trans Arachidonic acid isomers inhibit NADPH-oxidase activity by direct interaction with enzyme components

H. Souabni a,b, V. Thoma c,d, T. Bizouarn a,b, C. Chatgilialoglu c, A. Siafaka-Kapadai d, L. Baciou a,b, C. Ferreri d,
C. Houée-Levin a,b,*, M.A. Ostuni a,b,**

ª UMR8000, CNRS, 91405 Orsay, France

ᵇ Laboratoire de Chimie-Physique Bat. 350, Université Paris-Sud, 91405 Orsay, France

ᶜ I.S.O.F., Consiglio Nazionale delle Ricerche, Via Piero Gobetti 101, 40129 Bologna, Italy

ᵈ Department of Chemistry (Biochemistry), University of Athens, Panepistimioupolis, 15771 Athens, Greece

article in fo

Article history:

Received 30 November 2011

Received in revised form 23 April 2012

Accepted 24 April 2012

Available online 3 May 2012

Keywords:

NADPH oxidase

trans Fatty acid

Arachidonic acid

Protein complex assembly

Cell free system

Superoxide production inhibition

abstract

NADPH-oxidase is an enzyme that represents, when activated, the major source of non-mitochondrial reactive oxygen species. In phagocytes, this production is an indispensable event for the destruction of engulfed pathogens. The functional NADPH-oxidase complex consists of a catalytic membrane flavocytochrome *b* (*Cytb*<sub>558</sub>) and four cytosolic proteins p47<sup>phox</sup>, p67<sup>phox</sup>, Rac and p40<sup>phox</sup>. The NADPH-oxidase activity is finely regulated spatially and temporally by cellular signaling events that trigger the translocation of the cytosolic subunits to its membrane partner involving post-translational modifications and activation by second messengers such as arachidonic acid (AA). Arachidonic acid in its natural *cis*-poly unsaturated form (C20:4) has been described to be an efficient activator of the enzyme in vivo and in vitro. In this work, we examined in a cell-free system whether a change of the natural *cis* geometry to the *trans* configuration, which could occur either by diet or be produced by the action of free radicals, may have consequences on the functioning of NADPH-oxidase. We showed the inability of mono-*trans* AA isomers to activate the NADPH-oxidase complex and demonstrated the inhibitory effect on the *cis*-AA-induced NADPH oxidase activation. The inhibition is mediated by a direct effect of the mono-*trans* AA which targets both the membrane fraction containing the *cytb*<sub>558</sub> and the cytosolic p67<sup>phox</sup>. Our results suggest that the loss of the natural geometric feature (*cis*-AA) induces substantial structural modifications of p67<sup>phox</sup> that prevent its translocation to the complex.

1. Introduction

NADPH-oxidase is well known as a major source for non-mitochondrial superoxide radical in cells. Superoxide anion is rapidly transformed into toxic oxygen species such as hydrogen peroxide, hypochlorous acid, hydroxyl radical and peroxynitrite. In phagocytes, the generation of superoxide anion (O₂⁻•) initiated by the NADPH oxidase complex is triggered by the activation of these cells by

inflammatory stimuli (for a review see [1,2]). Superoxide anion and its derived Reactive Oxygen and Nitrogen Species contribute efficiently to the destruction of phagocytized infectious agents [3]. In other cells, NADPH oxidase-generated superoxide is recognized to play other crucial roles in events such as cellular communication [4]. A deregulation of NADPH-oxidase activity is associated with several pathologies involving inflammatory processes, cardiovascular diseases, renal damage, central nervous system diseases and immune system disorders (for a review see [5–9]).

The phagocyte NADPH oxidase complex consists of a heterodimeric, integral membrane protein, namely flavocytochrome *b*<sub>558</sub> (*Cytb*<sub>558</sub>, composed of p22<sup>phox</sup> and a heavily glycosylated gp91<sup>phox</sup> subunits) and four cytosolic subunits (p40<sup>phox</sup>, p47<sup>phox</sup>, p67<sup>phox</sup>, GTP-binding protein Rac). The *Cytb*<sub>558</sub> serves as the catalytic core of the NADPH oxidase complex allowing the electron transfer across the membrane. To prevent the high toxicity of reactive oxygen species under physiological conditions, the NADPH-oxidase activity is highly regulated spatially and temporally. Following receptor-mediated activation of phagocytes, the regulatory subunits p40<sup>phox</sup>, p47<sup>phox</sup>, p67<sup>phox</sup> and Rac translocate to the membrane bound *Cytb*<sub>558</sub> to form the active enzyme [10–13]. This activation process is tightly controlled by intracellular signaling

Abbreviations: cyt, cytochrome; AA, arachidonic acid; Ag-TLC, argentation thin layer chromatography; PMSF, phenylmethanesulfonyl fluoride; SOD, superoxide dismutase; bMF, bovine membrane fractions; yMF, yeast membrane fractions; Me AA, AA methyl ester; GC/MS, gas chromatography-mass spectrometry; NMR, nuclear magnetic resonance; BSA, bovine serum albumin; Cytc, cytochrome c; SH3, Src Homology 3; SDS, sodium dodecyl sulfate

◆ Correspondence to: C. Houée-Levin, Laboratoire de Chimie-Physique, UMR8000, Bat. 350 Université Paris-Sud, 91405 Orsay, France. Tel.: +33 1 6915 5549; fax: +33 1 6915 6188.

** Correspondence to: M.A. Ostuni, UMR-S945 Immunité et Infection, Faculté de Médecine, Université Pierre et Marie Curie, 91 Boulevard de l'Hôpital, 75013 Paris, France. Tel.: +33 1 40779894; fax: +33 1 40779734.

E-mail addresses: chantal.houec@u-psud.fr (C. Houée-Levin), mariano.ostuni@upmc.fr (MA. Ostuni).
<figure><img src="image_1.png" /></figure>
mechanisms that regulate phosphorylation events of the oxidase
components (for a review see [14,15]) and the action of lipid second mes-
sengers [16,17]. Lipid mediators like arachidonic acid (5c,8c,11c,14c-
C20:4, *cis*-AA), participate in the phosphorylation processes of the regula-
tory subunits through kinase activation, but it has been also shown that
long-chain polyunsaturated fatty acids are directly involved in the
NADPH oxidase assembly process and elicit superoxide anion production,
with arachidonic acid being the most potent activator [18].

