimoto, Y. et al. (1999) The p67phox activation domain regulates electron transfer flow from NADPH to flavin in flavocytochrome b558. J. Biol. Chem. 274, 22999 – 23005 31 Bokoch, G.M. et al. (1994) Guanine nucleotide exchange regulates membrane translocation of Rac/Rho GTP-binding proteins. J. Biol. Chem. 269, 31674 – 31679 32 Welch, H.C. et al. (2002) P-Rex1, a PtdIns(3,4,5)P3- and Gbg-regulated guanine nucleotide exchange factor for Rac. Cell 108, 809 – 821
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33 Di-Poi, N. et al. (2001) Mechanism of NADPH oxidase activation by the Rac/Rho-GDI complex. Biochemistry 40, 10014 – 10022 34 Freeman, J.L.R. et al. (1994) A Ras effector-homologue region on rac regulates protein associations in the neutrophil respiratory burst oxidase complex. Biochemistry 33, 13431 – 13435 35 Xu, S. et al. (1994) Differing structural requirements for GTPaseactivating protein responsiveness and NADPH oxidase activation by Rac. J. Biol. Chem. 269, 23569 – 23574 36 Diekmann, D. et al. (1994) Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity. Science 265, 531 – 533 37 Koga, H. et al. (1999) Tetratricopeptide repeat (TPR) motifs of p67phox participate in interaction with the small GTPase Rac and activation of the phagocyte NADPH oxidase. J. Biol. Chem. 274, 25051 – 25060 38 Lapouge, K. et al. (2000) Structure of the TPR domain of p67phox in complex with Rac-GTP. Mol. Cell 6, 899– 907 39 Joseph, G. and Pick, E. (1995) Peptide walking is a novel method for mapping functional domains in proteins. J. Biol. Chem. 270, 29079 – 29082 40 Diebold, B.A. and Bokoch, G.M. (2001) Molecular basis for Rac2 regulation of the phagocyte NADPH oxidase. Nat. Immunol. 2, 211 – 215 41 Freeman, J.L. et al. (1996) Rac ‘insert region’ is a novel effector region that is implicated in the activation of NADPH oxidase, but not PAK65. J. Biol. Chem. 271, 19794 – 19801 42 Toporik, A. et al. (1998) Mutational analysis of novel effector domains in Rac1 involved in the activation of nicotinamide adenine dinucleotide phosphate (reduced) oxidase. Biochemistry 37, 7147– 7156 43 Alloul, N. et al. (2001) Activation of the superoxide-generating NADPH oxidase by chimeric proteins consisting of segments of the cytosolic component p67phox and the small GTPase Rac1. Biochemistry 40, 14557 – 14566 44 Joneson, T. and Bar-Sagi, D. (1998) A Rac1 effector site controlling mitogenesis through superoxide production. J. Biol. Chem. 273, 17991 – 17994 45 Nimnual, A.S. et al. (2003) Redox-dependent downregulation of Rho by Rac. Nat. Cell Biol. 5, 236– 241 46 Bokoch, G.M. and Diebold, B.A. (2002) Current molecular models for NADPH oxidase regulation by Rac GTPase. Blood 100, 2692 – 2696 47 Lambeth, J.D. et al. (2000) Novel homologs of gp91phox. Trends Biochem. Sci. 25, 459 – 461 48 Lambeth, J.D. (2002) Nox/Duox family of nicotinamide adenine dinucleotide(phosphate) oxidases. Curr. Opin. Hematol. 9, 11 – 17 49 Keller, T. et al. (1998) A plant homolog of the neutrophil NADPH oxidase gp91phox subunit gene encodes a plasma membrane protein with Ca-binding motifs. Plant Cell 10, 255 – 266 50 Banfi, B. et al. (2001) A Ca(2 þ )-activated NADPH oxidase in testis, spleen and lymph nodes. J. Biol. Chem. 276, 37594 – 37601 51 Dupuy, C. et al. (1999) Purification of a novel flavoprotein involved in the thyroid NADPH oxidase. J. Biol. Chem. 274, 37265 – 37269 52 Edens, W.A. et al. (2001) Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with
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homology to the phagocyte oxidase subunit gp91phox. J. Cell Biol. 154, 879 – 891 De Deken, X. (2000) Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family. J. Biol. Chem. 275, 23227 – 23233 Cheng, G. et al. (2001) Homologs of gp91phox: cloning and tissue distribution of Nox3, Nox4, and Nox5. Gene 269, 131– 140 Geiszt, M. et al. (2000) Identification of Renox, an NAD(P)H oxidase in kidney. Proc. Natl. Acad. Sci. U. S. A. 97, 8010 – 8014 Shiose, A. et al. (2001) A novel superoxide-producing NAD(P)H oxidase in kidney. J. Biol. Chem. 276, 1417– 1423 Geiszt, M. et al. (2003) Dual oxidases represent novel hydrogen peroxide sources supporting mucosal surface host defense. FASEB J. doi 10.1096/fj.02-1104fje Suh, Y-S. et al. (1999) Cell transformation by the superoxidegenerating oxidase Mox1. Nature 401, 79 – 82 Banfi, B. et al. (2000) A mammalian Hþchannel generated through alternative splicing of the NADPH oxidase homolog NOH-1. Science 287, 138 – 142 Kawahara, T. et al. (2001) Type I Helicobacter pylori lipopolysaccharide stimulates Toll-like receptor 4 and activates mitogen oxidase 1 in gastric pit cells. Infect. Immun. 