Abstract 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.
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.
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).
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.
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.
Display omitted
•High temperatures promote high production rates of superoxide anions.•Production of reactive oxygen species is closely related to bilayer composition and membrane thickness.•Oxidase activation energy is correlated to the bilayer fluidity.•Sterols are negative regulators of NADPH oxidase activity.
•Nox2 is inhibited by Reactive Oxygen Species mostly because of oxidation of membrane lipids.•The activity of Nox2 in untreated hypercholesterolemic patients is lower than that of normal ...patients.•Addition or diminution of cholesterol in cell free system leads to a decrease of activity of Nox2.•Oxysterols and non-oxidized cholesterol have similar effects.
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.
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.
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 ATPhydrolysis with high efficiency. Contrary to the predictions of the currently prevailing "molecular bellows" model of MacB-operation, which assigns the power stroke to the ATPbinding 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.
Le complexe NADPH oxydase est un élément essentiel de l’immunité inné. Présent dans les cellules phagocytaires (neutrophile), sa fonction est de produire massivement, dans le phagosome, des anions ...superoxyde et générer ainsi des espèces encore plus réactives de l’oxygène qui vont détruire acides nucléiques, lipides et protéines des bactéries phagocytées. Le cœur membranaire catalytique du complexe NADPH oxydase est constitué d’un hétérodimère membranaire, le cytochrome b₅₅₈ (Cyt b₅₅₈). Après activation de celui-ci par les partenaires protéiques cytosoliques p47phox, p67phox, p40phox et Rac, une succession de réactions de transferts d’électron de part et d’autre de la membrane a lieu au sein du Cyt b₅₅₈ pour aboutir à la réduction du dioxygène de manière très contrôlée. Afin de mieux comprendre cette régulation, nous nous sommes d’abord intéressés aux stéreoisomères trans de l’acide arachidonique, activateur naturel de cet enzyme (cis), sur le fonctionnement de la NADPH oxydase et avons abordé cette étude parallèlement sur du Cytb₅₅₈ d’origine bovine présent dans des membranes de neutrophiles et dans des membranes de levures exprimant le Cytb₅₅₈ de manière hétérologue. Nous avons montré que la géométrie joue un rôle important sur l’activation du complexe enzymatique. Dans un deuxième temps, afin d’étudier le rôle de l’environnement membranaire sur le fonctionnement de la NADPH oxydase, nous avons déterminé les propriétés cinétiques et thermodynamiques de l’activité NADPH oxydase du Cytb₅₅₈ recombinant exprimé en levures, purifié, puis reconstitué en liposomes de composition lipidique variée. Après comparaison avec ces mêmes propriétés obtenues pour le Cytb₅₅₈ dans les membranes plasmiques et du réticulum endoplasmique de levures, nous avons montré que l’activité NADPH oxydase très sensible à la température peut être modulée par la composition et l’état physique de la membrane.
NADPH oxidase complex is a major actor of both antimicrobial host defense and inflammation by generating highly regulated superoxide anion, rapidly converted into reactive oxygen species (ROS). The NADPH oxidase complex consists of a heterodimeric integral membrane flavocytochrome b₅₅₈ and three cytosolic components p67phox, p47phox and p40phox, and the small GTP binding protein Rac. In response to a cellular stimulus, cytosolic proteins are recruited to the phagosomal membrane where they are assembled with the Cytb₅₅₈ to form the active NADPH oxidase. The aim of the work was to better understand the modulation of superoxide anion production by this enzyme. For this purpose, we performed experiments with both bovine neutrophil membranes and yeast membranes expressing the bovine recombinant Cytb₅₅₈. We first investigated the effect of the trans-isomerization of the cis-arachidonic acid, the activator of NADPH oxidase in vitro and showed that specific geometry of the activator plays an important role in the activation of the complex. We also studied the role of the membrane environment on the functioning of NADPH oxidase and determined the kinetics and thermodynamics of NADPH oxidase activity depending on the lipid composition of Cytb₅₅₈ proteoliposomes. Comparison with these properties obtained with recombinant Cytb₅₅₈ embedded into endoplasmic reticulum and plasma membranes, we showed that the NADPH oxidase activity is highly temperature dependent and can be modulated by the lipid environment and the physic state of the membrane.
PDF