The understanding of the mechanism and activation of NADPH oxidase has been greatly facilitated by the development of a cell-free system in which the enzyme in the resting state is activated by anionic amphiphiles, such as arachidonic or other fatty acids as well as SDS [18–22]. Evidence exists that the anionic amphiphiles induce conformational changes in the cytosolic subunits which are analogous to those induced in vitro by phosphorylation [23–26], leading to unmasking the SH3 regions interacting with Cytb<sub>558</sub> [27,28]. Anionic amphiphiles impact also the integral membrane Cytb<sub>558</sub> by eliciting conformational changes, which were proposed to be relevant for the activation of superoxide production [29–32]. Moreover, it has been proposed that cis-AA may facilitate the accessibility of the Cytb<sub>558</sub> hemes to cyanide derivatives allowing the transition of the spin state of the heme iron [33,34].

In eukaryotic cells, the prevalent geometry displayed by unsatu-
rated fatty acids is the *cis* configuration. The change of the *cis* geome-
try to the corresponding geometrical *trans* isomer is an unnatural
event which influences cellular responses [35]. This can happen in
an exogenous manner, with the intake of *trans* fatty acids through
foods containing partially hydrogenated or deodorized vegetal/fish
oils or processed at high temperature [36,37]. In addition to the up-
take from food, an endogenous pathway has been identified in trans-
free models and in humans through the occurrence of a free radical-
induced *cis-trans* double bond isomerization [38–40].

Due to the connection of NADPH-oxidase activity with arachidonic
acid and free radical production, it is important to address a first
question on the relationship between the fatty acid geometrical con-
figuration and the functioning of the NADPH-oxidase. In this paper,
we explored the events induced by free long unsaturated fatty acid,
that occur during NADPH oxidase activation using a cell-free system
approach that permits to strictly quantify the system components.
We used in parallel two cell free systems: i) the most frequent “ca-
nonical” semi-recombinant cell-free system in which mixed native
Cytb<sub>558</sub> was contained in membrane fractions (isolated from neutro-
phils) with purified cytosolic recombinant components, and ii) an en-
tire recombinant cell-free system in which the native neutrophil
membranes are replaced by recombinant Cytb<sub>558</sub> expressed in yeast
(Pichia pastoris) membrane [41]. In both systems, we compared the
NADPH oxidase activating ability of arachidonic acid (cis-AA) with
that of mono-trans arachidonic acid isomers (trans-AA, Fig. S1). We
demonstrated a specific inhibitory effect of the trans-AA on the cis-
AA-induced NADPH oxidase activation and performed experiments
to decipher the target(s) of the inhibition mediated by a direct effect
of trans-AA.

2. Materials and methods

2.1. Materials

Superoxide dismutase (SOD), reduced β-nicotinamide adenine dinucleotide phosphate (NADPH), cytochrome c (Cytc), arachidonic acid (cis-AA), phenylmethanesulfonyl fluoride (PMSF), and tert-butylisocyanide were from Sigma (Saint Quentin Fallavier, France).

2.2. *Synthesis of mono trans- and methyl ester arachidonic acid*

Synthesis of *trans*-AA was performed following the protocol for
free radical geometrical isomerization procedure [42]. Briefly, a

2.2 mL solution of 15 mM *cis*-AA in propan-2-ol (i-PrOH) was placed in a quartz photochemical reactor and bubbled with nitrogen for 20 min. The thiol (2-mercaptoethanol) in a 50% mole equivalent with respect to mole of *cis*-AA was added and the mixture was exposed to low pressure mercury lamp (5.5 W) at 25 °C under a slow flow of nitrogen. Reaction was monitored by the argentation-thin layer chromatography (Ag-TLC) that showed the formation of the *trans*-AA fraction in the first 15 min. The reaction was stopped and the mixture was dried under vacuum, followed by purification by Ag-TLC. The remaining fraction containing the starting material, the pure *cis* isomer, was used to reiterate the isomerization protocol, in order to increase the *trans*-AA yield up to an 85% (purity> 97%). The purity was checked by proton nuclear magnetic resonance (¹H NMR) as well as by GC/MS after transformation of the fraction to the corresponding fatty acid. The *trans*-AA fraction was isolated as a mixture of four mono-*trans* isomers in equal percentages as described [40] (Supplementary Fig. S1). Methyl ester fatty acids were obtained by treatment with an ethereal solution of diazomethane, as previously described [40]. 20 mg/mL stock solutions of *cis*-AA, *trans*-AA and methyl ester AA (*me*-AA) were prepared in ethanol.

2.3. Isolation of native membranes from neutrophil cells

Membrane fractions from bovine neutrophils were prepared as de-
scribed [43]. Briefly, isolated neutrophils were extracted from 8 to 10 L
of bovine blood by two successive hemolysis followed by Percoll sed-
imentation, and then they were disrupted by brief sonifications. The
membrane fraction (bMF) was separated from the cytosolic fraction
and other cell contents on sucrose gradient and was collected by cen-
trifugation at 150,000 ×g for 160 min at 4 °C. Pellets were resuspended
in phosphate buffer saline (PBS) pH 7.8 containing 1 mM EDTA, 1 mM
PMSF, and 0.5 mM leupeptin before storage at -80 °C for further ex-
periments. The protein concentration of each sample was estimated
using the Pierce BCA protein assay (Thermo Scientific, Courtabœuf,
France) with BSA as standard. Between 0.5 and 0.8 mg proteins/L of
blood were usually obtained.

2.4. Isolation of recombinant Cytb<sub>558</sub>-enriched plasma membrane from yeast cells

Recombinant Cytb<sub>558</sub> was expressed in *P. pastoris* and yeast mem-
brane fraction (yMF) was obtained as previously described [41]. Brief-
ly, cell pellets from a 72 h yeast culture were thawed and resuspended
in cell breaking buffer (CBB) containing 50 mM sodium phosphate, pH
7.4, 1 mM PMSF, 1 mM EDTA and 5% glycerol. All steps are carried out
at 4 °C. An equivalent volume of glass beads was added and cells were
disrupted by 8 cycles of 30 s vortexing/30 s ice bath. The clear super-
natant containing the crude cell lysate was separated from the cell de-
bris and unbroken cells by centrifugation at 500 ×g for 10 min at 4 °C.
The membrane fractions were collected by centrifugation at
100,000 ×g for 120 min and resuspended in 50 mM Tris/HCl pH 8,
120 mM NaCl, 10% glycerol, and 1 mM PMSF and loaded onto a discon-
tinuous sucrose gradient comprising 60, 40 and 20% sucrose. After
overnight centrifugation at 110,000 ×g, the essential pure plasma
membranes were removed from the 60 and 40% interface, diluted 4-
fold and pelleted at 30,000 ×g. The Cytb<sub>558</sub>-enriched membrane frac-
tion was resuspended in 1 mM EDTA, 30% sucrose, and 20 mM Tris/
HCl pH 8.0 and stored at -20 °C.

