Fused p47 and p67 Truncations Efficiently Reconstitute NADPH Oxidase with Higher Activity and Stability Than the Individual Components

Abstract

Activation of the neutrophil NADPH oxidase occurs via assembly of the cytosolic regulatory proteins p47phox , p67 phox , and Rac with the membrane-associated flavocytochrome b 558 . Following cell-free activation, enzymatic activity is highly labile (Tamura, M., Takeshita, M., Curnutte, J. T., Uhlinger, D. J., and Lambeth, J. D. (1992) J. Biol. Chem. 267, 7529 -7538). To try to stabilize the activity and investigate the nature of the complex, fusion proteins between p47N-(1-286) and p67N-(1-210) were constructed. In a cell-free system, a fusion protein, p67N-p47N, had an 8-fold higher efficiency and produced a higher activity than the individual proteins, and also resulted in an 8-fold improved efficiency for Rac and a lowered K m for NADPH. O 2 . generating activity was remarkably stabilized by using p67N-p47N. The cytosolic proteins fused in the opposite orientation, p47N-p67N, showed similar activity and stability as individual proteins, but with a 4-fold improved efficiency compared with the individual cytosolic factors. In the system efficiency for Rac and affinity for NADPH were also higher than those with the nonfused components. Interestingly, the p67N-p47N showed nearly full activation in the absence of an anionic amphifile in a cell-free system containing cytochrome b 558 relipidated with phosphatidylinositol-or phosphatidylserine-enriched phospholipid mixtures. From the results we consider multiple roles of anionic amphifiles in a cell-free activation, which could be substituted by our system. The fact that a fusion produces a more stable complex indicates that interactions among components determine the longevity of the complex. Based on the findings we propose a model for the topology among p47N, p67N, and cytochrome b 558 in the active complex.The superoxide-generating phagocyte NADPH oxidase (Phox/Nox-2) functions in host defense against microbial infection (1, 2). The enzyme is dormant in resting cells and becomes active upon cell stimulation. The activation is thought to occur via assembly of cytosolic components, p47 phox (p47), 1 p67 phox (p67), and Rac with the membrane-associated flavocytochrome b 558 (cyt. b 558 ), which consists of p22 phox (p22) and gp91 phox (gp91) although the structure of the complex has remained unclear (3-6). Other two factors p40 phox and rap1A are also assumed to be involved in the enzyme regulation although they are not essential for the activation.The activated NADPH oxidase is highly labile, complicating investigations of the subunit structure and preventing isolation of the active enzyme complex (7). In a previous study (8) using a cell-free system consisting of cytosol and plasma membrane (PM) we showed that the stability is dramatically improved by chemical cross-linking, and suggested that the deactivation is caused by dissociation of proteins from the complex. Crosslinking was useful in stabilizing the oxidase, but it was difficult to isolate the active complex because the cross-linked complex resisted solubilization (8). We have also attempted cross-linkin...

Dual Role of Rac in the Assembly of NADPH Oxidase, Tethering to the Membrane and Activation of p67

“…The advantages of such an approach are: 1) to enable the study of low affinity interactions between two oxidase components by increasing the probability of domain-domain encounters, consequent to fusion; and 2) to build models of complex assembly, by generating artificial proteins composed of selected domains involved in protein-protein interactions, derived from two or more components. So far, this methodological approach has been applied to the design of p47 phox -p67 phox chimeras (45) and p67 phox -Rac1 chimeras (27,28,33).…”

A Prenylated p47 -p67 -Rac1 Chimera Is a Quintessential NADPH Oxidase Activator

“…Fusion of cytosolic components into a single molecule supersedes the need for first stage interactions and should allow a direct approach to second and third stage processes. The first such chimera was a p47 phox -p67 phox fusion protein (23), and this was followed by the independent description by two groups of p67 phox -Rac1 chimeras (20,24), which could be prenylated at the C terminus (16). We recently described the design and bacterial expression of a tripartite chimera consisting of selected segments of p47 phox , p67 phox , and Rac1, which we called "trimera" (25).…”

Identification of an actin‐binding site in p47phox an organizer protein of NADPH oxidase

“…phox [11]. After expression, the cells were lysed in the presence of 0.2 mM diisopropyl fluorophosphate and purified with glutathione-Sepharose beads.…”

“…The activation mixture contained p47 phox or p47 phox (S303/304/328E) (1.3 lM), p67 phox (1-210) (6 lM), racQ61L (6 lM), and purified cytochrome b 558 [11] (0.1 lM) in 50 ll of buffer A containing 4mM MgCl 2 , 10 lM FAD and 10 lM GTP. The mixture was incubated with 200 lM SDS for 5 min at 25°C to activate NADPH oxidase.…”

“…The reactive oxygen species produced in these cells may have other function than host defense, such as redox regulation or cell proliferation [4,7]. p47 phox is assumed to play as an adapter for organization of the oxidase complex, since it reduces the required concentrations of other subunits [8,9] and stabilizes the oxidase [10,11]. It has been established that p47…”