SciCombinator

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Concept: NADPH oxidase

166

NADPH oxidases (Nox) are major enzymatic systems that generate reactive-oxygen species (ROS) in multicellular eukaryotes. In several fungi they have been shown to be involved in sexual differentiation and pathogenicity. However, in contrast to the well characterized mammalian systems, basic information on the composition, recruitment, and localization of fungal Nox complexes and on the molecular mechanisms of their cellular effects are still lacking. Here we give a detailed analysis of components of the Nox complexes in the gray mold fungus Botrytis cinerea. It had previously been shown that the two catalytic transmembrane subunits BcNoxA and B are important for development of sclerotia and for full virulence, with BcNoxA being involved in spreading of lesions and BcNoxB in penetration; BcNoxR functions as a regulator of both subunits. Here we present evidence (using for the first time a functional GFP fusion able to complement the ΔbcnoxA mutant) that BcNoxA localizes mainly to the ER and at the plasma membrane; BcNoxB shows a similar localization pattern, while the regulator BcNoxR is found in vesicles throughout the hyphae and at the hyphal tip. To identify possible interaction partners, which could be involved in the localization or recruitment of the Nox complexes, we functionally characterized the tetraspanin Pls1, a transmembrane protein, which had been suggested to be a NoxB-interacting partner in the saprophyte Podospora anserina. Knock-out experiments and GFP fusions substantiate a link between BcNoxB and BcPls1 because both deletion mutants have overlapping phenotypes (especially a defect in penetration), and the proteins show a similar localization pattern (ER). However, in contrast to the corresponding protein in P. anserina BcPls1 is important for female fertility, but not for ascospore germination.

Concepts: Protein, Cell, Cell membrane, Fungus, Yeast, NADPH oxidase, Transmembrane protein, Botrytis cinerea

163

Insulin resistance is defined as a reduced ability of insulin to stimulate glucose utilization. C57BL/6 mice fed with a high-fat diet (HFD) are a model of insulin resistance. In skeletal muscle, hydrogen peroxide (H2O2) produced by NADPH oxidase 2 (NOX2) is involved in signaling pathways triggered by insulin. We evaluated oxidative status in skeletal muscle fibers from insulin-resistant and control mice by determining H2O2 generation (HyPer probe), reduced-to-oxidized glutathione ratio and NOX2 expression. After eight weeks of HFD, insulin-dependent glucose uptake was impaired in skeletal muscle fibers when compared with control muscle fibers. Insulin-resistant mice showed increased insulin-stimulated H2O2 release and decreased reduced-to-oxidized glutathione ratio (GSH/GSSG). In addition, p47phox and gp91phox (NOX2 subunits) mRNA levels were also high (~3-fold in HFD mice compared to controls), while protein levels were 6.8- and 1.6-fold higher, respectively. Using apocynin (NOX2 inhibitor) during the HFD feeding period, the oxidative intracellular environment was diminished and skeletal muscle insulin-dependent glucose uptake restored. Our results indicate that insulin-resistant mice have increased H2O2 release upon insulin stimulation when compared with control animals, which appears to be mediated by an increase in NOX2 expression.

Concepts: Oxygen, Insulin, Glucose, Redox, Glycogen, Hydrogen peroxide, NADPH oxidase, Skeletal muscle

35

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47(phox)-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47(phox) encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47(phox)-subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47(phox) CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47(phox)-deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.

Concepts: Immune system, DNA, Gene, Genetics, Cell, Nicotinamide adenine dinucleotide, NADPH oxidase, Acute myeloid leukemia

31

Phagocytic immune cells kill pathogens in the phagolysosomal compartment with a cocktail of antimicrobial agents. Chief among them are reactive species produced in the so-called oxidative burst. Here, we show that bacteria exposed to a neutrophil-like cell line experience a rapid and massive oxidation of cytosolic thiols. Using roGFP2-based fusion probes, we could show that this massive breakdown of the thiol redox homeostasis was dependent on phagocytosis, presence of NADPH oxidase and ultimately myeloperoxidase. Interestingly, the redox-mediated fluorescence change in bacteria expressing a glutathione-specific Grx1-roGFP2 fusion protein or an unfused roGFP2 showed highly similar reaction kinetics to the ones observed with roGFP2-Orp1, under all conditions tested. We recently observed such an indiscriminate oxidation of roGFP2-based fusion probes by HOCl with fast kinetics in vitro. In line with these observations, abating HOCl production in immune cells with a myeloperoxidase inhibitor significantly attenuated the oxidation of all three probes in bacteria.