The protein concentration of each sample was estimated using the Pierce BCA protein assay with BSA as standard. About 6 mg proteins/L of yeast culture was usually obtained.

2.5. Production of recombinant cytosolic proteins

Recombinant p47<sup>phox</sup>, p67<sup>phox</sup> and Rac1-Q61L (Rac) were expressed and purified as described [44]. Here, the mutant Rac1Q61L was used
because it is constitutively in the active form, the GTP-bound form and thus greatly facilitates the superoxide anion production in vitro [45]. The cDNA plasmids were a generous gift from Dr. M.C. Dagher (Laboratoire TheRex, TIMC-IMAG, Grenoble, France) and Dr. M. Quinn (University of Montana, USA).

## 2.6. Cell-free NADPH-oxidase activity assay

Superoxide anion production was quantified by the initial rate of cytochrome c (Cytc) reduction, as described before [46]. The components of the cell-free system were added as follows: 5-8 nM *Cytb*<sub>558</sub>-enriched yeast or bovine membrane fractions (estimated from difference absorption spectra), 100 nM p67<sup>phox</sup>, 160 nM p47<sup>phox</sup> and 80 nM RacQ61L so that the membrane protein/cytosolic protein ratio was kept at around 1/20, and the chosen free fatty acid (*cis*-AA or *trans*-AA). After 5 min of incubation at 25 °C, 0.1 mM Cytc was added and the O₂⁻ production was initiated by the addition of 200 µM NADPH. The linear portion of the curves was used to determine the maximal rates of O₂⁻ production. The reaction buffers contain PBS pH 7.4 and 10 mM MgSO₄, for bovine cytb₅₅₈ activity assay or 20 mM Tris/HCl pH 8, 1 mM EDTA and 120 mM NaCl for recombinant yeast cytb₅₅₈ activity assay. The SOD-inhibitable reduction of Cytc was followed at 550 nm on double beam Uvikon UV946 spectrophotometer. The amount of superoxide was calculated using a molar absorption coefficient (Δε of the reduced minus oxidized form of cytochrome c) of 21 mM⁻¹ cm⁻¹. Control experiments were performed in the presence of 350 U mL⁻¹ of SOD. Negative control experiments were performed replacing the *cis*-AA by *me*-AA. Activation and inhibition curves were fitted using GraphPad Prism 5 following the equation:
$$A \frac{1}{4} A \left( \frac{\Delta A}{t} \right) \left( \frac{1}{10^{\log_{10}C} \cdot 50 - \Delta A \cdot n} \right) \text{ or } A \frac{1}{4} A \left( \frac{\Delta A}{t} \right) \left( \frac{1}{10^{\log_{10}C} \cdot 50 - \Delta A \cdot n} \right),$$
where A is the activity, ΔA is the difference between maximal and minimal activity, [AA] is the arachidonic acid concentration and n is the Hill constant.

## 2.7. Intrinsic fluorescence assays

Steady-state fluorescence measurements were performed on a Photon Technology International scanning fluorimeter at 25 °C. The tryptophan fluorescence spectra of protein were obtained by exciting the samples at 280 nm (1 nm bandwidth) and recorded between 300 and 450 nm (2 nm bandwidth). Although measurement of tryptophan fluorescence is usually performed using an excitation wavelength of 295 nm, we found that, as previously described [23], using an excitation wavelength of 280 nm optimized the signal to noise ratio without adding significant tyrosine fluorescence interference.

Various concentrations of *cis*-AA, *trans*-AA or *me*-AA were added as indicated to a final volume of 1 mL of buffer (10 mM potassium phosphate, pH 7.2, 130 mM NaCl,) in a quartz cuvette containing a magnetic stir bar, and recombinant p47<sup>phox</sup> (125 nM), or p67<sup>phox</sup> (45 nM) proteins. The measured fluorescence intensity was corrected for dilution effects or for inner-filter effect.

## 2.8. Spectral analysis of cytb<sub>558</sub>

Absorption spectra were recorded at room temperature using a Uvikon dual-beam spectrophotometer. The sodium dithionite-reduced *minus* oxidized difference of absorption spectra was recorded between 400 and 600 nm.

Yeast or bovine membrane fractions were suspended in 200 µL phosphate buffer, incubated at 25 °C for 5 min and supplemented with optimal concentrations of *cis*-AA, *trans*-AA or *me*-AA. The dithionite reduced membranes were treated with 5 mM or 30 mM tert-butylisocyanide for yeast or bovine membranes, respectively.

## 2.9. Co-sedimentation assays

*Cytb*<sub>558</sub> from bovine or yeast membrane fractions was mixed with the recombinant purified cytosolic subunits (same ratio as for activity assays) in the presence of 200 µM *cis*-AA, *trans*-AA, or *me*-AA (600 µM in the case of yeast membrane fractions). After 5 min incubation at 25 °C, the protein mixtures were layered onto a discontinuous gradient composed of 1.5 mL of 10% (w/v), 6.5 mL of 15% (w/v) and 2.0 mL of 50% (w/v) sucrose, all in 10 mM Tris-HCl pH 7.8, 1 mM EDTA, and 10 mM Na<sub>3</sub>NH<sub>2</sub> buffer, and centrifuged at 150,000 × g for 30 min at 15 °C in a SW32Ti rotor. After centrifugation, the essential soluble proteins were collected in the 0.5 mL aliquots from the top of the gradient ("supernatant") while the membrane or membrane-associated proteins were retrieved from the bottom of the gradient ("pellet").

## 2.10. SDS PAGE and immunoblotting

"Supernatant" and "pellet" from the co-sedimentation experiments were diluted in Laëmmli buffer, loaded onto a 10% ClearPAGE SDS-polyacrylamide precast gel (VWR International, Fontenay-Sous-Bois, France), and transferred to a nitrocellulose membrane. Nitrocellulose membranes were then immunoblotted with anti-p47 (1:500) or anti-p67 (1:500) polyclonal antibodies. Immune complexes were visualized with secondary peroxidase-conjugated antibodies (1:10,000 dilution), using a chemiluminescent kit (GE Healthcare, Saclay, France).

