Membrane Translocation Of P67phox Research Articles

The role of NADPH oxidase, neuronal nitric oxide synthase and poly(ADP ribose) polymerase in oxidative neuronal death induced in cortical cultures by brain-derived neurotrophic factor and neurotrophin-4/5.

Certain neurotrophins promote or induce oxidative neuronal death in cortical cultures. However, the effector mechanisms mediating this phenomenon have not been delineated. In this study, we investigated the possibility that NADPH oxidase and nitric oxide synthase (NOS) function as such effectors. Western blot analysis showed that treatment with brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-4/5 increased the levels of NADPH oxidase subunits. Moreover, neurotrophin treatment resulted in membrane translocation of p67phox, a characteristic feature of NADPH oxidase activation. Administration of the specific NADPH oxidase inhibitor, 4-(2-aminoethyl)benzenesulfonylfluoride (AEBSF), attenuated increases in oxygen free radicals thereby suggesting that NADPH oxidase contributes to the oxidative stress induced by neurotrophins. Furthermore, neuronal death induced by BDNF or NT-4/5 was significantly attenuated by AEBSF. Treatment with BDNF has previously been shown to induce neuronal NOS (nNOS). Our data indicated that inhibitors of nNOS attenuated neuronal death induced by BDNF or NT-4/5, consistent with an active role of nNOS in the mediation of neurotrophin neurotoxicity. As in other models of oxidative cell death, BDNF-induced neuronal death was accompanied by poly(ADP ribose) polymerase (PARP) activation. AEBSF or N-nitro-l-arginine (NNA) reduced BDNF-mediated PARP activation. PARP and poly(ADP ribose) glycohydrolase (PARG) are actively involved in mediating neurotrophin neurotoxicity since inhibitors of PARP and PARG significantly reduced levels of cell death. These results suggest that NADPH oxidase and nNOS contribute to increased oxidative stress, subsequent activation of PARP/PARG, and neuronal death induced by prolonged neurotrophin exposure.

Multiple SH3 domain interactions regulate NADPH oxidase assembly in whole cells.

Src homology 3 (SH3) domains mediate specific protein-protein interactions crucial for signal transduction and protein subcellular localization. Upon phagocyte stimulation, two SH3 domain-containing cytosolic components of the NADPH oxidase, p47phox and p67phox, are recruited to the membrane where they interact with flavocytochrome b558 to form an activated microbicidal oxidase. Deletion analysis of p47phox and p67phox in transfected K562 cells demonstrated multiple SH3-mediated interactions between p47phox and the transmembrane flavocytochrome b558 and also between the cytosolic components themselves. The core region of p47phox (residues 151-284), spanning both SH3 domains, was required for flavocytochrome-dependent translocation and oxidase activity in whole cells. Furthermore, translocation of p67phox occurred through interactions of its N-terminal domain (residues 1-246) with p47phox SH3 domains. Both of these interactions were promoted by PMA activation of cells and were influenced by the presence of other domains in both cytosolic factors. Deletion analysis also revealed a third SH3 domain-mediated interaction involving the C-termini of both cytosolic factors, which also promoted p67phox membrane translocation. These data provide evidence for a central role for p47phox in regulation of oxidase assembly through several SH3 domain interactions.

Inhibitory effect of porcine surfactant on the respiratory burst oxidase in human neutrophils. Attenuation of p47phox and p67phox membrane translocation as the mechanism.

Surfactant has been shown to inhibit the production of reactive oxygen intermediates by various cells including alveolar macrophages and peripheral blood neutrophils. Superoxide O2-. production by the respiratory burst oxidase in isolated plasma membranes prepared from PMA-treated human neutrophils was significantly attenuated by prior treatment with native porcine surfactant. The effect was concentration dependent with half-maximal inhibition seen at approximately 0.050 mg surfactant phospholipid/ml. Kinetic analyses of the membrane-bound enzyme prepared from neutrophils stimulated by PMA in the presence or absence of surfactant demonstrated that surfactant treatment led to a decrease in the maximal velocity of O2-. production when NADPH was used as substrate, but there was no effect on enzyme substrate affinity. Immunoblotting studies demonstrated that surfactant treatment induced a decrease in the association of two oxidase components, p47phox and p67phox, with the isolated plasma membrane. In contrast, surfactant treatment of the cells did not alter the phosphorylation of p47phox. A mixture of phospholipids (phosphatidylcholine and phosphatidylglycerol in a 7:3 ratio) showed similar inhibition of the PMA-induced O2-. generation. Taken together, these data suggest the mechanism of surfactant-induced inhibition of O2-. production by human neutrophils involves attenuation of translocation of cytosolic components of the respiratory burst oxidase to the plasma membrane. The phospholipid components of surfactant appear to play a significant role in this mechanism.