69, 4382– 4389 Arnold, R.S. et al. (2001) Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1. Proc. Natl. Acad. Sci. U. S. A. 98, 5550 – 5555 Arbiser, J.L. et al. (2002) Reactive oxygen species generated by Nox1 triggers the angiogenic switch. Proc. Natl. Acad. Sci. U. S. A. 99, 715– 720 Foreman, J. et al. (2003) Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422, 442– 446 Geiszt, M. et al. (2003) NAD(P)H oxidase 1, a product of differentiated colon epithelial cells, can partially replace glycoprotein 91phox in the regulated production of superoxide by phagocytes. J. Immunol. 170, 299– 306 Takeya, R. et al. (2003) Novel human homologyes of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases. J. Biol. Chem. 278, 25234 – 25246 Banfi, B. et al. (2003) Two novel proteins activate superoxide generation by the NADPH oxidase NOX1. J. Biol. Chem. 278, 3510– 3513 Geiszt, M. et al. (2003) Proteins homologous to p47phox and p67phox support superoxide production by NAD(P)H oxidase 1 in colon epithelial cells. J. Biol. Chem. 278, 20006 – 20012 Irani, K. et al. (1997) Mitogenic signaling mediated by oxidants in Rastransformed fibroblasts. Science 275, 1649 – 1652 Sundaresan, M. et al. (1996) Regulation of reactive-oxygen-species generation in fibroblasts by Rac1. Biochem. J. 318, 379 – 382 Kim, K-S. et al. (1998) Protection from reoxygenation injury by inhibition of rac1. J. Clin. Invest. 101, 1821 – 1826 Ozaki, M. et al. (2000) Inhibition of the Rac1 GTPase protects against nonlethal ischemia/reperfusion-induced necrosis and apoptosis in vivo. FASEB J. 14, 418– 429
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NADPH oxidases: not just for leukocytes anymore! Gary M. Bokoch and Ulla G. Knaus Departments of Immunology and Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
In addition to their role in bacterial killing by leukocytes, reactive oxygen species (ROS) have been increasingly recognized as important components of signaling and host defense in other cell types. The formation of ROS in both phagocytic- and non-phagocytic cells involves membrane-localized NADPH oxidases (Noxs). Nox proteins show structural homology to the cytochrome b558 of leukocytes but, until recently, their regulation has been poorly understood. Here, we describe our current understanding of Nox function, and discuss emerging paradigms for regulation of Nox activity by Rac GTPase and/or other cytosolic components. Phagocytic leukocytes play major roles in the innate immune response to pathogens. An important component of this response is the ability of leukocytes to generate reactive oxygen species (ROS) via a membrane-associated NADPH oxidase (Nox) [1,2]. This multi-component enzyme uses electrons derived from intracellular NADPH to generate superoxide anion, which subsequently dismutates to H2O2 and other ROS that are used for host defense against bacterial and fungal pathogens. Genetic defects in Nox result in chronic granulomatous disease, a group of inherited disorders in which innate immunity is impaired [1]. However, the inappropriate or excessive action of Nox has been implicated in the pathogenesis of inflammatory tissue injury and several additional disease states. This highlights the importance of tight regulation of the activity of this system [3]. The phagocyte Nox was the first identified (and remains one of the best-characterized) Rho GTPase-regulated biological systems. It was shown that addition of either of the Rho family members, Rac1 or Rac2, was essential for high-level activity in cell-free Nox systems [4,5], with Rac2 being the predominant active isoform in human neutrophils [6]. Substantial additional evidence has established that Rac2 is an integral and required component of Nox in intact leukocytes, including that rac22/2 murine neutrophils have significantly reduced or absent superoxide production in response to various stimuli [7– 10]. In support of the crucial role of Rac2 in Nox regulation and function, murine neutrophils with a conditional Rac1 deficiency showed no defects in superoxide production in response to the phorbol ester PMA (phorbol myristate acetate) or the chemoattractant fMLF (N-formyl-Met-LeuCorresponding author: Gary M. Bokoch ([email protected]).
