Abstract
In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40(phox) remains less well defined than those of p47(phox) and p67(phox). We investigated the biological role of p40(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40(phox) reduces both the maximal rate and total amount of ROS produced without altering the K(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40(phox) chimeras, and deletion constructs, we found that the p40(phox) PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67(phox) requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40(phox), however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40(phox) can increase oxidase activity approximately 2.5-fold above that of wild-type p40(phox) but maintain the requirement for PX and SH3 domain function. We present a model where p40(phox) translocates p67(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.
Highlights
The NADPH oxidase is a multisubunit enzyme made up of a membrane-spanning heterodimer of gp91phox and p22phox, which forms the catalytic core, as well as four cytosolic components, p47phox, p67phox, p40phox, and the small G-protein Rac2 [2, 6]
PLB-985 cells, which can differentiate into mature neutrophils [32] that express all of the endogenous phox proteins, including p40phox, and display robust NADPH oxidase activity
In contrast to p40phoxϪ/Ϫ neutrophils, which demonstrated an ϳ60% decrease in p67phox levels, the levels of p67phox double mutants were hyperactive (Fig. 7C), suggesting that open- in the PLB-985-derived knockdown neutrophils were ing p40phox does not overcome the requirement for the PX, Src homology 3 (SH3), unchanged, demonstrating a direct effect of endogenous and PB1 domain functions
Summary
Materials—Streptolysin-O, ATP, luminol, diisopropyl fluorophosphate, creatine kinase, creatine phosphate, and dimethyl formamide (DMF) were purchased from Sigma. PLB-985 Chemiluminescence Assays—For chemiluminescence assays following S. aureus infection, 2 ϫ 105 p40KD or LucKD differentiated PLB-985 cells were resuspended in DMPBS containing 100 g/ml bovine serum albumin and 0.15 mM luminol (PBS/B/L) and incubated in 75% human serum. Permeabilized PMN cores were prepared by incubating 1 ϫ 106 freshly isolated cells in 300 l of RB-EBL buffer containing 1000 units of reduced streptolysin-O for 5 min at 0 °C. PMN cytosol was prepared by nitrogen cavitation of 2 ϫ 108 cells/ml in RB buffer containing 0.3 mM EGTA and 5.6 mM diisopropyl fluorophosphate by pressurization to 400 p.s.i. for 20 min at 0 °C prior to release. Reconstitution Reactions and Chemiluminescence Assay— Reconstitution reactions were performed in 100 l of RB-EBL buffer containing 8 ϫ 104 freshly prepared neutrophil cores, 30 g of cytosol, 2 mM ATP, 200 M GTP, 200 M GTP␥S, 10 mM creatine phosphate, and 25 g/ml creatine kinase.
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