## 2.11. Membrane lipid composition

Fatty acid composition of yeast and bovine membrane fractions were determined after lipid extraction followed by trans-esterification to obtain fatty acid methyl esters. The methyl ester composition was analyzed by gas chromatography and compared to standard reference compounds profiles, as previously described [47]. Peaks are expressed as relative percentages of the total peak areas of the chromatograms (peak covering >99.9%). The main composition is described in Fig. 2.

# 3. Results

## 3.1. Activation of the NADPH oxidase complex in semi and total recombinant cell-free system

The NADPH oxidase activation by *cis*-AA was investigated in semi-recombinant and fully recombinant cell-free system. In both assays, the activity follows a characteristic bell shape activation profile (Fig. 1). As a negative control, the oxidase activities were quantified with *me*-AA that has been shown to competitively counteract *cis*-AA-mediated oxidase activation. In agreement with previous findings [18,34,48,49], *me*-AA was not able to activate NADPH-oxidase complex in both cell-free assays, supporting the evidence that the negative charge present in *cis*-AA is indispensable for the NADPH oxidase activation.

Similar to *me*-AA, the addition of *trans*-AA (an equimolar mixture of four mono-*trans* isomers) could not induce O₂⁻ production neither in the recombinant (Fig. 1A) nor in the semi-recombinant cell-free system (Fig. 1B). This result indicates that *trans*-AA is unable to activate the NADPH oxidase complex although it contains the negative charge similar to the *cis* form.

In the semi-recombinant cell free system, the maximal O₂⁻ production occurred at a *cis*-AA concentration of 200 µM (Fig. 1B), while in the yeast cell-free system, this value is significantly higher (600 µM, Fig. 1A) and the bell-shape is wider. The optimal *cis*-AA concentration for the oxidase activity in the semi-recombinant assay is in good agreement with several reported results [32,50,51], whereas lower values were also reported by other authors [20,23,52]. However no significant difference of the maximal activity values induced by *cis*-AA is observed when bovine or yeast membrane fractions were used as source of NOX. Both cell free systems produce about 20 mol Q⁻/s/mol heme.
<figure><img src="image_2.png" /><figcaption>Fig. 1. Activation of NADPH-oxidase complex by different arachidonic acid isomers. The superoxide production was measured spectrophotometrically by the rate of cytochrome c reduction at 550 nm as described in the Materials and methods section. In the reaction mixture, yeast (A) or bovine (B) membrane fractions were incubated 5 min in the presence of recombinant cytosolic proteins, and different concentrations of *cis* arachidonic acid (circles), *trans* arachidonic acid (diamonds) or methyl ester arachidonate (squares). The reaction was initiated by the addition of 200 µM NADPH in the presence of 50 µM cytochrome *c*. Results are presented as the mean±SD of four independent experiments. Visual fit of activation curves was performed using KaleidaGraph, Synergy Software, (Reading, PA, USA).</figcaption></figure>

The maximal activity obtained herein was lower compared to well characterized cell-free-AA-stimulated NADPH-oxidase activity [18] probably due to the presence of contaminant $Ca^{2+}$. Actually, we did not add EGTA in the NADPH-oxidase assays, working under contaminant $Ca^{2+}$ conditions (estimated to be lower than 30 µM). It was previously demonstrated that the presence of $Ca^{2+}$ inhibits the stimulatory action of fatty acid on NADPH-oxidase [18,53], with an $IC_{50}$ around 100 µM. This inhibitory effect is correlated with the raise of the fatty acid Krafft point in the presence of $Ca^{2+}$ [53].

## 3.2. Membrane lipid composition

Fatty acid compositions of the yeast and neutrophil membranes are also very different. As shown in Fig. 2, a prevalent presence of the saturated fatty acid C16:0 (palmitic acid) was observed in *P. pastoris* plasma membrane.

In neutrophils the C18 family was predominantly, with the saturated fatty acid C18:0 (stearic acid) and the polyunsaturated omega-6 fatty acid 9c,12c-C18 (linoleic acid) as the major components, with the relative percentages of 32% and 30% of the total composition, respectively. In *P. pastoris* membrane, only 3% of linoleic acid was detected. Thus the *P. pastoris* membrane is enriched in saturated fatty acids compared to neutrophil membranes.

## 3.3. NADPH-oxidase activity was inhibited by the addition of trans-AA

The *cis*-AA induced NADPH-oxidase activity was followed upon increased concentration of either *trans*-AA or *cis*-AA (Fig. 3A). The latter experiment serves as control in order to distinguish the inhibitory action of *trans*-AA from the concentration-related inhibition itself. As NADPH-oxidase activity follows a bell shape activation profile (Fig. 1), the NADPH oxidase activities decrease upon the addition of exogenous fatty acids. The *cis*-AA-induced NADPH-oxidase activity is reduced by a threefold upon the addition of *trans*-AA in a dose-dependent manner and only twice when *cis*-AA was added. These results indicated that *trans*-AA inhibitory effect resulted from two synergic actions: an unspecific, concentration-related inhibition and a specific inhibitory effect. The fits of the curves described in Fig. 3A give $IC_{50}$ values of 14.9±2.8 µM and 47.1±1.2 µM for *trans*-AA and *cis*-AA, respectively (Supplementary material Table S1). A residual activity of 25% remains even for higher amount of *trans*-AA. Comparable results were obtained using bovine membrane fraction (Supplementary material, Fig. S2).

A complementary experiment was performed in which activity was followed in the presence of constant 100 µM *trans*-AA concentration and variable *cis*-AA concentration up to 600 µM (Fig. 3B and Supplementary Table S2). The maximal NADPH-oxidase activity induced by *cis*-AA reached only 40% of the value obtained in the control experiment in which 100 µM *cis*-AA was added instead of 100 µM *trans*-AA. In that case, the *cis*-AA $EC_{50}$ value (Supplementary material Table S1B) is not affected by the presence of *trans* isomer. It might suggest that inhibition is competitive.

## 3.4. Deciphering of the NADPH oxidase target(s) of trans-AA

It is likely that *trans*-AA affects differently the membrane and cytosolic components. To investigate the targets of *trans*-AA, superoxide anion production rate was measured in recombinant cell-free assay in which membrane and cytosolic components were simultaneously or separately incubated with *cis*- or *trans*-AA isomers (Fig. 4A). Activity induced by simultaneous *cis*-AA incubation was considered as the 100% value.