Phe) [11]. Rac2 is a member of the Rho family of small GTPases, which control a variety of essential functions in eukaryotes, including those requiring regulated cytoskeletal dynamics. GTPases function as molecular switches that are active when bound to GTP and inactive when bound to GDP. It is only in the active GTP form that GTPases interact with and regulate the function of downstream effector molecules to mediate their cellular effects. Activation of Rho GTPases is thought to occur through the regulated action of guanine nucleotide exchange factors (GEFs) that catalyze the release of bound GDP to enable GTP loading and activation of the GTPase. Conversely, inactivation of the Rho GTPase is mediated via GTPase activating proteins (GAPs), which stimulate their intrinsic ability to hydrolyze GTP to GDP. Components and regulation of the phagocyte NADPH oxidase The Nox system of stimulated phagocytic leukocytes catalyzes the one-electron reduction of oxygen to produce superoxide anion using NADPH as substrate. When the phagocyte is activated through the action of soluble chemoattractants and chemokines, or phagocytic particles, the cytosolic components (Rac2, p47phox and p67phox) of the oxidase are induced to assemble at the level of the membrane-associated flavocytochrome b558 (cyt b) to form the active enzyme. Cyt b has two subunits, gp91phox (Nox2) and p22phox, as well as an NADPH binding site, two heme groups, and bound FAD [1,2]. The formation of this minimal complex enables the flow of electrons via a two-step mechanism, from NADPH to FAD (Step 1), then from FAD to the heme of cyt b, and finally to molecular oxygen (Step 2), whose one-electron reduction leads to the formation of superoxide anion. The cytosolic p47phox regulatory component exists both in a free form and in complexes with the other cytosolic oxidase components, p67phox and p40phox. p67phox appears to serve as the bridging molecule that links p47phox with p40phox [12]. p47phox is phosphorylated on multiple sites through the action of several kinases, resulting in translocation of the p47phox – p67phox (and possible p40phox) complex to the membrane, where it interacts via multiple binding sites with both subunits of the integral cyt b membrane protein to form an active enzyme complex [2,13]. Available biochemical and genetic evidence suggests that phosphorylation of p47phox induces conformational changes that disrupt an inhibitory intramolecular interaction between internal SH3 domains
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and a C-terminal polyproline domain, thereby exposing the SH3 domains in p47phox for binding to proline-rich regions of other NADPH oxidase components (reviewed in [1,2,13]). This notion is supported by the recently solved X-ray structure of both auto-inhibited and active p47phox by Rittinger and colleagues [14]. They showed that the tandem SH3 domains of p47phox form a ‘super-SH3’ domain in which the ligand-binding surface is formed by a combination of the two individual SH3-ligand-binding domains. The flexible linkage of the two SH3 domains ensures a high local-domain concentration that drives an otherwise low-affinity interaction, while a conserved GlyTrp-Trp motif in each SH3 domain contributes unique interdomain interactions. The super-SH3 domain can then bind to the non-conventional C-terminal polyproline SH3 binding motif. Additional residues outside or at the border of the ligand-binding surface, including Ser303, Ser304 and Ser328, contribute substantially to the auto-inhibited conformation. Substitution of these Ser residues with glutamate residues (to mimic phosphorylation) induces cumulative disruption of the auto-inhibited conformation, enabling p47phox to bind with enhanced affinity to a p22phox peptide containing the p47phox binding site. The ability of multiple Ser phosphorylations within p47phox to destabilize and relieve auto-inhibition by this unique super-SH3 domain provides an important control mechanism to prevent inappropriate activation of Nox activity. An additional level of control of the activation process is achieved through the regulated release of Rac GTPase from cytosolic complexes with Rho GDP dissociation inhibitor (GDI) (see later; Fig. 1), with subsequent activation and translocation to the membrane. Translocation of Rac occurs via an independent mechanism from (but in a