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

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(558)) and four cytosolic proteins p47(phox), p67(phox), Rac and p40(phox). 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(558) and the cytosolic p67(phox). Our results suggest that the loss of the natural geometric feature (cis-AA) induces substantial structural modifications of p67(phox) that prevent its translocation to the complex.

Contribution of lipid environment to NADPH oxidase activity: Influence of sterol

“…Subcellular fractionation of yeast cells 0.7 ml of total membrane fractions containing 1.5e6 mg/ml membrane proteins was loaded on top of a discontinuous sucrose gradient as described in Ref. [22]. Briefly, in an SW32 centrifuge tube 3.7 ml of STE 60 (60% sucrose), STE sucrose, 20 mM TriseHCl (pH 7.4), 1 mM EDTA, 1 mM NaN 3 ).…”

Arachidonic Acid Induces Direct Interaction of the p67 -Rac Complex with the Phagocyte Oxidase Nox2, Leading to Superoxide Production

“…By contrast, the endogenous PMA analog diacylglycerol acts transiently because of its fast metabolism (41). Nox2 has been shown to be activated by cis-unsaturated fatty acids but not by trans-unsaturated or saturated ones (8,42). Indeed, oleic acid activated the Nox2-based oxidase reconstituted in CHO cells (Fig.…”

NOX Modifiers—Just a Step Away from Application in the Therapy of Airway Inflammation?

“…The study shows that p67phox integrity is crucial for the oxidase activity and that irradiation of p67phox drastically decreases its interaction with arachidonic acid (AA) and destabilizes the p47phox-p67phox complex (93,110). The precise role played by AA still remains unclear, though Souabni et al have shown that trans-arachidonic acid (trans-AA), in contrast to cis-AA, cannot activate the NADPH oxidase but is able to inhibit cis-AA-activated generation of O 2 -through a direct interaction with p67phox and with the membrane fraction, probably indirectly via modification of the membrane physical properties (138).…”