When membrane and cytosolic fractions were incubated separately with *cis*-AA and then mixed, the activity dropped to about 71% (Fig. 4B). Surprisingly, when *cis*-AA was added only to the cytosolic or to the membrane fraction, the activity measured was similar (~79%, Fig. 4B). It should be noted that the initial activity was measured immediately after fractions were mixed in the presence of NADPH, reducing but not eliminating the possibility of a cross activation phenomena. Indeed the activity measured in the presence of *cis*-AA but without pre incubation time (simultaneously added with NADPH to a whole mixture) did not result in a significant activity (Fig. 4A). This underlines the fact that when the NADPH oxidase components are separately incubated with *cis*-AA and subsequently mixed, they partially lose their capacity to efficiently assemble afterwards. It is consistent with the importance of the numerous protein-protein and protein-phospholipid interactions for the assembly of a functional oxidase complex during the period of incubation prior to the onset of oxidase activity.

Consistent with our above results, incubation of both fractions with *trans*-AA (Fig. 4A) was unable to activate the NADPH oxidase complex leading to a very small $O_2^{•−}$ production rate (less than 10% of that in the presence of *cis*-AA, similar to baseline activity in the absence of AA, or in the presence of *cis*-AA but without pre incubation
<figure><img src="image_3.png" /><figcaption>Fig. 2. Fatty acids composition in the lipids extracted from yeast strain *P. pastoris* (gray bars) and bovine neutrophil (black bars) membrane fractions. Lipid identification was performed using reference compounds. Peaks are expressed as relative percentages of the total peak areas of the chromatograms (peak covering> 99.9%).</figcaption></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 3. NADPH-oxidase activity inhibition by *trans* arachidonic acid using the yeast membrane fractions. Yeast membrane fractions together with cytosolic proteins pre-incubated in the presence of (A) 600 µM *cis*AA supplemented with increasing concentrations of *trans*AA (filled triangles) or *cis*AA (open triangles), or (B) 100 µM *trans*AA (filled triangles) or *cis*AA (open triangles) supplemented with increasing concentrations of *cis*AA. Oxidase activity was expressed as the percent of activity measured in the absence of *trans*AA (20.3 mol O₂⁻/s/mol heme). Results are presented as the mean±SEM of three independent experiments.</figcaption></figure>

time). In order to identify whether *trans*-AA affected the membrane and/or cytosolic components, we performed cell-free NADPH-oxidase activity assays where cytosolic and membrane fractions were pre-incubated separately (Fig. 4B). Adding *trans* isomers to only one or to both counterparts resulted in a lower level of O₂⁻ production rate (below 12%). When added only to the membrane fraction, the production of superoxide was almost undetectable. To summarize, these results let us hypothesize that *trans*-AA, in contrast to *cis*-AA, did not act as oxidase activator at the level of the membrane component of NADPH-oxidase complex. Moreover, a major result of these experiments is a decreased O₂⁻ production of 45% when one of the components was activated with *cis*-AA and the counterpart with *trans*-AA. These decreased activities not only confirm the inability of *trans*-AA to activate the NADPH oxidase complex but also show a capacity of *trans*-AA to inhibit the oxidase activation process, through its binding on both the cytosolic and the membrane fraction. Comparable results were obtained using bovine membrane fraction (Supplementary Fig. S3).

### 3.5. Effect of the *trans*AA on the tryptophan intrinsic fluorescence of *p47phox* and *p67phox*

To probe the effect of AA isomers on the protein conformational state, we followed the evolution of intrinsic tryptophan fluorescence emission spectra when AA isomers were added to the regulatory proteins p47phox and p67phox (Figs. 5 and 6). Five from the seven total tryptophan residues of p47phox are located in the protein region containing Src Homology 3 (SH3) domains [28,54]. It was previously demonstrated that activation of the NADPH oxidase induced changes in the micro-environment of these tryptophan residues, reflected by measurable variations of the intrinsic tryptophan emission fluorescence level [23,24]. p67phox, a potential target for AA, contains a total of eight tryptophan residues and was described as a highly sensitive protein to the action of oxidant agents during the assembly process [55,56].

As was previously reported [23], the addition of *cis*-AA isomer induced a final decrease of 72% of the tryptophan fluorescence

($\lambda_{max}$ = 331 ± 1 nm) for p47phox (Fig. 5A) and of 58% for p67phox (Fig. 6A). The fluorescence decrease was dependent of cis-AA concentration up to 50 µM.

For both proteins, $\lambda_{max}$ was independent of *cis*-AA concentration and no spectral shift was observed up to the maximal concentration of fatty acid used (328 µM).

Addition of *trans*-AA produced a similar effect, resulting in a decrease of the fluorescence level of about 65% for p47phox (Fig. 5B)
<figure><img src="image_6.png" /><figcaption>Fig. 4. Oxidase activity in cell free system with different preincubation conditions.(A) The whole mix of yeast membrane fraction and cytosolic proteins was preincubated for 5 min in the presence of 600 µM *cis*-AA or *trans*-AA or without the addition of AA. An additional control experiment was performed adding 600 µM *cis*-AA to the whole mixture and measuring activity immediately after NADPH addition without pre-incubation time (B) Yeast membrane fraction and cytosolic proteins were separately preincubated in the absence or in the presence of *cis* or *trans* arachidonic acid as indicated. Activities were expressed as the percent of control activity (16.7 mol O₂*/s/mol heme, measured when whole mix was incubated in the presence of 600 µM *cis*AA, left bar, panel A). Results are presented as the mean±SEM of three independent experiments. \*Pb0.05; \*\*Pb0.01.</figcaption></figure>

and 60% for p67^phox^ (Fig. 6B). Addition of me-AA diminished the λ^max^ peak intensity to a lower extent. The fluorescence levels of p47^phox^ and p67^phox^ dropped only 40% (Fig. 5C) and 32% (Fig. 6C) of the initial peak value, respectively. Although we cannot exclude that the carboxylate groups of *cis* and *trans* AA interact with Trp residue(s) resulting in a quenching of their fluorescence, we can conclude that the absence of the anionic charge on the me-AA significantly reduces their impacts not only on p47^phox^, as was previously described [23], but also with p67^phox^.

Surprisingly, in the case of p67phox but not for p47phox, the de-
crease of fluorescence intensity was associated with a red-shift from
331 nm in the absence of trans-AA to 348 nm upon the addition of a
concentration of 50 µM *trans*-AA (Fig. 6B and D), indicating a signifi-
cant change in the tryptophan environment. This can be explained
by structural changes in extremely buried tryptophan(s) to fully ex-
posed to solvent.

