Role of the phospholipid binding sites, PX of p47phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2.

In phagocytes, superoxide anion (O2-), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b558 (cyt b558) and four cytosolic components: p40phox, p47phox, p67phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b558 to form the active enzyme. To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47phox. We also used a fused chimera consisting of p47phox(aa 1-286), p67phox(aa 1-212) and Rac1Q61L, as well as mutated versions in the p47phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b558. They also have an impact on O2.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.

Splice variants of protein disulfide isomerase - identification, distribution and functional characterization in the rat.

BACKGROUND: Protein Disulfide Isomerase (PDI) enzyme is an emerging therapeutic target in oncology and hematology. Although PDI reductase activity has been studied with isolated fragments of the protein, natural structural variations affecting reductase activity have not been addressed. METHODS: In this study, we discovered four coding splice variants of the Pdi pre-mRNA in rats. In vitro Michaelis constants and apparent maximum steady-state rate constants after purification and distribution in different rat tissues were determined. RESULTS: The consensus sequence was found to be the most expressed splice variant while the second most expressed variant represents 15 to 35% of total Pdi mRNA. The third variant shows a quasi-null expression profile and the fourth was not quantifiable. The consensus sequence splice variant and the second splice variant are widely expressed (transcription level) in the liver and even more present in males. Measurements of the reductase activity of recombinant PDI indicate that the consensus sequence and third splice variant are fully active variants. The second most expressed variant, differing by a lack of signal peptide, was found active but less than the consensus sequence. GENERAL SIGNIFICANCE: Our work emphasizes the importance of taking splice variants into account when studying PDI-like proteins to understand the full biological functionalities of PDI.

Impacts of vesicular environment on Nox2 activity measurements in vitro.

BACKGROUND: The production of superoxide anions (O2•-) by the phagocyte NADPH oxidase complex has a crucial role in the destruction of pathogens in innate immunity. Majority of in vitro studies on the functioning of NADPH oxidase indirectly follows the enzymatic reaction by the superoxide reduction of cytochrome c (cyt c). Only few reports mention the alternative approach consisting in measuring the NADPH consumption rate. When using membrane vesicles of human neutrophils, the enzyme specific activity is generally found twice higher by monitoring the NADPH oxidation than by measuring the cyt c reduction. Up to now, the literature provides only little explanations about such discrepancy despite the critical importance to quantify the exact enzyme activity. METHODS: We deciphered the reasons of this disparity in studying the role of key parameters, including. cyt c and arachidonic acid concentrations, in conjunction with an ionophore, a detergent and using Clark electrode to measure the O2 consumption rates. RESULTS: Our results show that the O2•- low permeability of the vesicle membrane as well as secondary reactions (O2•- and H2O2 disproportionations) are strong clues to shed light on this inconsistency. CONCLUSION AND GENERAL SIGNIFICANCE: These results altogether indicate that the cyt c reduction method underestimates the accurate Nox2 activity.

Biomarkers Potency to Monitor Non-target Fauna Poisoning by Anticoagulant Rodenticides.

The widespread use of pesticides to control agricultural pests is a hot topic on the public scene of environmental health. Selective pest control for minimum environmental impact is a major goal of the environmental toxicology field, notably to avoid unintended poisoning in different organisms. Anticoagulant rodenticides cause abnormal blood coagulation process; they have been widely used to control rodents, allowing inadvertent primary and secondary exposure in domestic animals and non-target predatory wildlife species through direct ingestion of rodenticide-containing bait or by consumption of poisoned prey. To report toxic effect, the most common approach is the measurement of liver or plasma residues of anticoagulant rodenticides in dead or intoxicated animals showing clinical symptoms. However, one major challenge is that literature currently lacks a hepatic or plasma concentration threshold value for the differentiation of exposure from toxicity. Regarding the variation in pharmacology properties of anticoagulant rodenticides inter- and intra-species, the dose-response relationship must be defined for each species to prejudge the relative risk of poisoning. Beyond that, biomarkers are a key solution widely used for ecological risk assessment of contaminants. Since anticoagulant rodenticides (AR) have toxic effects at the biochemical level, biomarkers can serve as indicators of toxic exposure. In this sense, toxicological knowledge of anticoagulant rodenticides within organisms is an important tool for defining sensitive, specific, and suitable biomarkers. In this review, we provide an overview of the toxicodynamic and toxicokinetic parameters of anticoagulant rodenticides in different animal species. We examine different types of biomarkers used to characterize and differentiate the exposure and toxic effects of anticoagulant rodenticide, showing the strengths and weaknesses of the assays. Finally, we describe possible new biomarkers and highlight their capabilities.

A Close-Up View of the Impact of Arachidonic Acid on the Phagocyte NADPH Oxidase.

