NADPH oxidase

NADPH oxidase Bernard M Babior NADPH oxidase is an enzyme that catalyzes the production of superoxide from oxygen and NADPH. It is a complex enzyme consisting of two membrane-bound components and three components in the cytosol, plus rac 1 or rac 2. Activation of the oxidase involves the phosphorylation of one of the cytosolic components. Recent crystallography data indicate that the tail of this cytosolic component lies in a groove between two Src homology 3 domains and, when phosphorylated, the tail leaves the groove and is replaced by the tail of one of the membranebound components. Chronic granulomatous disease is an inherited immune deficiency caused by the absence of one of the components of the oxidase. The most important recent advances in the field have been the crystallographic analysis of the oxidase and the use of antifungal agents in the prophylaxis of chronic granulomatous disease. Addresses The Scripps Research Institute, Department of Molecular and Experimental Medicine, Division of Biochemistry, La Jolla, California 92037, USA e-mail: [email protected]

Current Opinion in Immunology 2004, 16:42–47 This review comes from a themed issue on Innate immunity Edited by Bruce Beutler and Jules Hoffmann 0952-7915/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2003.12.001

Abbreviations IFN interferon NADP nicotinamide adenine dinucleotide phosphate NADPH reduced NADP SH3 Src homology 3

Introduction Reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme that catalyzes the production of superoxide (O2
) from oxygen and NADPH, according to the following reaction [1]: NADPH þ 2O2 ! NADPþ þ Hþ þ 2O2
: This enzyme, which makes very large amounts of superoxide, is found in professional phagocytes (neutrophils [2], eosinophils, monocytes and macrophages [3]) at certain stages of their development. There is also a small group of superoxide-producing enzymes each known as a ‘nox’ [4,5], which are more widespread (found in endothelium, kidney and spleen [5]) and which make Current Opinion in Immunology 2004, 16:42–47

superoxide in small amounts, apparently for purposes of signaling. The function of NADPH oxidase in professional phagocytes, however, is to provide agents that kill organisms that are in contact with the phagocytes. These organisms can be ingested, in the case of neutrophils, monocytes and macrophages, and applied, in the case of eosinophils, which kill metazoans such as worms [6]. The oxidizing agents generated by NADPH oxidase include H2O2, which is produced by the dismutation of superoxide [7]: 2O2
þ 2Hþ ! O2 þ H2 O2 ½8: Other oxidizing agents generated by NADPH oxidase include HOCl, which is generated by the H2O2-mediated oxidation of Cl
, a reaction catalyzed by myeloperoxidase (HOBr [9] or HOSCN [10] in the case of eosinophils, which have a unique peroxidase), 1 O2 [11,12], which is derived by the reaction of HOCl and H2O2, ozone [13,14,15], whose origin is at present mysterious, and OH , which is postulated to arise from the oxidation of reduced metals (Fe2þ or Cuþ) by H2O2 [16]. However, free reduced metals are very scarce in biological systems, although Fe2þ can be released from iron–sulfur proteins by O2
[17–19]. A more probable source of OH is the reaction between ozone and H2O2.

Structure of NADPH oxidase The structure of NADPH oxidase is quite complex, consisting of two membrane-bound elements (gp91PHOX and p22PHOX), three cytosolic components (p67PHOX, p47PHOX and p40PHOX), and a low-molecular-weight G protein (either rac 2 or rac 1) [8]. The racs are kept inactive by binding to a guanine nucleotide dissociation inhibitor, which prevents the exchange of guanine nucleotides from the rac proteins [20]. Activation of NADPH oxidase is associated with, and probably caused by, the migration of the cytosolic components to the cell membrane so that the complete oxidase can be assembled. p91PHOX

The essential element of NADPH oxidase is gp91PHOX, to which are bound the electron carrying components of the oxidase. These include flavine adenine dinucleotide [21], which, according to bioinformatics, associates about halfway down the cytosolic tail of the component, and a pair of hemes that are located in the membraneassociated portion of the component [22]. The two hemes, whose redox potentials are very low, are both hexacoordinate (i.e. all six coordination positions on the heme iron are occupied) [23,24], which questions whether they could participate in electron transfer to oxygen in the www.sciencedirect.com

