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Exploring the arachidonic acid‐induced structural changes in phagocyte NADPH oxidase p47 phox and p67 phox via thiol accessibility and SRCD spectroscopy
Author(s) -
Bizouarn Tania,
Karimi Gilda,
Masoud Rawand,
Souabni Hager,
Machillot Paul,
Serfaty Xavier,
Wien Frank,
Réfrégiers Matthieu,
HouéeLevin Chantal,
Baciou Laura
Publication year - 2016
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13779
Subject(s) - nadph oxidase , superoxide , arachidonic acid , chemistry , oxidase test , phospholipase a2 , cytosol , phagocyte , biochemistry , thiol , enzyme , stereochemistry , biology , microbiology and biotechnology , phagocytosis
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.