Concepts: Immune system, Antibody, Photosynthesis, Cell, Adenosine triphosphate, Enzyme, Nitrogen, NADPH oxidase

28

Gene repair of CD34(+) hematopoietic stem and progenitor cells (HSPCs) may avoid problems associated with gene therapy, such as vector-related mutagenesis and dysregulated transgene expression. We used CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated 9) to repair a mutation in the CYBB gene of CD34(+) HSPCs from patients with the immunodeficiency disorder X-linked chronic granulomatous disease (X-CGD). Sequence-confirmed repair of >20% of HSPCs from X-CGD patients restored the function of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and superoxide radical production in myeloid cells differentiated from these progenitor cells in vitro. Transplant of gene-repaired X-CGD HSPCs into NOD (nonobese diabetic) SCID (severe combined immunodeficient) γc(-/-) mice resulted in efficient engraftment and production of functional mature human myeloid and lymphoid cells for up to 5 months. Whole-exome sequencing detected no indels outside of the CYBB gene after gene correction. CRISPR-mediated gene editing of HSPCs may be applicable to other CGD mutations and other monogenic disorders of the hematopoietic system.

Concepts: Immune system, DNA, Gene, Genetics, Biotechnology, NADPH oxidase, Hematopoietic stem cell, Chronic granulomatous disease

28

The effect of arsenic (25 and 50μM As for 1 and 5d) was analysed in wild type (WT) and Arabidopsis thaliana (L.) Heynh plants deficient in NADPH oxidase C (AtrbohC). The content of H(2)O(2) and malondialdehyde (MDA) increased with the As concentration, while the opposite effect was found for NO in WT and AtrbohC plants. The As treatment reduced catalase and increased glutathione reductase activities to the same extent in WT and AtrbohC plants, although the induction of all SOD isoforms (mainly CuZn-SODs) was observed in WT plants, the opposite effects being found in AtrbohC plants. Glycolate oxidase (H(2)O(2) producers) considerably increased with the concentration and time of treatment with As in WT and AtrbohC mutants. Arsenic induced the uptake and translocation of P, S, Cu, Zn, and Fe in WT plants, while in AtrbohC plants the opposite trend was noted and the uptake of As became considerably lower than in WT plants. These results suggest that As causes oxidative stress by inducing glycolate oxidase, while NADPH oxidase does not appear to participate in ROS overproduction but could be critical in regulating antioxidant defences as well as the transport and translocation of As and macro/micronutrients.

Concepts: Photosynthesis, Antioxidant, Oxidative stress, Reactive oxygen species, Superoxide dismutase, NADPH oxidase, Arabidopsis, Glutathione

27

During phagocytosis, neutrophils internalize pathogens in a phagosome and produce reactive oxygen species (ROS) by the NADPH oxidase to kill the pathogen. The cytosolic NADPH oxidase subunits p40(phox) , p47(phox) , p67(phox) and Rac2 translocate to the phagosomal membrane to participate in enzyme activation. The kinetics of this recruitment and the underlying signalling pathways are only partially understood. Anionic phospholipids, phosphatidylserine (PS) and phosphoinositides (PPI) provide important attachment site for numerous proteins, including several oxidase subunits.