coordinated manner with) translocation of the p47phox –p67phox complex [6,15,16]. Price et al. [16] showed that transient expression of constitutively active Rac derivatives was sufficient to induce the membrane translocation of cytosolic oxidase components and assembly of an active oxidase. These data suggest that Rac GTPase must act to coordinate translocation of the p47phox –p67phox complex, consistent with the simultaneous translocation kinetics observed for these three cytosolic factors by Quinn et al. [17]. Addition of activated Rac (added as Rac-GTPgS or constitutively active mutant) is insufficient to induce effective oxidase assembly under standard cell-free assay conditions unless an active conformation of p47phox is induced by the addition of an anionic amphiphile. Therefore, these results suggest that a regulatory action of Rac on p47phox is probable. This might occur through the ability of Rac to modulate the phosphorylation of p47phox through the action of target kinases such as p21-activated kinases (PAKs). p21activated kinases are abundant in neutrophils and their activity is stimulated by chemoattractant [18] and Fc receptors [19]. Moreover, PAK phosphorylates p47phox on several sites of known physiological significance [18,20] and inhibition of PAK activity can impair Nox-mediated ROS generation (K.D. Martyn, U.G. Knaus et al., unpublished). p47phox is now known to be dispensable for Nox activity under cell-free conditions, and appears to serve primarily http://tibs.trends.com
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Fig. 1. Phagocyte NADPH oxidase (Nox) complexes in the context of the intact phagosome. Nox complexes in various stages of assembly are shown on the membrane of the phagosome. Rac2, GDP dissociation inhibitor (GDI), p40phox, p47phox and p67phox components are color coded as indicated; the transmembrane cyt b is depicted as the red gp91phox and the blue p22phox helices. Ingested bacterium (not to scale) is shown in purple.
in the intact cell to: (1) provide a regulatory response element for Nox assembly induced by extracellular activators, and (2) act as an adaptor to facilitate binding of p67phox with cyt b [21,22]. It should be emphasized that this adaptor function of p47phox is crucial for normal Nox activation in response to stimuli in intact leukocytes, as shown by the marked impairment of superoxide formation in chronic granulomatous disease patients lacking p47phox [1,2]. Similar to p47phox, the cytosolic component p40phox might facilitate complex assembly under certain circumstances [23], but is not required for Nox activity in the cellfree system. Both p47phox and p40phox contain conserved ‘phox homology’ or PX domains that preferentially bind PtdIns(3,4)P2 and PtdIns(3)P, respectively [24,25]. It has been suggested that these domains facilitate the translocation of p47phox and p40phox to both plasma membrane and internal membranes to help assemble a functional enzyme complex [24 – 27]. Although this remains a tempting speculation, the physiological significance of these domains in oxidase assembly and regulation remains to be elucidated. In contrast to p47phox and p40phox, p67phox is an essential regulatory component of the enzyme. Deletional and mutational analysis was used to identify a putative activation domain in p67phox (aa 199– 210 and perhaps aa 187– 193) that is required for stimulation of oxidant
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formation by cyt b in cell-free systems [28,29]. This domain appears to interact directly with cyt b to regulate the transfer of electrons from NADPH to cyt-b-bound flavin (step 1) through as-yet undefined mechanisms [30]. Considering its crucial importance to the regulation of electron flow in Nox, little is known about the mechanism of action of p67phox. Rac GTPase function in Nox regulation As indicated earlier, Rac2 GTPase is also a required component of the active phagocyte Nox. Upon cell activation, Rac2 dissociates from a pre-existing cytosolic complex with GDI by an as-yet undetermined mechanism. GDP is exchanged for GTP through the action of membrane-localized GEFs [31] possibly including P-Rex1, a leukocyte-specific PtdIns(3,4,5)P3- and G-protein bg subunit-regulated Rac GEF [32] and Vav1 [16]. Rac – now in its GTP-bound active form – becomes membraneassociated. Rac supports Nox activity only in its GTPbound active form. (There might be an