NADPH oxidase Babior 43

oxidase, particularly as flavins are remarkably efficient at one-electron reductions of oxygen. Nevertheless, it is generally believed that both the hemes and the flavin are involved in electron transfer by NADPH oxidase. p67PHOX

p67PHOX is generally thought of as an ‘accessory protein’, whose exact function is unclear, although it is required for the activity of the oxidase. It contains two Src homology 3 (SH3) domains, one in the middle of the protein and one near the carboxyl terminus. Although originally thought to be an accessory protein with a mysterious function, p67PHOX is now known to be inactivated by NADPH dialdehyde with kinetics that are similar to the kinetics shown by NADPH in the catalytic reaction of NADPH oxidase [25–27]. Furthermore, p67PHOX catalyzes the transfer of electrons from NADPH to electron acceptor dyes (although not to oxygen) [28]. These findings suggest that p67PHOX might be involved in the transfer of electrons directly from NADPH to oxygen to form superoxide. p47PHOX

p47PHOX is the protein that carries the cytosolic proteins to the membrane proteins to assemble the active oxidase. It is not absolutely required, because at sufficiently high concentrations of p67PHOX, superoxide production takes place in the absence of p47PHOX [29]. It is essential in the neutrophil, however, because patients whose neutrophils are deficient in p47PHOX have chronic granulomatous disease, a disease in which neutrophils are unable to produce superoxide. Chronic granulomatous disease also occurs in patients with deficiencies in p67PHOX and deficiencies in the two membrane components of the oxidase: gp91PHOX, discussed above, and p22PHOX, discussed below. Mutant analysis of serine phosphorylation

In the leukocyte, p47PHOX has to be phosphorylated to carry out its function of assembling the oxidase. Considerable work has defined the phosphorylation sites necessary for oxidase activity, using Epstein–Barr virustransformed B lymphocytes from patients with p47PHOXdeficient chronic granulomatous disease. The outcomes are that all the phosphorylation takes place on serine residues, and when phosphorylation is completed with protein kinase C (as determined by activating the cells with phorbol myristate acetate), serines S303 or S304, and S358 or S370 are essential [30,31]. S358 or S370 are phosphorylated first, and, surprisingly, these results further showed that if S358 and S370 were converted to alanines, there was no phosphorylation at all [31]. Oxidase activity in the mutant S303A, S304A could be restored by replacing the serines with glutamates [30]. In a cell-free system, p47PHOX could be activated with Akt, the essential serines in this case being S304 and S328 [32]. The mutant p47PHOX (S303D, S304D, S328D) could activate the oxidase in the cell-free system, similar to Akt-phosphorylated p47PHOX, but the requirement for www.sciencedirect.com

three substituents is not consistent with earlier work [1]. It may be that, for steric reasons, aspartate is not as good a replacement for serine phosphate as glutamate [1]. When p47PHOX is phosphorylated, it binds to p22PHOX [33] an interaction that is probably responsible for activating the oxidase. Other components

p22PHOX is located in the membrane, together with gp91PHOX, and has a tail in the cytosol [34]. Under the appropriate circumstances (specifically, when p47PHOX is phosphorylated) it binds to p47PHOX, as described above, bringing the entire cytosolic oxidase complex to the membrane to assemble the active oxidase. p40PHOX is a protein without a clear function. It forms part of the cytosolic oxidase subunit complex, which consists of p40PHOX, p47PHOX and p67PHOX [35]. It does not seem to be absolutely essential for oxidase activity, because there is no form of chronic granulomatous disease in which it is lacking. Furthermore, its function is not clear; some researchers claim that it activates the oxidase [36] and others claim that it has an inhibitory action [37]. More study is necessary to work out its real function. The nox proteins have been identified by their homology to gp91PHOX. The first nox protein was thought to be carcinogenic, but this is controversial [4,38,39]. Several additional nox proteins have now been discovered [5], and various functions of nox have been described [40]. Others will no doubt be discovered, together with their functions and locations, in the future. X-ray crystallographic studies

