The physicochemical properties of membranes correlate with the NADPH oxidase activity.

BACKGROUND: Phagocytes kill ingested microbes by exposure to high concentrations of toxic reactive species generated by NADPH-oxidases. This membrane-bound electron-transferring enzyme is tightly regulated by cellular signaling cascades. So far, molecular and biophysical studies of the NADPH-oxidase were performed over limited temperature ranges, which weaken our understanding of immune response or inflammatory events. In this work, we have inspected the influence of temperature and lipid membrane properties on the NADPH-oxidase activity using a system free of cell complexity. METHODS: We have extended the experimental conditions of the accepted model for NADPH-oxidase activity, the so-called cell-free assay, to a large temperature range (10-40°C) using different membrane compositions (subcellular compartments or liposomes). RESULTS: A remarkable increase of superoxide production rate was observed with rising temperature. Synchrotron radiation circular dichroism data showed that this is not correlated with protein secondary structure changes. When lipid bilayers are in fluid phase, Arrhenius plots of the oxidase activity showed linear relationships with small activation energy (Ea), while when in solid phase, high Ea was found. The sterol content modulates kinetic and thermodynamic parameters. CONCLUSION: High temperature promotes the rate of superoxide production. The key element of this enhancement is related to membrane properties such as thickness and viscosity and not to protein structural changes. Membrane viscosity that can be driven by sterols is a paramount parameter of Ea of NADPH oxidase activity. The membrane bilayer state modulated by its sterol content may be considered locally as an enzyme regulator. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Recombinant form of mammalian gp91(phox) is active in the absence of p22(phox).

The flavocytochrome b558 of the phagocyte NADPH oxidase complex comprises two membrane proteins, a glycosylated gp91phox and a non-glycosylated p22phox. Gp91phox contains all of the redox carriers necessary to reduce molecular oxygen to superoxide using NADPH. The capacity of gp91phox to produce superoxide in the absence of its membrane partner p22phox has been little studied. In the present study, we have generated in Pichia pastoris for the first time an active form of bovine gp91phox able to carry out the entire NADPH oxidase activity in the absence of p22phox. Collected information on the maturation and the activity of the recombinant gp91phox and the participation of individual cytosolic subunits in the active complex allowed us to propose, in the absence of p22phox, an unconventional stabilized complex compared with the heterodimer.

Corseting a tripartite ABC transporter to make it fit for transport.

ABC transporters have long been known to mediate resistance phenotypes in all kingdoms of life, and ATP-driven tripartite efflux pump from Gram-negative bacteria have attracted increasing interest. We give a special focus on MacAB TolC, a prototypical member of the recently described Type VII ABC transporter superfamily, from Escherichia coli. We provide original experimental evidence for the in vitro, substrate-induced ATPase activity and show a maximal activity when the tripartite pump is fully assembled in lipid nanodiscs. These results are evaluated and interpreted in the context of the structural and functional data that have accumulated over the years.

Quantitative real-time analysis of the efflux by the MacAB-TolC tripartite efflux pump clarifies the role of ATP hydrolysis within mechanotransmission mechanism.

Tripartite efflux pumps built around ATP-binding cassette (ABC) transporters are membrane protein machineries that perform vectorial export of a large variety of drugs and virulence factors from Gram negative bacteria, using ATP-hydrolysis as energy source. Determining the number of ATP molecules consumed per transport cycle is essential to understanding the efficiency of substrate transport. Using a reconstituted pump in a membrane mimic environment, we show that MacAB-TolC from Escherichia coli couples substrate transport to ATP-hydrolysis with high efficiency. Contrary to the predictions of the currently prevailing "molecular bellows" model of MacB-operation, which assigns the power stroke to the ATP-binding by the nucleotide binding domains of the transporter, by utilizing a novel assay, we report clear synchronization of the substrate transfer with ATP-hydrolysis, suggesting that at least some of the power stroke for the substrate efflux is provided by ATP-hydrolysis. Our findings narrow down the window for energy consumption step that results in substrate transition into the TolC-channel, expanding the current understanding of the efflux cycle of the MacB-based tripartite assemblies. Based on that we propose a modified model of the MacB cycle within the context of tripartite complex assembly.

Rationale for the Quantitative Reconstitution of Membrane Proteins into Proteoliposomes.

Proteoliposome reconstitution is a method of choice for the investigation of membrane proteins as it allows their manipulation in the desired hydrophobic environment and allows one to tackle their study from both functional and structural points of view. Methods for their rapid and efficient reconstitution have been known for a long time but the quality and dispersity of the resulting suspensions is often overlooked. Here we describe our routine for the obtention of monodisperse populations of proteoliposomes as well as for the quantitation of protein per liposome.

BAmSA: Visualising transmembrane regions in protein complexes using biotinylated amphipols and electron microscopy.

Membrane protein (MP) complexes play key roles in all living cells. Their structural characterisation is hampered by difficulties in purifying and crystallising them. Recent progress in electron microscopy (EM) have revolutionised the field, not only by providing higher-resolution structures for previously characterised MPs but also by yielding first glimpses into the structure of larger and more challenging complexes, such as bacterial secretion systems. However, the resolution of pioneering EM structures may be difficult and their interpretation requires clues regarding the overall organisation of the complexes. In this context, we present BAmSA, a new method for localising transmembrane (TM) regions in MP complexes, using a general procedure that allows tagging them without resorting to neither genetic nor chemical modification. Labels bound to TM regions can be visualised directly on raw negative-stain EM images, on class averages, or on three-dimensional reconstructions, providing a novel strategy to explore the organisation of MP complexes.

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.

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.

Contribution of lipid environment to NADPH oxidase activity: influence of sterol.

The NADPH-oxidase complex, which plays beneficial or detrimental role in the inflammatory and degenerative diseases, is a membrane multi-subunit complex tightly regulated in order to produce superoxide anions, precursor of oxygen reactive species (ROS), in cells. The flavocytochrome b(558) (Cytb(558)) is the catalytic core of the NADPH oxidase which consists of two membrane proteins gp91(phox) (highly glycosylated) and p22(phox). In this work we took advantage of heterologous yeast cells engineered to express wild-type bovine Cytb(558) to analyze the properties of the NADPH oxidase activity during the biosynthesis processing steps of gp91(phox) and p22(phox) within endoplasmic reticulum (ER) and plasma membrane (Pmb). Our data showed that, in yeast, the heterodimerization at the endoplasmic reticulum membranes was concomitant with high level glycosylation of gp91(phox) and the heme acquisition. This study also demonstrated that the phagocyte NADPH oxidase was active at ER membranes and that this activity was surprisingly higher at the ER compared to the Pmb membranes. We have correlated these findings with the presence of sterols in the plasma membranes and their absence in ER membranes. This correlation was confirmed by decreased superoxide anion production rates in proteoliposomes supplemented with ergosterol or cholesterol. Our data support the idea that membrane environment might be determinant for ROS regulation and that sterols could directly interact with the membrane proteins of the NADPH oxidase constraining its capacity to produce superoxide anions.