Concepts: Oxygen, Bacteria, Signal transduction, Mitochondrion, Cell membrane, Reactive oxygen species, NADPH oxidase, Mycobacterium tuberculosis

27

NADPH oxidases transport electrons from cytosolic NADPH through biological membranes to generate reactive oxygen species. NADPH oxidase 4, broadly expressed in humans, is an interesting pharmacological target, since its activity is deregulated in several diseases, including pulmonary fibrosis, diabetic nephropathy, and cardiac hypertrophy. Whereas several candidate NADPH oxidase 4 inhibitors were recently described, most of these compounds are either unspecific or toxic. Here we set out to identify new NADPH oxidase 4 inhibitors from edible plants, in an attempt to decrease the number of hits with toxic side effects. We screened a compound library prepared from edible plants for new bioactives with the ability to inhibit the activity of NADPH oxidase 4. Using both cell-based and cell-free assays, we identified several compounds with significant inhibitory activity towards NADPH oxidase 4. For selected compounds, the activity profile towards NADPH oxidase 2 and NADPH oxidase 5 was established, and controls were carried out to exclude general reactive oxygen species scavengers. A number of promising NADPH oxidase 4 inhibitors from edible plants was identified and characterised. Several new chemical entities are disclosed which act as NADPH oxidase 4 inhibitors, and the efficacies of our best hits, in particular several diarylheptanoids and lignans, are comparable to the best available pharmacological NADPH oxidase 4 inhibitors. These findings will provide valuable tools to study mechanisms of NADPH oxidase inhibition.

Concepts: Oxygen, Mitochondrion, Enzyme inhibitor, NADPH oxidase, Oxidase, Inhibitor, Chemical compound, New chemical entity

27

The direct effect of the four catecholamines (adrenaline, noradrenaline, dopamine and isoproterenol) on superoxide anion radicals (O2-•) was investigated. The reaction between 18-crown-6-ether and potassium superoxide in dimethylsulfoxide was used as a source of O2-•. The reactivity of catecholamines with O2-• was examined using chemiluminescence, reduction of nitroblue tetrazolium and electron paramagnetic resonance spin-trapping techniques. 5,5-Dimethyl-1-pyrroline-N-oxide was included as the spin trap. The results showed that the four catecholamines were effective and efficient in inhibiting chemiluminescence accompanying the potassium superoxide/18-crown-6-ether system in a dose-dependent manner over the range 0.05-2 mm in the following order: adrenaline > noradrenaline > dopamine > isoproterenol, with, IC(50)  = 0.15 ± 0.02 mm 0.21 ± 0.03 mm, 0.27 ± 0.03 mm and 0.50 ± 0.04 mm, respectively. The catecholamines examined also exhibited a strong scavenging effect towards O2-• when evaluated this property by the inhibition of nitroblue tetrazolium reduction (56-73% at 1 m concentration). A very similar capacity of O2-• scavenging was monitored in the 5,5-dimethyl-1-pyrroline-N-oxide spin-trapping assay. The results suggest that catecholamines tested may involve a direct effect on scavenging O2- radicals. Copyright © 2013 John Wiley & Sons, Ltd.

Concepts: Redox, NADPH oxidase, Epinephrine, Superoxide, Chronic granulomatous disease, Dopamine, Norepinephrine, Potassium superoxide

24

Physiological levels of ROS support neurite outgrowth and axonal specification, but the mechanisms by which ROS are able to shape neurons remain unknown. Ca(2+), a broad intracellular second messenger, promotes both Rac1 activation and neurite extension. Ca(2+) release from the endoplasmic reticulum, mediated by both the IP3R1 and ryanodine receptor (RyR) channels, requires physiological ROS levels that are mainly sustained by the NADPH oxidase (NOX) complex. In this work, we explore the contribution of the link between NOX and RyR-mediated Ca(2+) release toward axonal specification of rat hippocampal neurons. Using genetic approaches, we find that NOX activation promotes both axonal development and Rac1 activation through a RyR-mediated mechanism, which in turn activates NOX through Rac1, one of the NOX subunits. Collectively, these data suggest a feedforward mechanism that integrates both NOX activity and RyR-mediated Ca(2+) release to support cellular mechanisms involved in axon development.

Concepts: Neuron, Endoplasmic reticulum, Receptor, Ryanodine receptor, Axon, NADPH oxidase, Feed-forward