Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella.

Activation of the superoxide forming respiratory burst oxidase of human neutrophils, crucial in host defence, requires the cytosolic proteins p47phox and p67phox which translocate to the plasma membrane upon cell stimulation and activate flavocytochrome b558, the redox centre of this enzyme system. We have previously demonstrated the presence of proteins (67 and 47kDa) in hemocytes of the insect Galleria mellonella homologous to proteins of the superoxide-forming NADPH oxidase complex of neutrophils. The work presented here illustrates for the first time translocation of homologous hemocyte proteins, 67 and 47kDa from the cytosol to the plasma membrane upon phorbol 12-myristate 13 acetate (PMA) activation. In hemocytes, gliotoxin (GT), the fungal secondary metabolite significantly suppressed PMA-induced superoxide generation in a concentration dependent manner and reduced translocation to basel nonstimulated levels. Primarily these results correlate translocation of hemocyte 47 and 67kDa proteins with PMA induced oxidase activity. Collectively results presented here, demonstrate further cellular and functional similarities between phagocytes of insects and mammals and further justify the use of insects in place of mammals for modelling the innate immune response to microbial pathogens.

Protein kinase Cdelta regulates p67phox phosphorylation in human monocytes.

Phosphorylation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components p67phox and p47phox accompanies the assembly and activation of this enzyme complex. We have previously reported that activation of human monocytes with opsonized zymosan (ZOP), a potent stimulator of NADPH oxidase activity, results in the phosphorylation of p67phox and p47phox. In this study, we investigated the regulation of p67phox phosphorylation. Although protein kinase C (PKC)alpha has previously been shown to regulate NADPH oxidase activity, we found that inhibition of PKCalpha had no effect on p67phox phosphorylation. Our studies demonstrate that pretreatment of monocytes with antisense oligodeoxyribonucleotides specific for PKCdelta or rottlerin, a selective inhibitor for PKCdelta, inhibited the phosphorylation of p67phox in monocytes, and Go6976, a specific inhibitor for conventional PKCs, PKCalpha and PKCbeta, had no such inhibitory effect. Additional studies indicate that ZOP stimulation of monocytes induces PKCdelta and p67phox to form a complex. We also demonstrate that lysates from activated monocytes as well as PKCdelta immunoprecipitates from activated monocytes can phosphorylate p67phox in vitro and that pretreatment of monocytes with rottlerin blocked the phosphorylation in each case. We further show that recombinant PKCdelta can phosphorylate p67phox in vitro. Finally, we show that PKCdelta-deficient monocytes produce significantly less superoxide anion in response to ZOP stimulation, thus emphasizing the functional significance of the PKCdelta regulation of p67phox phosphorylation. Taken together, this is the first report to describe the requirement of PKCdelta in regulating the phosphorylation of p67phox and the related NADPH oxidase activity in primary human monocytes.

NADPH oxidase immunoreactivity in the mouse brain.

Superoxide production via NADPH oxidase has been shown to play a role in neurotoxicity, ischemic stroke, and possibly Parkinson's and Alzheimer's diseases. In addition, NADPH oxidase-dependent production of superoxide may be necessary for normal brain functions, including neuronal differentiation and neuronal plasticity. To improve our understanding of NADPH oxidase in the brain, we studied the localization of the various protein components of NADPH oxidase in the central nervous system of the adult mouse using immunohistochemistry. We detected staining for the cytoplasmic NADPH proteins, p40(phox), p47(phox), and p67(phox), as well as the membrane-associated NADPH oxidase proteins, p22(phox) and gp91(phox) in neurons throughout the mouse brain. Staining of each of the NADPH oxidase proteins was observed in neurons in all regions of the neuraxis, with particularly prominent localizations in the hippocampus, cortex, amygdala, striatum, and thalamus. The expression of NADPH oxidase proteins in neurons suggests the possibility that enzymatic production of superoxide by a NADPH oxidase may play a role in both normal neuronal function as well as neurodegeneration in the brain.

Superoxide production in Galleria mellonella hemocytes: identification of proteins homologous to the NADPH oxidase complex of human neutrophils.