The most recent discoveries are the X-ray crystal structures of parts of the NADPH oxidase proteins [41]. The 1–210 lengths of active p67PHOX have been described (Figure 1). The crystal structure of 1–210 of p67PHOX consists of tetratricopeptides. It is not yet clear how it works. Figure 2 is a stereoview of a PX domain from p40PHOX. This domain binds to the phosphatidylinositol phosphate polyphosphodomain [42]. Figure 3a shows the nearby pair of SH3 domains from p47PHOX with the tail of the protein lying in the groove between the two SH3 domains, held there without phosphates. Finally, the groove between the SH3 domains, which now contains the tail of p22PHOX is shown in Figure 3b. The tail of the p47PHOX was displaced from the groove by phosphorylation [33], creating a binding site for p22PHOX that allows the oxidase to be activated.

Chronic granulomatous disease Chronic granulomatous disease is the condition caused by a deficiency of one of the four PHOX subunits (PHOX subunits are the subunits that comprise the NADPH oxidase) [43–45]. Accordingly, cells from patients with chronic granulomatous disease are unable to make Current Opinion in Immunology 2004, 16:42–47

44 Innate immunity

Figure 1

N C

TPR4

TPR1

TPR3

TPR2

N B2 B1 A2 C

A1

B3 A3 A4

B4

superoxide [46], and the disease is characterized by severe infections that are very hard to treat [47,48]. These infections begin very early in life and are frequently fatal [49,50], although experience has shown that the severity of the infections tends to decrease when the patient enters his or her 20s. In the past, the greatest problems were with staphylococci. More recently, however, Burkholdaria cepacea has been a major source of trouble in chronic granulomatous disease patients, causing a pneumonia that is difficult to treat [51]; this is a surprising infection in humans, because it is normally a pathogen of onions. In addition to B. cepacea, chronic granulomatous disease patients are infected by a wide variety of other microorganisms. The real problem, however, is Aspergillus of various species [52,53]. These cause an intractable pneumonia and sometimes septicemia in chronic granulomatous disease patients, and are probably the most frequent cause of death in these patients. The patients sometimes develop chronic inflammation at various sites, leading to strictures affecting the gastric outlet, the ureter and other hollow organs, the most serious of which is chronic inflammation of the lungs, which over a few years can lead to destruction, resulting in death. Treatment of chronic granulomatous disease

Structure of p67PHOX (1–210) as determined by X-ray crystallography. The structure is made up almost entirely of a group of tetratricopeptides. Reproduced from [41] with permission.

Over the past few decades it has become clear that it is necessary to treat chronic granulomatous disease patients prophylactically [47,54]. Initially, they were treated only with antibiotics, specifically a mixture of sulfonamides (Septra1; trimethoprim-sulfamethoxazole, GlaxoSmithKline, Middlesex, UK). This treatment resulted in a decrease in mortality and in days of hospitalization, but was not ideal because there was still significant morbidity

Figure 2

β

β

α

α α

α

α

10

α

10

α

α

β β

α

β β

α II

II

Stereoview of the PX domain of p40PHOX. PX domains, actually named after the PHOX proteins, bind phosphoinositide polyphosphates. The affinities of the PX domains vary from domain to domain depending on how many phosphates are on the inositol. A 3-phosphate on the inositol residue is essential. Reproduced from [42] with permission. Current Opinion in Immunology 2004, 16:42–47

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NADPH oxidase Babior 45

Figure 3

(a)

(b)

Distal loop SH3B

βD

SH3 A-B linker

βC

RT loop

SH3A

βA

βB βA

βE

βE

n-Src loops

P157

SH3A

βB

P155

βC βD

P160

SH3B

RT loop Polybasic region (a) The two SH3 domains of p47PHOX and the C-terminal tail, as determined by X-ray crystallography. The structure shows how the C-terminal portion of the protein lies in the groove between the SH3 domains. (b) The two SH3 domains of p47PHOX in which the C-terminal portion has been phosphorylated. The C-terminal tail no longer resides in the groove, having been replaced by the cytosolic portion of p22PHOX, one of the two oxidase components that are located in the membrane. Figure 3a,b probably represents the manner in which the oxidase is activated. Reproduced from [65] with permission.

