RESUMO
Neutrophils provide the first line of defense against microbial invasion in part through production of reactive oxygen species (ROS) which is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generating superoxide anion (O2-). The phagocyte oxidase (phox) has multiple protein components that assemble on the plasma membrane in stimulated neutrophils. We recently described a protein in neutrophils, peroxiredoxin 6 (Prdx6), which has both peroxidase and phospholipase A2 (PLA2) activities and enhances oxidase activity in an SDS-activated, cell-free system. The function of Prdx6 in phox activity is further investigated. In reconstituted phox-competent K562 cells, siRNA-mediated suppression of Prdx6 resulted in decreased NADPH oxidase activity in response to formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). In neutrophils stimulated with PMA, Prdx6 translocated to plasma membrane as demonstrated by Western blot and confocal microscopy. Translocation of Prdx6 in phox competent K562 cells required both p67phox and p47phox. In addition, plasma membrane from PMA-stimulated, oxidase competent K562 cells with siRNA-mediated Prdx6 suppression contained less p47phox and p67phox compared to cells in which Prdx6 was not decreased. Cell-free oxidase assays showed that recombinant Prdx6 did not alter the Km for NADPH, but increased the Vmax for O2- production in a saturable, Prdx6 concentration-dependent manner. Recombinant proteins with mutations in Prdx (C47S) and phospholipase (S32A) activity both enhanced cell-free phox activity to the same extent as wild type protein. Prdx6 supports retention of the active oxidase complex in stimulated plasma membrane, and results with mutant proteins imply that Prdx6 serves an additional biochemical or structural role in supporting optimal NADPH oxidase activity.
Assuntos
Membrana Celular/metabolismo , NADPH Oxidases/metabolismo , Ativação de Neutrófilo , Neutrófilos/metabolismo , Peroxirredoxina VI/metabolismo , Humanos , Células K562 , NADPH Oxidases/química , NADPH Oxidases/genética , Neutrófilos/citologia , Peroxirredoxina VI/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , TransgenesRESUMO
Peroxiredoxin 6-phospholipase A(2) (Prdx6-PLA(2) ) is a bi-functional enzyme with peroxi-redoxin (Prdx) and phospholipase A(2) (PLA(2) ) activities. To investigate its impact on phagocyte NADPH oxidase (phox) activity in a neutrophil model, the protein was knocked down in PLB-985 cells using stable expression of a small hairpin RNA (shRNA) and phox activity was monitored after cell differentiation. The knockdown cells had reduced oxidase activity in response to stimulation with the formylated peptide fMLF, but the response to the phorbol ester PMA was unchanged. Reintroduction of shRNA-resistant Prdx6-PLA(2) into the knockdown cells by stable transfection with a Prdx6-PLA(2) expression plasmid restored the fMLF response, as did reintroduction of Prdx6-PLA(2) mutated in the Prdx active site; reintroduction of PLA(2) active site mutants, however, failed to restore the response. Thus, the PLA(2) activity of Prdx6-PLA(2) in intact cells mediates its ability to enhance phox activity in response to fMLF. In combination with previous publications by other groups, our work indicates that various PLA(2) isoforms can enhance oxidase activity but they are differentially important in different cell types and in the response to different agonists.
Assuntos
Leucemia Mieloide/enzimologia , NADPH Oxidases/metabolismo , Peroxirredoxina VI/metabolismo , Fosfolipases A2/metabolismo , Linhagem Celular Tumoral , Humanos , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Tretinoína/farmacologiaRESUMO
Chemoattractant priming and activation of PMNs results in changes in cytosolic Ca2+ concentration, tyrosine kinase activity, and gene expression. We hypothesize that the initial signaling for the activation of a 105kDa protein (Rel-1) requires Ca2+-dependent tyrosine phosphorylation. A rapid and time-dependent tyrosine phosphorylation of Rel-1 occurred following formyl-Met-Leu-Phe (fMLP) stimulation of human PMNs at concentrations that primed or activated the NADPH oxidase (10(-9) to 10(-6)M), becoming maximal after 30s. Pretreatment with pertussis toxin (Ptx) or tyrosine kinase inhibitors abrogated this phosphorylation and inhibited fMLP activation of the oxidase. The fMLP concentrations employed also caused a rapid increase in cytosolic Ca2+ but chelation negated the effects, including the cytosolic Ca2+ flux, oxidase activation, and the tyrosine phosphorylation of Rel-1. Conversely, chelation of extracellular Ca2+ decreased the fMLP-mediated Ca2+ flux, had no affect on the oxidase, and augmented tyrosine phosphorylation of Rel-1. Phosphorylation of Rel-1 was inhibited when PMNs were preincubated with a p38 MAP kinase (MAPK) inhibitor (SB203580). In addition, fMLP elicited rapid activation of p38 MAPK which was abrogated by chelation of cytosolic Ca2+. Thus, fMLP concentrations that prime or activate the oxidase cause a rapid Ca2+-dependent tyrosine phosphorylation of Rel-1 involving p38 MAPK activation.
Assuntos
Cálcio/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Fatores de Transcrição/metabolismo , Tirosina/metabolismo , Citosol , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Humanos , Proteínas I-kappa B/metabolismo , Imidazóis/farmacologia , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peso Molecular , N-Formilmetionina Leucil-Fenilalanina/antagonistas & inibidores , NADPH Oxidases/efeitos dos fármacos , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Toxina Pertussis/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/farmacologia , Piridinas/farmacologia , Fatores de Transcrição/genética , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
INTRODUCTION: Newborn infants are prone to develop life-threatening pyogenic infections. Alterations in the function of neonatal phagocytes, including the activity of the neutrophil NADPH oxidase, have been suggested as one cause of increased susceptibility to such infections. METHODS: In the present study, comprehensive analysis of NADPH oxidase enzyme system was performed in cord blood neutrophils from vaginally and cesarean section (CS) delivered, healthy, full-term infants. RESULTS: Superoxide anion (O(2) (-)) production by intact neutrophils from cord blood in response to soluble stimuli was equal to or increased compared to levels generated by cells from adult controls. In the sodium dodecyl sulfate (SDS) cell-free system, cytosol and plasma membrane from cord blood neutrophils generated O(2) (-) at comparable rates to subcellular fractions from healthy adults. However, mixing experiments demonstrated higher O(2) (-) generation with combination of cytosol from adult controls and membrane from cord blood neutrophils and lower O(2) (-) production with combination of cytosol from cord blood neutrophils and membrane from adult controls. Kinetic parameters for cord blood specimens were no different from those obtained for fractions from adult controls. Quantitative analysis of cytosolic components showed moderately reduced amount of p40-phox, p47-phox, and p67-phox in neutrophils from cord blood. In contrast, cytochrome b(558) content of plasma membrane of cord blood neutrophils was approximately 2-fold higher compared to adult controls. CONCLUSION: The normal to increased respiratory burst of intact cord blood neutrophils is the result of alterations to oxidase components: increased content of cytochrome b(558) in the plasma membrane and decreased levels of cytosolic components p47-phox, p67-phox, and p40-phox.