Bacterial LPS is a pluripotent agonist for PMNs. Although it does not activate the NADPH-dependent oxidase directly, LPS renders PMNs more responsive to other stimuli, a phenomenon known as "priming." Since the mechanism of LPS-dependent priming is incompletely understood, we investigated its effects on assembly and activation of the NADPH oxidase. LPS pretreatment increased superoxide (O2-) generation nearly 10-fold in response to N-formyl methionyl leucyl phenylalanine (fMLP). In a broken-cell O2--generating system, activity was increased in plasma membrane-rich fractions and concomitantly decreased in specific granule-rich fractions from LPS-treated cells. Oxidation-reduction spectroscopy and flow cytometry indicated LPS increased plasma membrane association of flavocytochrome b558. Immunoblots of plasma membrane vesicles from LPS-treated PMNs demonstrated translocation of p47-phox but not of p67-phox or Rac2. However, PMNs treated sequentially with LPS and fMLP showed a three- to sixfold increase (compared with either agent alone) in plasma membrane-associated p47-phox, p67-phox, and Rac2, and translocation paralleled augmented O2- generation by intact PMNs. LPS treatment caused limited phosphorylation of p47-phox, and plasma membrane-enriched fractions from LPS- and/or fMLP-treated cells contained fewer acidic species of p47-phox than did those from cells treated with PMA. Taken together, these studies suggest that redistribution of NADPH oxidase components may underlie LPS priming of the respiratory burst.

Myeloperoxidase (MPO), a lysosomal heme protein found exclusively in neutrophils and monocytes, is necessary for efficient oxygen-dependent microbicidal activity. Acquisition of heme by the heme-free MPO precursor apopro-MPO appears to be a prerequisite for its subsequent proteolytic processing and advancement along the biosynthetic pathway to mature MPO. We present data indicating that calreticulin (CRT), a high capacity calcium-binding protein residing in the lumen of the endoplasmic reticulum of a wide variety of cells, interacts specifically with fully glycosylated apopro-MPO. Biosynthetically radiolabeled CRT (60 kDa) and apopro-MPO (90 kDa) were coprecipitated from PLB 985 cells by monospecific antiserum against CRT when the immunoprecipitations were performed either under nondenaturing conditions or following reversible crosslinking. Nonglycosylated MPO precursors synthesized in the presence of tunicamycin did not interact with CRT. The CRT-apopro-MPO interaction was restricted to an early phase of MPO biosynthesis, and CRT did not interact with the later appearing, heme-containing species of MPO, i.e. pro-MPO or the heavy subunit of mature MPO. These data show that CRT participates in the post-translational processing of MPO, perhaps by maintaining apopro-MPO in a conformation competent to accommodate insertion of the heme group. In this general way, CRT shares certain functional properties with the structurally homologous transmembrane calcium-binding endoplasmic reticulum protein calnexin. Both interact with glycosylated biosynthetic precursors of proteins selectively expressed in specialized cells.

BACKGROUNDCelastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions, and shows benefits in models of neurodegenerative disease, cancer and arthritis, although its mechanism of action is incompletely understood. EXPERIMENTAL APPROACHCelastrol was tested on human NADPH oxidases (NOXs) using a panel of experiments: production of reactive oxygen species and oxygen consumption by NOX enzymes, xanthine oxidase activity, cell toxicity, phagocyte oxidase subunit translocation, and binding to cytosolic subunits of NOX enzymes. The effect of celastrol was compared with diphenyleneiodonium, an established inhibitor of flavoproteins. KEY RESULTSLow concentrations of celastrol completely inhibited NOX1, NOX2, NOX4 and NOX5 within minutes with concentration-response curves exhibiting higher Hill coefficients and lower IC50 values for NOX1 and NOX2 compared with NOX4 and NOX5, suggesting differences in their mode of action. In a cell-free system, celastrol had an IC50 of 1.24 and 8.4 mM for NOX2 and NOX5, respectively. Cytotoxicity, oxidant scavenging, and inhibition of p47 phox translocation could not account for NOX inhibition. Celastrol bound to a recombinant p47 phox and disrupted the binding of the proline rich region of p22 phox to the tandem SH3 domain of p47 phox and NOXO1, the cytosolic subunits of NOX2 and NOX1, respectively. CONCLUSIONS AND IMPLICATIONSThese results demonstrate that celastrol is a potent inhibitor of NOX enzymes in general with increased potency against NOX1 and NOX2. Furthermore, inhibition of NOX1 and NOX2 was mediated via a novel mode of action, namely inhibition of a functional association between cytosolic subunits and the membrane flavocytochrome.

Human neutrophil microbicidal activity is largely mediated by reactive species generated by the oxygen-dependent myeloperoxidase (MPO) system. Peroxidase-negative neutrophils from many patients with hereditary MPO deficiency possess a 90-kDa MPO-related protein. We recently identified a missense mutation, R569W, in the MPO gene of many subjects with MPO deficiency. In these studies we examined the consequences of R569W on MPO biosynthesis and processing, using stably transfected K562 cells expressing normal MPO or the R569W mutation. K562 cells expressing normal MPO mimicked faithfully many features of MPO biosynthesis in myeloid cells. 1) apopro-MPO was synthesized; 2) a functional heme group was inserted into apopro-MPO, and enzymatically active pro-MPO was thereby generated; 3) pro-MPO underwent proteolytic processing to mature MPO; and 4) hemin augmented the processing of pro-MPO. pREP-R569W cells synthesized apopro-MPO, but heme was not inserted. Neither enzymatically active pro-MPO nor mature MPO was synthesized by transfectants expressing mutated cDNA, confirming our hypothesis that the R569W mutation results in a form of apopro-MPO which does not undergo post-translational processing to enzymatically active MPO species. In addition, these data support previous suggestions that heme insertion into apopro-MPO is necessary for its subsequent proteolytic processing into mature MPO subunits.

The heterodimeric flavocytochrome b 558 , comprised of the two integral membrane proteins p22 phox and gp91 phox , mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22 phox required for maturation of gp91 phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22 phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91 phox and two other NADPH oxidase subunits, p47 phox , and p67 phox . The results demonstrate that p22 phox -dependent maturation of gp91 phox carbohydrate, cell surface expression of gp91 phox , and the enzymatic function of flavocytochrome b 558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22 phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22 phox (amino acids 142-195), maturation and cell surface expression of gp91 phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47 phox . Antibody binding studies indicate that the extreme N terminus of p22 phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N-and C-terminal domains of p22 phox extend into the cytoplasm, anchored by two membrane-embedded regions.