The NADPH oxidase NOX2 complex consists of assembled cytosolic and redox membrane proteins. In mammalian cells, natural arachidonic acid (cis-AA), released by activated phospholipase-A2, plays an important role in the activation of the NADPH oxidase, but the mechanism of action of cis-AA is still a matter of debate. In cell-free systems, cis-AA is commonly used for activation although its structural effects are still unclear. Undoubtedly cis-AA participates in the synergistic multi-partner assembly that can be hardly studied at the molecular level in vivo due to cellular complexity. The capacity of this anionic amphiphilic fatty acid to activate the oxidase is mainly explained by its ability to disrupt intramolecular bonds, mimicking phosphorylation events in cell signaling and therefore allowing protein-protein interactions. Interestingly the geometric isomerism of the fatty acid and its purity are crucial for optimal superoxide production in cell-free assays. Indeed, optimal NADPH oxidase assembly was hampered by the substitution of the cis form by the trans forms of AA isomers (Souabni et al., BBA-Biomembranes 1818:2314-2324, 2012). Structural analysis of the changes induced by these two compounds, by circular dichroism and by biochemical methods, revealed differences in the interaction between subunits. We describe how the specific geometry of AA plays an important role in the activation of the NOX2 complex.

Phagocyte NADPH oxidase, oxidative stress and lipids: Anti- or pro ageing?

The role of NADPH oxidase in ageing is debated because of the dual roles of free radicals, toxic though necessary. In this paper we summarize some results about two aspects linked to the regulation of the activity of phagocyte NADPH oxidase (Nox2), encountered frequently in elderly people: inflammation and hypercholesterolemia. In the presence of a high amount of reactive oxygen species (ROS) created by itself or by any other source, the enzyme activity is mostly lowered. Oxidation of the membrane and/or of one of the cytosolic partners could be responsible for this loss of activity. However using a cell free system, we had also shown that a low amount of ROS could activate this enzyme. Similarly, cholesterol has a similar dual role, either activating or inhibiting. In in vitro cell free system with neutrophil membranes from healthy donors, the addition, as well as the removal of cholesterol, diminishes the Nox2 activity. The activity of Nox2 is lowered in neutrophils of untreated hypercholesterolemic patients. Finally oxysterols (25-hydroxy-cholesterol or 5α, 6α - epoxy-cholesterol) do not induce effects different from that of non-oxidized cholesterol. These findings are in agreement with the Janus role of NADPH oxidase, the main source of non-mitochondrial ROS.

Conversion of NOX2 into a constitutive enzyme in vitro and in living cells, after its binding with a chimera of the regulatory subunits.

During the phagocytosis of pathogens by phagocyte cells, the NADPH oxidase complex is activated to produce superoxide anion, a precursor of microbial oxidants. The activated NADPH oxidase complex from phagocytes consists in two transmembrane proteins (Nox2 and p22phox) and four cytosolic proteins (p40phox, p47phox, p67phox and Rac1-2). In the resting state of the cells, these proteins are dispersed in the cytosol, the membrane of granules and the plasma membrane. In order to synchronize the assembly of the cytosolic subunits on the membrane components of the oxidase, a fusion of the cytosolic proteins p47phox, p67phox and Rac1 named trimera was constructed. The trimera investigated in this paper is composed of the p47phox segment 1-286, the p67phox segment 1-212 and the mutated Rac1(Q61L). We demonstrate that the complex trimera-cyt b558 is functionally comparable to the one containing the separated subunits. Each of the subunits p47phox, p67phox and Rac1Q61L has kept its own activating property. The trimera is produced in an activated conformation as seen by circular dichroism. However, the presence of amphiphile is still necessary in a cell-free system to trigger superoxide anion production. The COS7gp91-p22 cells expressing the trimera produce continuously superoxide anion at high rate. This constitutive activity in cells can be of particular interest for understanding the NADPH oxidase functioning independently of signaling pathways.

Exploring the arachidonic acid-induced structural changes in phagocyte NADPH oxidase p47(phox) and p67(phox) via thiol accessibility and SRCD spectroscopy.

The NADPH oxidase is the sole enzymatic complex that produces, in a controlled way, superoxide anions. In phagocytes, it is constituted by the assembly of four cytosolic (p67(phox) , p47(phox) , p40(phox) and Rac) and two membrane (p22(phox) and Nox2) proteins. In response to pro-inflammatory mediators, the NADPH oxidase is activated. In cells, arachidonic acid (cis-AA), released by activated phospholipase A2, also plays a role in activation of the NADPH oxidase complex, but the mechanism of action of cis-AA is still a matter for debate. In cell-free systems, cis-AA is commonly used for activation. We have shown previously that trans-AA isomers were unable to activate the NADPH oxidase complex. Here, we aim to evaluate the structural changes in p47(phox) and p67(phox) induced by AA. The structural impact of both AA isomers on both cytosolic proteins was investigated by the accessibility of the thiol group and by circular dichroism in the far-UV for global folds. cis-AA induces secondary structure changes of p47(phox) and p67(phox) , while the trans isomer does not, suggesting that the changes observed are of importance for the activation process of these proteins. While five of the nine thiol groups in p67(phox) and all of them in p47(phox) have low access to the solvent when proteins are alone in solution, all of them become fully accessible when proteins are together. In conclusion, the secondary structures of p47(phox) and p67(phox) are both dependent on the presence of the partner protein in solution and on the presence of the activator molecule cis-AA.