and mortality among patients with chronic granulomatous disease. The next modality of treatment used for prophylaxis was IFN-g [55]. Treatment with this agent resulted in an improvement in their clinical status, with a further reduction in mortality and hospital days. Presently, antifungal agents are being added to the prophylactic armamentarium; the effect of these agents remains to be determined, but it seems likely that they will be of further help [56]. When it comes to the treatment of infected patients, antibiotics and antifungal agents are the mainstay. They have to be given in high doses over a long period of time. Occasionally it may be necessary to carry out bone marrow transplantation; for example, in a chronic granulomatous disease patient with severe chronic pulmonary aspergillosis [57,58]. This has been a successful treatment modality in several patients, resulting in cure of the disease, but leaving the patient with the immunological problems of the transplant (e.g. graft-versus-host disease). Genetics of chronic granulomatous disease

Approximately 80% of the patients with chronic granulomatous disease are male. This is due to the distribution of the various PHOX genes on the chromosomes. Specifically, the gene for gp91PHOX is on the X chromosome, and accounts for 60% of cases. There is a wide range of genetic defects in these patients, including missense mutations, nonsense mutations and splicing defects, amongst others [59–61]. The rest of the PHOX proteins are autosomal and explain the remainder of the cases of chronic granulomatous disease — half in males and half in females. At first glance it would appear that the genetic www.sciencedirect.com

defect in the 40% of patients with autosomal chronic granulomatous disease should be distributed evenly between the remaining PHOX genes. However, this is not the situation. Studies have shown that defects in p67PHOX and p22PHOX [62] each account for about 5% of patients with chronic granulomatous disease. The remaining 30% of patients have defects in p47PHOX. What is the explanation for this anomaly? Almost all the patients with p47PHOX deficiency have the same mutation — the elimination of a guanine–thymine dinucleotide in exon 2 [63]. This arises because of the presence of a nearby pseudogene. On very rare occasions, the pseudogene and the p47PHOX gene recombine, causing the loss of the guanine–thymine dinucleotide and the inactivation of p47PHOX, resulting in chronic granulomatous disease due to a functional deficiency of p47PHOX [64].

Conclusions Since the discovery of NADPH oxidase in the 60s, a great deal has been learned about the enzyme. Its subunits have been characterized, its mode of activation has been elucidated, at least in part, and crystallographic studies are yielding interesting and important information about how the enzyme operates. At the same time, studies on patients with chronic granulomatous disease are furnishing important information as to the genetics of the enzyme, and are providing advances in the treatment of this rare but devastating disease. It is to be hoped that future studies will reveal further details about the operation of the oxidase and will produce further advances in the treatment of chronic granulomatous disease, the disease caused by the failure of oxidase activity. Current Opinion in Immunology 2004, 16:42–47

46 Innate immunity

Acknowledgements Supported in part by US Public Health Service Grants AI-24227, AI-28479 and AI-44434. With thanks to Carol Fedoryszyn for invaluable help.

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Activation of the oxidase by a kinase other than protein kinase C. This work brings the oxidase into the domain of phosphoinositide 3-kinase.

48. Johnston RB, Newman SL: Chronic granulomatous disease. Pediatr Clin North Am 1977, 24:365-376.

33. Groemping Y, Lapouge K, Smerdon MJ, Rittinger K: Molecular  basis of phosphorylation-induced activation of the NADPH oxidase. Cell 2003, 113:343-355. A highly original crystallographic study that reveals a great deal about how the oxidase is activated.

49. Babior BM, Woodman RC: Chronic granulomatous disease. Semin Hematol 1990, 27:247-259.

34. Dinauer MC, Pierce EA, Bruns GAP, Curnutte JT, Orkin SH: Human neutrophil Cytochrome b light chain (p22-phox). Gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease. J Clin Invest 1990, 86:1729-1737.

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