SH3-dependent assembly of the phagocyte NADPH oxidase

F ~hagocytic cells contain a complicated enzyme system, termed NADPH oxidase, that is responsible for the production of toxic oxygen species (1). The enzyme transfers electrons from NADPH to 02, forming O2-, which then can dismutate into H202. Subsequently, other oxygen derivatives, such as hydroxyl radical and hypochlorous acid, may be formed. Collectively, these oxygen products are toxic to components in their environment, either phagocytosed or extracellular microorganisms or surrounding tissue (1, 2). Thus, the NADPH oxidase of phagocytic cells is an important participant in both host defense and inflammatory mechanisms. This is well-illustrated by the genetic disorder, chronic granulomatous disease (CGD), in which the NADPH oxidase enzyme is defective. Patients with CGD suffer from frequent and severe infections, as well as noninfectious complications, such as lymphadenopathy and hepatomegaly (3). Components of NADPH Oxidase. The NADPH oxidase enzyme consists of at least four polypeptide components (1, 3, 4). Two of the components, termed gp91-phox and p22phox, form an unusual heterodimeric cytochrome b, cytochrome bsss. The other two components, termed p47-phox and p67-phox, are cytosolic proteins that assemble with cytochrome bsss during activation of the enzyme. The nomenclature used designates the size of each protein by SDS-PAGE and indicates that each protein is a component of the phagocyte oxidase ("gp" denotes glycoprotein, "p" denotes protein). At least one additional protein, the small GTP-binding protein Rac (either Racl or Rac2), is needed for NADPH oxidase activation (5-7). The mechanism by which GTP-bound Rac influences the enzyme is unknown, but is under intense investigation (8, 9). A sixth oxidase-related protein, termed p40-phox, recently has been identified (10, 11), which has sequence similarity to p47-phox and p67-phox and appears to physically associate with p67-phox. Defects in any of the four genes that code for components of the NADPH oxidase enzyme system can cause CGD (3). In most cases, the gene defect results in the absence of the protein product and, thus, the absence of NADPH oxidase activity. Both gp91-phox and p22-phox are usually absent if either gene is defective, suggesting each is unstable without the other. A few cases have been reported where cytochrome bsss is present, but nonfunctional, and these are usually caused by point mutations in the gene for either gp91-phox or p22-phox. These have been particularly informative for gaining insights into structure/ function relationships, as discussed below. Additional information on the molecular genetics of CGD can be found in recent excellent reviews (3, 12). Activation ofNADPH Oxidas~ NADPH oxidase is inactive until the cell is stimulated by phagocytosis or various inflammatory mediators (e.g., chemoattractants, cytokines). Binding of an agonist to its cell-surface receptor triggers various signal transduction pathways (13). While a number of signaling intermediates (e.g., phospholipases, protein kinases) have been implicated as regulators of NADPH oxidase activation, a complete pathway has not been defined. Complexity is increased by the likelihood that multiple pathways are involved (14, 15). However, it is dear that activation of NADPH oxidase culminates in assembly ofp47-phox and p67-phox with cytochrome bsss in the membrane (16-21). With the development of a cell-free system that models the assembly/activation process (3, 22-26) and the cloning of the NADPH oxidase components, it has been possible to begin addressing the structural features of the components involved. Structure of NADPH Oxidase Components. Cloning of the four NADPH oxidase components (27-32) has revealed that the predicted sequence of each protein is unique, with only limited regions of similarity to other known proteins. The gp91-phox subunit of cytochrome bsss has weak homology to NADPH and FAD binding sites found in the ferredoxin reductase family of flavoproteins (33-36). This suggests that cytochrome bsss may be a flavocytochrome, capable of cartying out the entire electron transfer from NADPH to O2. Recent modds (36, 37) postulate that a conformational change in gp91-phox is needed to either enhance NADPH binding and/or fadlitate dectron transfer between NADPH and FAD. Such a conformational change could be induced by assembly of the cytosolic components with the cytochrome. Experimental support for cytochrome bsss as a flavocytochrome has appeared from several laboratories (20, 33-35, 38, 39), although the modal is not universally accepted (40, 41). Several predicted structural features of p47-phox and p67phox also are of interest. The COOH-terminal region ofp47phox contains six to eight clustered putative phosphorylation sites. It has been shown that p47-phax is phosphoryhted during stimulation of intact neutrophils, yidding up to eight phosphospecies ranging from •pI 6.8 to 10 (42-44). Phosphorylation of the dustered sites could dramatically alter the conformation of the protein (43). However, direct evidence that phosphorylation of p47-phox is required for NADPH oxidase activation is lacking (45-48). Both p47-phox and p67phox contain another important structural feature, Src homology 3 (SH3) domains. Each protein contains two of these regions (Fig. 1). The newly described p40-phox also contains one SH3 region (10). SH3 domains were originally described