interesting exception to this – it has been proposed that Rac-GDP bound in the complex with RhoGDI might exhibit a conformational state similar to that of the active GTPbound state; see [33].) The GTP-dependence for oxidase activation by Rac indicates that the Rac switch I domain is involved (the switch II domain only undergoes minimal conformational changes upon GTP binding). Consistent with this, point mutations within the switch I or effector domain of Rac were unable to support oxidase activity [34,35]. The finding that Rac binds directly to p67phox (but not to p47phox) via the switch I domain provided important insights into Rac function in the oxidase [36]. It was shown that the tetratricopeptide repeat (TPR) in the N-terminus of p67phox was the site of Rac binding [37], and this was confirmed upon determination of the crystal structure of the Rac– p67(TPR) complex [38]. The structure revealed specific stabilizing interactions between p67phox and Rac, including amino acids A27 and G30 of Rac, and the TPR domain of p67phox, and suggested the availability of the insert domain present in members of the Rho GTPase subfamily for possible additional protein interactions. Peptide-walking experiments indicated that blocking the insert domain of Rac abrogated Nox activation [39]. However, studies using insert-domain deletion mutants of Rac gave conflicting results, suggesting that the insert domain was either essential [40,41] or unnecessary [42,43] for Rac oxidase activity. Interestingly, it has been shown that the insert region of Rac1 is essential for ROS formation and mitogenesis in HeLa cells [44,45]. Mechanisms of Rac action in Nox regulation Currently, there are three major molecular models describing how Rac GTPase regulates Nox activity (see [46] for a more complete discussion of the data for each). Both Lambeth et al. and Pick et al. (Fig. 2a,b) suggest that p67phox, which contains the defined activation domain for cyt b, is the only protein influencing the rate-limiting electron-transfer step (step1) of Nox. Rac is considered to act solely as an adaptor molecule that binds to p67phox through the switch I region and aids in the correct http://tibs.trends.com
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orientation of this active regulator of the system to cyt b. The two models differ in that Lambeth et al. consider that the Rac insert domain might bind to cyt b but – as with p47phox – this interaction serves only to position and facilitate binding of p67phox to cyt b. The model favored by Pick et al. proposes that Rac does not interact directly with cyt b, but rather interacts solely with membrane phospholipids via the prenylated C-terminus to carry p67phox into its correct position with cyt b. The regulatory model proposed by Diebold and Bokoch [40] differs from the previous two models (Fig. 2c). They propose that Rac differentially regulates the two steps involved in electron transfer from NADPH to molecular oxygen. Rac is required to act separately from, and in addition to, p67phox to regulate the Step 1 reaction. However, Rac must subsequently interact with p67phox for Step 2 of the reaction to occur. Fluorescent binding data indicate that Rac interacts directly with cyt b to support the Step 1 reaction. Consistent with this model, we have recently demonstrated that recombinant Rac2 can specifically bind cyt b purified from neutrophils in pull-down assays (B.A. Diebold and G.M. Bokoch, unpublished). It is clear that the three models differ in how they view oxidase regulation by Rac GTPase: adaptor versus active participant. At least part of the discrepancy between these models might be related to the use of prenylated Rac (Model C) versus non-prenylated Rac (Models A and B), or chimeric proteins (Model B) in the cell-free system. The concentration of prenylated Rac required in the cell-free assay is at least 100-fold lower than non-prenylated Rac. Using high concentrations of unprocessed GTPase probably obscures relevant high-affinity protein–protein interactions that normally occur at physiological concentrations of reactants within the plane of the membrane. Identification of non-phagocyte Nox homologs Earlier observations describing stimulus-dependent ROS formation at modest levels in non-phagocytic cells, as well as the presence of the known Nox component p22phox in almost all cell types examined, prompted a search for gp91phox (also