Mechanism of p47phox-induced increase of reactive oxygen species in peripheral blood mononuclear cells from premature infants on oxygen therapy.

OBJECTIVE: This study aimed to explore the mechanism of p47phox-induced increase of reactive oxygen species (ROS) in peripheral blood mononuclear cells (PBMCs) from premature infants after oxygen therapy, and determine a new target for oxidative stress injury alleviation in clinical setting. METHODS: First, ROS levels as well as p47phox translocation and expression in PBMC samples were evaluated after treatment of premature infants with different concentrations of oxygen. Then, changes of all various parameters were detected after in vitro treatment of PBMCs with diphenyleneiodonium (DPI), apocynin, and high oxygen levels. RESULTS: In premature infants, ROS levels increased significantly after treatment with oxygen, in a concentration-dependent manner (p < 0.05); meanwhile, p47phox translocation and expression were significantly enhanced (p < 0.05) as well. In agreement, PBMCs cultured in vitro showed increased ROS levels after treatment with high oxygen concentrations; p47phox translocation, and expression increased as well (p < 0.05). However, treatment with DPI or apocynin resulted in opposite effects. CONCLUSION: Treatment with oxygen increases p47phox translocationand expression, which in turn induce ROS production. DPI and apocynin have the opposite effects.

Subcellular localization and function of alternatively spliced Noxo1 isoforms.

Nox organizer 1 (Noxo1), a p47(phox) homolog, is produced as four isoforms with unique N-terminal PX domains derived by alternative mRNA splicing. We compared the subcellular distribution of these isoforms or their isolated PX domains produced as GFP fusion proteins, as well as their ability to support Nox1 activity in several transfected models. Noxo1alpha, beta, gamma, and delta show different subcellular localization patterns, determined by their PX domains. In HEK293 cells, Noxo1beta exhibits prominent plasma membrane binding, Noxo1gamma shows plasma membrane and nuclear associations, and Noxo1alpha and delta localize primarily on intracellular vesicles or cytoplasmic aggregates, but not the plasma membrane. Nox1 activity correlates with Noxo1 plasma membrane binding in HEK293 cells, since Noxo1beta supports the highest activity and Noxo1gamma and Noxo1alpha support moderate or low activities, respectively. In COS-7 cells, where Noxo1alpha localizes on the plasma membrane, the activities supported by the three isoforms (alpha, beta, and gamma) do not differ significantly. The PX domains of beta and gamma bind the same phospholipids, including phosphatidic acid. These results indicate that the variant PX domains are unique determinants of Noxo1 localization and Nox1 function. Finally, the overexpressed Noxo1 isoforms do not affect p22(phox) localization, although Nox1 is needed to transport p22(phox) to the plasma membrane.

P67-phox-mediated NADPH oxidase assembly: imaging of cytochrome b558 liposomes by atomic force microscopy.

NADPH oxidase activity depends on the assembly of the cytosolic activating factors, p67-phox, p47-phox, p40-phox, and Rac with cytochrome b(558). The transition from an inactive to an active oxidase complex induces the transfer of electrons from NADPH to oxygen through cytochrome b(558). The assembly of oxidase complex was studied in vitro after reconstitution in a heterologous cell-free assay by using true noncontact mode atomic force microscopy. Cytochrome b(558) was purified from neutrophils and Epstein-Barr virus-immortalized B lymphocytes and incorporated into liposomes. The effect of protein glycosylation on liposome size and oxidase activity was investigated. The liposomes containing the native hemoprotein purified from neutrophils had a diameter of 146 nm, whereas after deglycosylation, the diameter was reduced to 68 nm, although oxidase activity was similar in both cases. Native cytochrome b(558) was used after purification in reconstitution experiments to investigate the topography of NADPH oxidase once it was assembled. For the first time, atomic force microscopy illustrated conformational changes of cytochrome b(558) during the transition from the inactive to the active state of oxidase; height measurements allow the determination of a size of 4 nm for the assembled complex. In the processes that were studied, p67-phox displayed a critical function; it was shown to be involved in both assembly and activation of oxidase complex while p47-phox proceeded as a positive effector and increased the affinity of p67-phox with cytochrome b(558), and p40-phox stabilizes the resting state. The results suggest that although an oligomeric structure of oxidase machinery has not been demonstrated, allosteric regulation mechanisms may be proposed.