Analysis of the curves according to the standard Stern–Volmer re-
lationship indicates a static quenching in the presence of an inacces-
sible population of tryptophan residues. The deduced Kd (1/Ksv)
values and the proportion of accessible tryptophan (fα) are shown
in Table 1. me-AA presents lower affinity for both cytosolic proteins
compared to *trans*-AA and *cis*-AA. Interestingly p67phox has a smaller
Kd value for *trans*-AA than for *cis*-AA while for p47phox, it is the oppo-
site. Tryptophans of p67phox are equally accessible for *cis*- and *trans*
isomers, while p47phox presents higher accessibility for *cis*- than for
*trans*-AA isomers.

3.6. *Effect of the trans-AA on the membrane component Cytb<sub>558</sub> of the*
*NADPH oxidase complex*

Here we probe the direct effect of trans-AA on the Cytb<sub>558</sub>. Anionic amphiphiles have been shown to influence the conformational changes during the transition from the inactive to the active form of Cytb<sub>558</sub>. It has been demonstrated that cis-AA facilitated the accessibility of the hemes to tert-butyl-isocyanide and the transition of the spin state of the heme iron [34]. A binding site for cis-AA on Cytb<sub>558</sub> was suggested [57]. It has also been reported that the structural changes induced in Cytb<sub>558</sub> not only by cis-AA (but also by SDS and lithium dodecyl sulfate), at concentrations where the maximal

activity of NADPH oxidase is observed, are specific and might partic-
ipate in oxidase activation [30,32]. To directly test the hypothesis
that *trans*-AA does not target the membrane component, we used
the same protocol as the one validated by Doussière's group to dis-
criminate the spin heme iron transition using tert-butyl-isocyanide.
The dithionite reduced minus oxidized absorption spectra were mea-
sured on native Cytb<sub>558</sub>-containing neutrophil membrane fraction
and recombinant Cytb<sub>558</sub>-containing yeast membrane fraction. The
direct effect of the AA isomers on the reduced minus oxidized absorp-
tion spectrum of both membrane fractions was tested (data not
shown). All AA isomers failed to modify the difference absorption
spectrum. In the same way, no spectral modification was observed
by the addition of tert-butyl-isocyanide (at the indicated concentra-
tion) in the absence of any AA isomer. As previously observed [34],
the addition of *cis*-AA (Fig. 7, trace “a”) to the dithionite-reduced
membrane fraction treated by tert-butyl isocyanide shifted the Soret
peak from 429 to 432 nm (yeast membrane fraction, Fig. 7A) and
from 427 to 434 nm (bovine membrane fraction, Fig. 7B). In contrast
to *cis*-AA, neither the addition of *trans*-AA (trace “b”) nor that of *me*
AA (trace “c”) changed the absorption difference spectra. These re-
sults indicate that, in contrast to *cis*-AA, *trans*-AA and *me*-AA cannot
modulate the tert-butyl isocyanide binding to the membrane Cytb<sub>558</sub>.

3.7. *Effect of the trans-AA on the NADPH-oxidase complex assembly*

Although the assembly merges with oxidase activity, we analyzed
the impact of *cis*-, *trans*- and *me*-AA on steps that bring to assembly
completion. It has been previously described that ca. 5 min period of
preincubation is necessary to allow the cytosolic and membrane com-
ponent to completely assemble and to remain stable for several mi-
nutes [48]. We found also that 5 min incubation time was necessary
to assure a constant rate of production of superoxide, i.e. full activation
in our cell-free systems, indicating a complete assembly [56]. We per-
formed yeast membrane and cytosolic component co-sedimentation
by ultracentrifugation in the presence of the different AA isomers
and analyzed the distribution by immunoreaction using antibodies
against anti-p47^phox^ or anti-p67^phox^. Cytosolic proteins that had trans-
located to the membrane were co-localized on a sucrose gradient in
the membrane pellet, while those not assembled remained in the
supernatant of the gradient. In the pellet compartment, only samples incubated in the presence of cis-AA presented positive signal to anti-p47phox (Fig. 8A) and anti-p67 (Fig. 8B). Concomitantly, the signals in the corresponding supernatant compartments were diminished. These results are consistent with the notion that cis-AA is a potent activator by favoring the assembly step. In contrast, preincubation with trans-AA, me-AA or without any fatty acid leads to anti-p47phox and

anti-p67phox recognitions mainly in the supernatant compartment indicating that no translocation to the membrane of these proteins occurred in those cases.

## 4. Discussion

Polyunsaturated fatty acids such as cis-AA are usual targets for a variety of free radicals [39,58,59] leading to generation of endogenous trans fatty acids by radical-induced cis-trans isomerization [36,38,60–64]. It has been shown that trans unsaturated fatty acids were ineffective on the release of superoxide from human neutrophils [65], but other authors working with cell-free system, have suggested that both cis and trans unsaturated fatty acids might activate NADPH-oxidase [66]. Although the necessity for anionic amphiphiles such as arachidonic acid to trigger the NADPH oxidase activation in cell-free system is well documented, the precise role played by arachidonic acid is still uncertain. Evidences have been reported on amphiphile-induced conformational changes in p47phox similar to those required to induce oxidase activity by phosphorylation [23,24] favoring the binding of the SH3 domain of p47phox to the p22phox subunit of the Cytb<sub>558</sub> [27]. CisAA, SDS and lithium dodecyl sulfate (LDS) have also been shown to induce conformational changes in flavocytochrome b<sub>558</sub> [30,32,34]. All activating molecules possess similar characteristics that define the requirement for effective action: an anionic head group and either an unsaturated or a restricted length of the saturated fatty acid part [18,19,30,67]. Thus, it was of particular interest to clarify whether trans-AA isomers are able to activate the NADPH-oxidase complex in a cell free system with similar efficiency to cis-AA and whether its targets are among the cytosolic and membrane oxidase components.

The major result that we have shown here is that, in contrast to cis-AA, trans-AA was not able to activate the NADPH-oxidase complex but moreover can inhibit the cis-AA-elicited superoxide production by interacting directly with the cytosolic component p67phox and with the membrane fraction, probably indirectly by modifying the membrane physical properties.