known as Nox2) homologs. Within the past three years, multiple mammalian Nox2 homologs have been identified in various tissues and can be classified into three groups. (1) Nox1, Nox3, Nox4, and a short form of Nox5 resemble Nox2 in that they consist of six transmembrane a-helices containing conserved histidines implicated in heme ligation in Nox2, and also exhibit consensus sequences comprising putative flavin- and NAD(P)H-binding sites in the carboxyl termini [47,48]. (2) The longer forms of Nox5 have additional similarities with plant oxidases, such as the Atrbohs gene identified in Arabidopsis [49], with two calcium-binding EF hand motifs in their N-terminal elongation [50]. (3) The third class, consisting of Duox1 and Duox2 (also known as Thox and Lnox), features an N-terminal peroxidase domain that is separated from dual EF hands by an additional transmembrane segment [51 – 53]. Duox homologs have been identified in Caenorhabditis elegans and Drosophila [48].
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Fig. 2. Comparison of proposed models of NADPH oxidase (Nox) regulation by Rac GTPase. Shown are the three major models proposed for phagocyte Nox regulation by Rac GTPase (see main text). Protein components are as indicated. The p67phox activation domain and the Rac insert domain are dark green and turquoise, respectively. The wavy line indicates the Rac isoprenyl group. Reproduced, with permission, from Ref. [46].
Biological functions of Nox proteins The biological functions of ROS generated by Nox/Duox proteins are currently based on many hypotheses and little solid data. Their roles might vary depending on the Nox/Duox isoform and the cell type involved. The tissue distribution of the mammalian Nox homologs appears to be restricted, perhaps reflecting specific biological roles of these enzymes. The expression of Nox3 is limited to fetal tissues, where it might play a role in developmental signaling [54]. Nox4 (also known as Renox) has been found primarily in kidney, uterus and testis, whereas the EFhand-containing Nox5 protein is restricted to B lymphocytes and mantel cells in lymph nodes [54 –56]. Although Duox proteins have been cloned from their predominant tissue source (the thyroid), they have also been detected in lung, placenta and testis [52]. Presumably, the presence of the additional peroxidase domain in the Duox enzymes reflects some specific contributions of the coupled Nox– peroxidase tandem to the physiological roles of these enzymes in these tissues. In this respect, a role for Duox in stabilization of the cuticle in C. elegans through the generation of stabilizing oxidized tyrosine cross-links has been described [52]. Recently, Geiszt et al. postulated a Duox – lactoperoxidase system, present in mucosal surfaces and lung tissues, as a microbicidal host defense mechanism [57]. Nox1, which is structurally most closely related to Nox2 [58,59], is ideally located in the colon and intestinal barrier tissues to come into close contact with both normal and pathogenic bacteria, where it could potentially serve as a component of innate immune defense. It is unclear, however, if mechanisms based upon the specific patternrecognition of pathogens exist that could trigger a Nox1dependent ROS defense system. Kawahara and colleagues [60] observed a Toll-like receptor-mediated pathway triggered by Helicobacter pylori lipopolysaccharide that initiated transcriptional upregulation of oxidant production in Nox1-expressing gastric pit cells, suggesting Nox1 activities might be transcriptionally regulated rather than acutely activated. In the near future, the development of Nox1 knockout mice will certainly shed more light on the potential roles of Nox1 function in innate immunity. http://tibs.trends.com
