Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.6.3.1 (NADPH oxidase)
 11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chloride anion is essential for myeloperoxidase (MPO) to produce hypochlorous acid (HOCl) in polymorphonuclear neutrophils (PMNs). To define whether chloride availability to PMNs affects their HOCl production and microbicidal capacity, we examined how extracellular chloride concentration affects killing of Pseudomonas aeruginosa (PsA) by normal neutrophils. PMN-mediated bacterial killing was strongly dependent on extracellular chloride concentration. Neutrophils in a chloride-deficient medium killed PsA poorly. However, as the chloride level was raised, the killing efficiency increased in a dose-dependent manner. By using specific inhibitors to selectively block NADPH oxidase, MPO, and cystic fibrosis transmembrane conductance regulator (CFTR) functions, neutrophil-mediated killing of PsA could be attributed to three distinct mechanisms: CFTR-dependent and oxidant-dependent; chloride-dependent but not CFTR- and oxidant-dependent; and independent of any of the tested factors. Therefore, chloride anion is involved in oxidant- and nonoxidant-mediated bacterial killing. We previously reported that neutrophils from CF patients are defective in chlorination of ingested bacteria, suggesting that the chloride channel defect might impair the MPO-hydrogen peroxide-chloride microbicidal function. Here, we compared the competence of killing PsA by neutrophils from normal donors and CF patients. The data demonstrate that the killing rate by CF neutrophils was significantly lower than that by normal neutrophils. CF neutrophils in a chloride-deficient environment had only one-third of the bactericidal capacity of normal neutrophils in a physiological chloride environment. These results suggest that CFTR-dependent chloride anion transport contributes significantly to killing PsA by normal neutrophils and when defective as in CF, may compromise the ability to clear PsA.
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PMID:The role of chloride anion and CFTR in killing of Pseudomonas aeruginosa by normal and CF neutrophils. 1835 29

Cystic fibrosis (CF), is the most common life-shortening autosomal recessive disorder in Caucasians. It is caused by mutations in a single gene on the long arm of chromosome 7 that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CF is characterized by abnormal Na+ and Cl- ion transport in several tissues, including the lungs, pancreas, gastrointestinal tract, liver, sweat glands, and male reproductive system. Progressive pulmonary disease is the dominant clinical feature of CF and accounts for morbidity and mortality. The inflammation characterized by an overabundance of activated neutrophils and macrophages on the respiratory epithelial surface is associated to a high production of reactive oxygen species (ROS) which contribute to the pathogenesis of cystic fibrosis. ROS could have different origins but the role of the NADPH oxidase system is essential. The "NADPH oxidases" (NOX/DUOX) family is an enzymatic complex formed by cytosolic and membrane subunits. Until now several homologues of the phagocytic NADPH oxidase have been identified in different tissues and it has been shown that the lungs preferentially expressed DUOX1-2. Thus, DUOX1-2 could be implicated in the anti-infectious defense system. The role of DUOX enzymes as a source of ROS in cystic fibrosis is examined as they could contribute to a better understanding of molecular mechanisms in CF. Moreover they could be a potential target for a new therapeutic approach.
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PMID:Potential role of the "NADPH oxidases" (NOX/DUOX) family in cystic fibrosis. 1909 60

To understand the role of reactive oxygen species in mechanosensory control of lung development a new approach to interfere with protein-protein interactions by means of a short interacting peptide was developed. This technology was used in the developing rodent lung to examine the role of NADPH oxidase (NOX), casein kinase 2 (CK2), and the cystic fibrosis transmembrane conductance regulator (CFTR) in stretch-induced differentiation. Interactions between these molecules was targeted in an in utero system with recombinant adeno-associated virus (rAAV) containing inserted DNA sequences that express a control peptide or small interfering peptides (siPs) specific for subunit interaction or phosphorylation predicted to be necessary for multimeric enzyme formation. In all cases only siPs with sequences necessary for a predicted normal function were found to interfere with assembly of the multimeric enzyme. A noninterfering control siP to nonessential regions or reporter genes alone had no effect. Physiologically, it was shown that siPs that interfered with the NOX-CFTR-CK2 complex that we call an "interactonome" affected markers of stretch-induced lung organogenesis including Wnt/beta-catenin signaling.
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PMID:Small interfering peptide (siP) for in vivo examination of the developing lung interactonome. 1916 Dec 44