### 4.1. Effects on cytosolic proteins

We have followed the changes in the intrinsic tryptophan fluorescence of isolated p47phox and p67phox treated with either cis-AA, trans-AA or me-AA. Both anionic AA isoforms quenched the tryptophan fluorescence signal of p47phox down to about 70–65%. The effect of me-AA is undoubtedly different and indicates that the conformational changes associated with the methyl ester of fatty acid are only partial. The absence of major differences between the cis- and trans-isomers suggests that inhibition of the NADPH-oxidase activity is not due to a direct effect of trans-AA on p47phox. It is postulated that cis-AA mimics phosphorylation events from the cellular signalization by adding negative charges. It is known that in vivo p47phox is phosphorylated sequentially. Three serine residues (S345, S359, S370; [68]) are first phosphorylated followed by additional phosphorylations on seven other serines, all situated on the C-terminal domain. The phosphorylation of these residues is necessary for the translocation to the membrane, in unveiling the SH3 domains-mediated binding of p47phox, which contain five of the seven tryptophan residues. Because of the absence of negative

Fig. 5. Intrinsic fluorescence of p47phox treated with different arachidonic acid (AA) isoamers. 125 nM p47phox was treated with raising concentration of cis-AA (panel A), trans-AA (panel B) or me-AA (panel C), and the emission spectrum was measured using an excitation wavelength of 280 nm as described in the Materials and methods section. In seek of clarity, only the first 7 concentrations were included in the figure. They correspond from top to bottom to the following concentrations of the 3 different AA isomers: 0 (blue thick line), 8.2; 16.4; 24.6; 32.8; 41.1 and 57.5 µM. Background buffer signal (black thin line) and 8 M urea treated protein spectrum (red thick line) were also included. Results are representative of at least three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /><figcaption>Fig. 6. Intrinsic fluorescence of p67phox treated with different arachidonic acid (AA) isomers. 45 nM p67phox was treated with increasing concentrations of cis-AA (panel A), trans AA (panel B) or me AA (panel C), and the emission spectrum was measured using an excitation wavelength of 280 nm as described in the Materials and methods section. In seek of clarity, only the first 7 concentrations were included in the figure. They correspond from top to bottom to the following concentrations of the 3 different AA isomers (in µM): 0 (blue thick line), 8.2; 16.4; 24.6; 32.8; 41.1 and 57.5. Background buffer signal (black thin line) and 8 M urea treated protein spectrum (red thick line) were also included. Peak wavelength value (panel D), was plotted as a function of arachidonic acid concentration. Results are representative of at least three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

charge, *me*-AA cannot contribute to the repulsive effect known to induce the active form of p47phox protein and therefore is unable to totally mimic the phosphorylation events. In the case of *me*-AA addition, the structural changes are limited to hydrophobic interactions involving the fatty acyl chain.

The effects of AA isomer addition on isolated p67phox were surprising. Indeed, the tryptophan fluorescence intensities were lowered as observed with p47phox. Conversely, in addition to the fluorescence quenching, *trans*-AA led to a progressive red shift (up to 20 nm) of the maximum fluorescence wavelength of p67phox close to the wavelength maxima determined for denaturated protein. This indicates that the environment of tryptophan residues was strongly affected by the addition of *trans*-AA, bringing tryptophans from non polar to polar environment. *Trans*-AA is able to induce very large structural changes in p67phox that *cis*-AA cannot perform. These results

underline the importance of the conformation of the acyl chain of fatty acids given by different double bond geometries (Fig. S1). The *trans*-AA induced “denaturation” of p67phox could be the major cause of the inhibition of *cis*-AA-induced NADPH-oxidase activity. It means that the hydrophobic part of amphiphilic molecules contains the capacity to inhibit the system whereas the anionic part is crucial to modify the soluble proteins to their active forms.

## 4.2. Effects on protein translocation

Our results show that *trans*-AA can bind p47phox and p67phox but does not allow the NADPH complex assembly. The co-sedimentation experiments showed that no translocation to the membrane occurs in the presence of *me*-AA and *trans*-AA. If the absence of co-translocation to the membrane with *me*-AA is due to the absence of

<table><caption>Table 1

Characteristics of p47phox and p67phox intrinsic fluorescence quenching by arachidonic acid isomers.</caption><thead><tr><th rowspan="2"></th><th colspan="3">p47phox</th><th colspan="3">p67phox</th></tr><tr><th>1/Ksv<br>(µM)</th><th>fa</th><th>F<sub>min</sub><br>(%)</th><th>1/Ksv<br>(µM)</th><th>fa</th><th>F<sub>min</sub><br>(%)</th></tr></thead><tbody><tr><td>cisAA</td><td>21.93 ± 0.63</td><td>0.87 ± 0.03</td><td>29.79 ± 0.73</td><td>42.74 ± 0.30</td><td>0.69 ± 0.02</td><td>42.41 ± 0.61</td></tr><tr><td>transAA</td><td>31.45 ± 1.28</td><td>0.71 ± 0.01*</td><td>37.63 ± 0.72*</td><td>23.98 ± 0.39**</td><td>0.63 ± 0.03</td><td>42.53 ± 0.87</td></tr><tr><td>meAA</td><td>204.08 ± 38.22**</td><td>0.45 ± 0.10**</td><td>60.92 ± 2.54**</td><td>79.37 ± 4.17**</td><td>0.39 ± 0.02**</td><td>67.99 ± 0.83**</td></tr></tbody></table>

Quenching data from Figs. 5 and 6 were analyzed according to the standard Stern–Volmer relationship, F0/F= 1 + Ksv[AA], where F0 is the intensity of fluorescence at 335 nm in the absence of AA, F is the intensity of fluorescence at the same wavelength in the presence of different AA isomer concentrations, and Ksv is the Stern–Volmer quenching constant obtained from the slope of a plot of F0/F vs [AA] (Supplementary Fig. S4A and B). To estimate the accessible fraction *fa*, modified Stern–Volmer plots were used, according to the relation F0/(ΔF)= 1/fa+ 1/(fa Ksv [AA]). The *fa* values were extrapolated from the plot of F0/(ΔF) (Supplementary Figs. S4C and D).

◆ Pb 0.05.