Conversely, Nox1 had been initially implicated in tumorigenesis by virtue of its growth-stimulating and angiogenic properties [58,61,62]. Overexpression of Nox1 in NIH3T3 and DU435 cells induced the formation of highly vascularized tumors when introduced into nude mice [61,62]. These were abolished by co-expression of the ROS-degrading enzymes catalase and superoxide dismutase. Interestingly, it has been observed that multiple cancer cell lines are capable of constitutively releasing large amounts of ROS, whose enzymatic source(s) are unknown. A role for Nox proteins in these processes is reasonable to suggest, because nearly all of these cancer cell lines express several Nox isoforms, and their oxidant production is sensitive to the flavoenzyme inhibitor diphenylene iodonium (DPI). Consistent with a potential growth-stimulating role of Nox proteins, a recent report suggested that the localized generation of ROS by the calcium-dependent plant Nox, RHD2/AtrbohC, is required for root-cell expansion in plants [63]. Conversely, Geiszt and colleagues used human tumor tissue array analysis to show that Nox1 expression was enhanced in moredifferentiated colon cancer samples [64]. Modification of Nox1 mRNA levels by antisense treatment or induced differentiation did not affect proliferation of colon cancer cell lines. A more general effect of Nox-generated oxidants on intracellular signaling pathways is also probable. Several important intracellular signaling proteins, for example, tyrosine phosphatases and transcription factors, are redox-sensitive, and their inhibition and/or activation in response to changes in oxidation state has the potential to affect cellular function in many ways. Reactive oxygen species generated by Nox proteins might also play a role in the ability of cells to sense and respond to changes in oxygen levels by modulating the oxidation state of the transcription factor HIF-1, thereby inducing hypoxiaregulated gene expression. Regulation of non-phagocyte Nox isoforms Although the regulation of the phagocyte Nox2 is welldocumented, very little is known about the molecular mechanisms involved in Nox isoform regulation. Although the Nox homologs resemble gp91phox (Nox2) in their basic
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overall structure, the formation of physical and/or functional complexes with p22phox for ROS production has not yet been established. Indirect evidence indicates that for Nox4 – the only Nox protein constitutively active in various epithelial cell types – p22phox is essential for ROS generation and association on internal cell membranes (L.M. Frederick, U.G. Knaus et al., unpublished). Takeya and colleagues reported enhanced Nox1-mediated superoxide production by elevating p22phox levels in CHO cells [65]. In general, regulation of Nox homologs, including Nox1, appears not to follow the existing paradigm established for the phagocyte Nox. For example, treatment of Nox1-expressing epithelial cell lines with phorbol esters and/or arachidonic acid – both potent activators of Nox2 in phagocytes – elicited no elevation of intracellular ROS levels or ROS release. Recently, some light has been shed on this issue by the identification of two novel Nox regulatory proteins, p41 [Nox Organizer 1 (NOXO1)] and p51 [Nox activator 1 (NOXA1)], with significant homology to the Nox regulatory components p47phox and p67phox, respectively. The p47phox homolog, p41, and its splice variants in the N-terminal PX domain, were initially identified through sequence homology searches of existing databases by Geiszt and Leto, and subsequently characterized by several groups [65,66,67]. They conserve several p47phox features, such as two SH3 domains and a proline-rich protein interaction motif. Absent, however, is the polybasic domain at the carboxyl terminus of p47phox, which is surrounded by several serine-containing regulatory phosphorylation sites. As discussed earlier, this polybasic domain mediates the basal auto-inhibited conformation of p47phox, which is relieved through the stimulusdependent phosphorylation of the adjacent regulatory serine residues. As the p41 protein lacks most of this autoinhibitory region, it spontaneously binds to p22phox in vitro [65] and forms a stimulus-independent regulatory complex (Fig. 3). Consistent with this, co-expression of Nox1 with p41 and p51 leads to stimulus-independent superoxide formation in HEK293, and Cos7 or CaCo2 cells, respectively [65– 67] (Fig. 3d). Substitution of p47phox for p41 led to stimulus (PMA)-dependent activity in HEK293 cells [66] (Fig. 3c). Conversely, two groups reported Nox1dependent ROS production in the presence of p41, and p51 was enhanced fourfold by PMA stimulation [65,67]. Substitution of p41 or p51 with p47phox or p67phox, respectively, abolished both constitutive and PMA-stimulated oxidant generation in NIH3T3 cells. The Nox component p67phox was partially able to substitute for p51 in Nox1-transfected Cos7 cells (Fig. 3b). Overall, these data suggest that the activity of Nox1 when associated with p41 and p51 might be regulated by mechanisms other than stimulus-dependent phosphorylation, for example, through a change in regulatory protein levels via a transcriptional process and/or protein degradation. Indeed, the expression profile of p41 closely reflects that of Nox1, being expressed mainly in colon, testis, pancreas, small intestine and liver, and both proteins might colocalize on endosomes [65– 67]. One can envision mixed modes of Nox regulation depending upon the cell type and http://tibs.trends.com