Burkholderia cenocepacia causes chronic lung infections in patients suffering from cystic fibrosis and chronic granulomatous disease. We have previously shown that B. cenocepacia survives intracellularly in macrophages within a membrane vacuole (BcCV) that delays acidification. Here, we report that after macrophage infection with live B. cenocepacia there is a approximately 6 h delay in the association of NADPH oxidase with BcCVs, while heat-inactivated bacteria are normally trafficked into NADPH oxidase-positive vacuoles. BcCVs in macrophages treated with a functional inhibitor of the cystic fibrosis transmembrane conductance regulator exhibited a further delay in the assembly of the NADPH oxidase complex at the BcCV membrane, but the inhibitor did not affect NADPH oxidase complex assembly onto vacuoles containing heat-inactivated B. cenocepacia or live Escherichia coli. Macrophages produced less superoxide following B. cenocepacia infection as compared to heat-inactivated B. cenocepacia and E. coli controls. Reduced superoxide production was associated with delayed deposition of cerium perhydroxide precipitates around BcCVs of macrophages infected with live B. cenocepacia, as visualized by transmission electron microscopy. Together, our results demonstrate that intracellular B. cenocepacia resides in macrophage vacuoles displaying an altered recruitment of the NADPH oxidase complex at the phagosomal membrane. This phenomenon may contribute to preventing the efficient clearance of this opportunistic pathogen from the infected airways of susceptible patients.
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PMID:Delayed association of the NADPH oxidase complex with macrophage vacuoles containing the opportunistic pathogen Burkholderia cenocepacia. 1933 3

Previous studies have identified abnormalities in the oxidative responses of the neutrophil in cystic fibrosis (CF), but it is unclear whether such changes relate to loss of membrane cystic fibrosis transmembrane conductance regulator (CFTR) or to the inflammatory environment present in this disease. The aim of the present study was to determine whether neutrophils from CF patients demonstrate an intrinsic abnormality of the respiratory burst. The respiratory burst activity of neutrophils isolated from stable DeltaF508 homozygote CF patients and matched healthy controls was quantified by both chemiluminscence and cytochrome C reduction. Expression of NADPH oxidase components and CFTR was determined by Western blotting and RT-PCR. The oxidative output from neutrophils from CF in response to receptor-linked and particulate stimuli did not differ from that of controls. Expression of NADPH oxidase components was identical in CF and non-CF neutrophils. While low levels of CFTR mRNA could be identified in the normal human neutrophil, we were unable to detect CFTR protein in human neutrophil lysates or immunoprecipitates. CFTR has no role in controlling neutrophil oxidative activity; previously reported differences in neutrophil function between CF and non-CF subjects most likely relate to the inflammatory milieu from which the cells were isolated.
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PMID:Cystic fibrosis neutrophils have normal intrinsic reactive oxygen species generation. 1984 Sep 64

Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on the generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that 95-99% of lysosomal-associated membrane protein 1 (LAMP-1)-positive mature phagosomes were CFTR positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed enhanced green fluorescent protein (EGFP) alone, EGFP-wt-CFTR and EGFP-DF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and colocalize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-DF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, the CFTR corrector compound VRT-325 facilitated the recruitment of DF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function.
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PMID:Cystic fibrosis transmembrane conductance regulator recruitment to phagosomes in neutrophils. 2354 24

Myeloperoxidase is a major neutrophil antimicrobial protein, but its role in immunity is often overlooked because individuals deficient in this enzyme are usually in good health. Within neutrophil phagosomes, myeloperoxidase uses superoxide generated by the NADPH oxidase to oxidize chloride to the potent bactericidal oxidant hypochlorous acid (HOCl). In this study, using phagocytosis assays and LC-MS analyses, we monitored GSH oxidation in Pseudomonas aeruginosa to gauge their exposure to HOCl inside phagosomes. Doses of reagent HOCl that killed most of the bacteria oxidized half the cells' GSH, producing mainly glutathione disulfide (GSSG) and other low-molecular-weight disulfides. Glutathione sulfonamide (GSA), a HOCl-specific product, was also formed. When neutrophils phagocytosed P. aeruginosa, half of the bacterial GSH was lost. Bacterial GSA production indicated that HOCl had reacted with the bacterial cells, oxidized their GSH, and was sufficient to be solely responsible for bacterial killing. Inhibition of NADPH oxidase and myeloperoxidase lowered GSA formation in the bacterial cells, but the bacteria were still killed, presumably by compensatory nonoxidative mechanisms. Of note, bacterial GSA formation in neutrophils from patients with cystic fibrosis (CF) was normal during early phagocytosis, but it was diminished at later time points, which was mirrored by a small decrease in bacterial killing. In conclusion, myeloperoxidase generates sufficient HOCl within neutrophil phagosomes to kill ingested bacteria. The unusual kinetics of phagosomal HOCl production in CF neutrophils confirm a role for the cystic fibrosis transmembrane conductance regulator in maintaining HOCl production in neutrophil phagosomes.
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PMID:Exposure of Pseudomonas aeruginosa to bactericidal hypochlorous acid during neutrophil phagocytosis is compromised in cystic fibrosis. 3134 Oct 24