The insect immune response has a number of structural and functional similarities to the innate immune response of mammals. The objective of the work presented here was to establish the mechanism by which insect hemocytes produce superoxide and to ascertain whether the proteins involved in superoxide production are similar to those involved in the NADPH oxidase-induced superoxide production in human neutrophils. Hemocytes of the greater wax moth (Galleria mellonella) were shown to be capable of phagocytosing bacterial and fungal cells. The kinetics of phagocytosis and microbial killing were similar in the insect hemocytes and human neutrophils. Superoxide production and microbial killing by both cell types were inhibited in the presence of the NADPH oxidase inhibitor diphenyleneiodonium chloride. Immunoblotting of G. mellonella hemocytes with antibodies raised against human neutrophil phox proteins revealed the presence of proteins homologous to gp91phox, p67phox, p47phox, and the GTP-binding protein rac 2. A protein equivalent to p40phox was not detected in insect hemocytes. Immunofluorescence analysis localized insect 47-kDa and 67-kDa proteins throughout the cytosol and in the perinuclear region. Hemocyte 67-kDa and 47-kDa proteins were immunoprecipitated and analyzed by matrix-assisted laser desorption ionization--time of flight analysis. The results revealed that the hemocyte 67-kDa and 47-kDa proteins contained peptides matching those of p67phox and p47phox of human neutrophils. The results presented here indicate that insect hemocytes phagocytose and kill bacterial and fungal cells by a mechanism similar to the mechanism used by human neutrophils via the production of superoxide. We identified proteins homologous to a number of proteins essential for superoxide production in human neutrophils and demonstrated that significant regions of the 67-kDa and 47-kDa insect proteins are identical to regions of the p67phox and p47phox proteins of neutrophils.

PX domain takes shape.

In recent years, a number of protein domains have been identified that bind phosphoinositides and direct proteins to membrane targets. A recent addition to this group is the Phox homology or PX domain, a 120-amino acid domain conserved from yeast to humans, which is present in proteins involved in cell signaling, protein sorting, vesicle fusion, and the assembly of components of the superoxide generating system of neutrophils. These domains have varying affinities for phosphatidylinositol-3-phosphate (PI(3)P), and PI(3,4) and (4,5) bisphosphates, which couple the PI kinase and phosphatase signaling networks to the assembly of proteins at membrane surfaces. These PX domains also contain a PXXP motif, allowing them to bind to proteins containing Src homology 3 (SH3) domains.

Cytosolic phospholipase A2 (cPLA2) regulation of human monocyte NADPH oxidase activity. cPLA2 affects translocation but not phosphorylation of p67(phox) and p47(phox).

The NADPH oxidase of human monocytes is activated upon exposure to opsonized zymosan and a variety of other stimuli to catalyze the formation of superoxide anion. Assembly of the NADPH oxidase complex is believed to be a highly regulated process, and molecular mechanisms responsible for this regulation have yet to be fully elucidated. We have previously reported that cytosolic phospholipase A(2) (cPLA(2)) expression and activity are essential for superoxide anion production in activated human monocytes. In this study, we investigated the mechanisms involved in cPLA(2) regulation of NADPH oxidase activation by evaluating the effects of cPLA(2) on translocation and phosphorylation of p67(phox) and p47(phox). We report that translocation and phosphorylation of p67(phox), as well as p47(phox), occur upon activation of human monocytes and that decreased cPLA(2) protein expression, mediated by antisense oligodeoxyribonucleotides (AS-ODN) specific for cPLA(2) mRNA, blocked the stimulation-induced translocation of p47(phox) and p67(phox) from the cytosol to the membrane fraction. Inhibition of translocation of both p47(phox) and p67(phox) by cPLA(2) AS-ODN was above 85%. Arachidonic acid (AA), a product of cPLA(2) enzymatic activity, completely restored translocation of both of these oxidase components in the AS-ODN-treated, cPLA(2)-deficient human monocytes. These results represent the first report that cPLA(2) activity or AA is required for p67(phox) and p47(phox) translocation in human monocytes. Although cPLA(2) was required for translocation of p47(phox) and p67(phox), it did not influence phosphorylation of these components. These results suggest that one mechanism of cPLA(2) regulation of NADPH oxidase activity is to control the arachidonate-sensitive assembly of the complete oxidase complex through modulating the translocation of both p47(phox) and p67(phox). These studies provide insight into the mechanisms by which activation signals are transduced to allow the induction of superoxide anion production in human monocytes.

Protein kinase C delta is required for p47phox phosphorylation and translocation in activated human monocytes.

Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKCdelta, -epsilon, -theta;, and -eta) and conventional PKC (including PKCalpha and -beta), inhibited both phosphorylation and translocation of p47phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKCepsilon, did not affect the phosphorylation or translocation of p47phox, suggesting that PKCdelta, -theta;, or -eta is required. Furthermore, rottlerin (at doses that inhibit PKCdelta activity) inhibited the phosphorylation and translocation of p47phox. Rottlerin also inhibited O2 production at similar doses. In addition to pharmacological inhibitors, PKCdelta-specific antisense oligodeoxyribonucleotides were used. PKCdelta antisense oligodeoxyribonucleotides inhibited the phosphorylation and translocation of p47phox in activated human monocytes. We also show, using the recombinant p47phox-GST fusion protein, that p47phox can serve as a substrate for PKCdelta in vitro. Furthermore, lysate-derived PKCdelta from activated monocytes phosphorylated p47phox in a rottlerin-sensitive manner. Together, these data suggest that PKCdelta plays a pivotal role in stimulating monocyte NADPH oxidase activity through its regulation of the phosphorylation and translocation of p47phox.