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the relative presence of the various cytosolic regulatory proteins (Fig. 3). The p51 Nox regulatory component is more ubiquitously expressed, albeit at fairly low levels, in pancreas, colon, testis, kidney, small intestine, muscle, heart, prostate, thyroid and lung [65– 67]. Comparison with the p67phox sequence reveals that this protein contains most of the p67phox domains known to be of functional importance, except for the N-terminal SH3 domain and the p40phoxbinding site. The presence of the cyt b activation domain and the four N-terminal TPRs that mediate interaction with Rac GTPase suggests that p51 – as with p67phox – can directly interact with Nox homologs and activated Rac GTPase. These structural features strongly suggest that Rac GTPase might constitute an essential element for Nox regulation. In fact, constitutively active Rac1 and Rac2 mutants associate with p51 in vitro [65]. A role for Rac in the regulation of Nox function is also supported by the observation of direct regulatory interactions of Rac with Nox2 [40]. GTPase-mediated regulation of Nox isoforms It has been known for some time that Ras and Rac GTPases regulate oxidant-signaling pathways that are crucial for mitogenesis and oncogenesis. Transient expression of a constitutively activated form of Ras in NIH3T3 cells induced a significant increase in intracellular ROS that could be inhibited by expression of a dominant negative allele of Rac1 [68,69]. Reactive oxygen species production was suppressed by treatment with the flavoprotein inhibitor diphenylene iodonium, suggesting that a Nox protein might be involved. Rac1 also regulates ROS production leading to reperfusion injury during reoxygenation of vascular smooth muscle [70,71]. Recombinant adenoviral expression of a dominant-negative Rac1 suppressed tissue damage in an in vivo model of mouse hepatic ischemia/reperfusion injury. This was also observed in mice deficient for the gp91phox of phagocytic Nox2, suggesting that the Rac mutant inhibited ROS production controlled by a Nox family member other than Nox2 [71]. Thus, it appears that ROS production in nonphagocytic cells involves the regulation of a Nox-like enzyme by Rac GTPase. Concluding remarks Although the observations outlined here strongly indicate a regulatory role for Rac GTPase in Nox function, unfortunately there are little data at this time that directly link Rac to regulation of the activity of the nonphagocytic Nox proteins. Observations from several laboratories, mainly based upon yeast two-hybrid studies and in vitro binding assays using recombinant proteins, point to the potential association of active Rac with Nox carboxyl-terminal residues and with p51, presumably through the TPR motifs. Conversely, a requirement for Rac in ROS generation by any Nox homolog apart from the phagocyte Nox2 has yet to be demonstrated. Clearly, the mechanisms of cellular regulation of Nox proteinmediated ROS formation and the role(s) of Rac GTPase in this process remain an area for further investigation.
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Fig. 3. Potential modes of NADPH oxidase (Nox) isoform regulation. Shown are potential models for interaction and regulation of Nox isoforms in comparison with the phagocyte NADPH oxidase component Nox2. Reactive oxygen species generation via Nox isoforms can be either stimulus-dependent (a,b,c,f) or constitutive (d,e). Models b– f are based on results from experiments using Nox-transfected epithelial cells (HEK293, Cos7 and CHO) and are described in detail in the text. Color coding indicates a particular class of Nox-associated regulatory proteins, and variations in color shades represent homology between components. Question marks represent hypothetical interactions that have not been demonstrated experimentally.
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