** Pb 0.01.
<figure><img src="image_11.png" /><figcaption>Fig. 7. Effect of arachidonic acid isomers and tert-butyl isocyanide on the dithionite-reduced minus oxidized absorption spectra of membrane fraction. Yeast (panel A) or bovine (panel B) membrane fractions supplemented with 600 µM (yeast) or 200 µM (bovine) *cis*-AA (a, blue), *trans*-AA (b, red) or *me*-AA (c, green) were suspended in 200 µL phosphate buffer and incubated at 25 °C for 5 min. The dithionite reduced membranes were treated with tert-butyl-isocyanide as indicated in the Materials and methods section. Control spectra were performed in the absence of arachidonic acid isomers and with (e, orange) or without (d, black) tert-butyl-isocyanide. Results are representative of at least three separate experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

the anionic part, thereby impeding the formation of the active form of
cytosolic proteins, the effect of *trans*-AA is more subtle. The absence
of co-translocation to the membrane fraction in the presence of
*trans*-AA can be attributed to the dramatic structural changes of
p67<sup>phox</sup>.

## 4.3. Effects on the membrane fraction

The effect of *trans*-AA isomers on the NADPH-oxidase membrane
fraction is more complex. In contrast to *cis*-AA, neither *trans*-AA
isoforms nor *me*-AA facilitates the accessibility of the hemes to tert-
butyl-isocyanide as revealed by the unmodified the dithionite reduced
minus oxidized absorption spectra of *cytb*<sub>558</sub> (Fig. 7). It is likely that
the spin transition does not occur correctly in the presence of *trans*-
AA, supporting the hypothesis that the activation process of the
NADPH-oxidase is dependent of the heme iron spin state.

As was previously demonstrated the change of the natural geo-
metrical configuration of lipid unsaturations could also affect
NADPH-oxidase activity by modifying basic membrane physical prop-
erties [65]. This indirect effect of AA isomers depends on membrane

lipid composition since the NADPH oxidase activity could be modified
by the constitution of the lipid environment [41,69].

Indeed we found that the optimal *cis*-AA concentration for the ox-
idase activity is different depending on the source of *Cytb*<sub>558</sub> (bovine
vs. yeast membrane fraction), which present different lipid composi-
tions and *Cytb*<sub>558</sub>/total protein ratios.

At one hand, yeast membranes contain lower proportion of *Cytb*<sub>558</sub>
compared to neutrophil membranes (0.4% vs 7% of total membrane pro-
teins, respectively). Since activation experiments were performed using
similar amounts of *Cytb*<sub>558</sub>, yeast membrane assays contribute by
higher quantities of membrane lipids into the enzymatic reaction mix-
ture than neutrophil assays. The consequence is that the added
arachidonic acid was distributed proportionally to the amount of lipid
content leading to a decrease of the effective concentration of *cis*-AA
for yeast membranes compared to neutrophils.

It is known that the difference of fatty acid is due to the very dif-
ferent lipid biosynthesis pathways in yeast and mammalian. Howev-
er, if a strictly biophysical comparison is concerned, the results also
indicate that saturated fatty acids in yeast contribute to a membrane
environment with properties such as permeability and fluidity

<figure><img src="image_12.png" /><figcaption>Fig. 8. Effect of arachidonic acid isomers on NADPH-oxidase complex assembly. Recombinant *Cytb*<sub>558</sub>-enriched yeast membrane fraction, p47<sup>phox</sup>, p67<sup>phox</sup> and RacQ61L were pre-incubated together 5 min in the absence (w/o) or in the presence of *cis* (*cis*-AA), *trans* (*trans*-AA) or methyl ester (*me*-AA) arachidonic acid. Samples were then centrifuged 30 min at 150,000 g and supernatant and pellet were separated. SDS-PAGE and western blotting were performed using anti p47<sup>phox</sup> (A) or anti p67<sup>phox</sup> (B) polyclonal antibodies. Molecular weight markers (MM) were seeded as indicated.</figcaption></figure>
significantly lower compared to neutrophil membranes. In addition, yeast membranes contain ergosterol instead of cholesterol present in mammal cells that leads to a more condensed and thicker membrane and lipid domain formation as computed by molecular dynamics simulation studies [70]. Taken as a whole, the different membrane properties furnish here a coherent explanation to the differences observed for the optimal fatty acid concentrations that have to be added to activate the complex. Higher concentration of arachidonic acid is necessary to activate the highly ordered yeast membrane fraction, rich of saturated fatty acids, than to activate bovine membrane fraction. However it is still not clear how cis-AA activates the NADPH oxidase complex at a concentration above its critical micellar concentration (60 µM; [71]). Taken as a whole, our results will need additional studies to separate direct effects on cytb558 from effects on membrane properties.

Further studies should be performed to better understand the role of different fatty acid compositions, which can vary also in correlation with dietary intakes. The relative abundance of *cis* and *trans* arachidonic acid isomers has been described in models, such as cells and animals, as well as in human with pathological conditions and the relative abundance of *cis*-AA and its *mono-trans* isomers can vary from model to model. For example, in rat erythrocytes fed a *trans*-free diet it was found to be ca. 2% (0.4% relative to *cis*-AA). The presence of *trans*-AA in humans has been reported in the case of dermatological diseases (around 1% relative to *cis*-AA in [72]) or smokers [73], however an extrapolation to the in vivo impairment of NADPH oxidase activity is premature.

## 5. Conclusion

Taken as a whole, our results underlined the importance of the chemical characteristics of the activating molecule and especially its cis-trans configuration for the activation of the NADPH oxidase components. The role of AA and its geometrical transformations under cellular stress deserves further investigations taking into account that, from the effects found on the NADPH oxidase, this isomerization appears to contribute to a down regulation of oxidative stress by lowering the superoxide production. The nature of fatty acids ingested in food might thus have consequences at the level of non-specific immune defenses, which could be considered for potential pharmacological applications. It has been reported that free radical cis/trans isomerization of membrane lipids is connected with inflammation and allergy process [72–75]. Our results (if extrapolable to in vivo conditions) suggest that the *trans* fatty acids would act on these processes also by other mechanisms (at the level of membrane protein regulation). It is likely that the massive production of free radicals generated by the activated NADPH oxidase could contribute to the in situ concentration increase of *trans*-AA isomers and this aspect is worth further in vivo investigations.

Overall, the present work intends to highlight the importance of the lipid geometry in living systems, which can be considered either as a signal or damage, and to stimulate further interest for this relevant molecular aspect.

## Acknowledgements

This work was supported by the EDF Grant [no. RB 2011-14], the ANRjc [JCJC06-137200] and the ANR-02010-BLAN-1536-01 grants from the French National Agency for Research, and the European COST action CM0603. We would like to express our grateful thanks to Drs P. Deterre, J-J Lacapere and F. Lederer for their interesting and encouraging discussions.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbamem.2012.04.018.

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