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.
Laboratoire de Chimie Physique UMR 8000, Univ. Paris-Sud, CNRS, Université Paris Saclay, Orsay Cedex, France.
Tania
T
Bizouarn
Laboratoire de Chimie Physique UMR 8000, Univ. Paris-Sud, CNRS, Université Paris Saclay, Orsay Cedex, France.
Gilda
G
Karimi
Laboratoire de Chimie Physique UMR 8000, Univ. Paris-Sud, CNRS, Université Paris Saclay, Orsay Cedex, France.
Rawand
R
Masoud
Laboratoire de Chimie Physique UMR 8000, Univ. Paris-Sud, CNRS, Université Paris Saclay, Orsay Cedex, France.
Hager
H
Souabni
Laboratoire de Chimie Physique UMR 8000, Univ. Paris-Sud, CNRS, Université Paris Saclay, Orsay Cedex, France.
Paul
P
Machillot
Laboratoire de Chimie Physique UMR 8000, Univ. Paris-Sud, CNRS, Université Paris Saclay, Orsay Cedex, France.
Xavier
X
Serfaty
Synchrotron SOLEIL, Campus Paris-Saclay, Gif-sur-Yvette Cedex, France.
Frank
F
Wien
Synchrotron SOLEIL, Campus Paris-Saclay, Gif-sur-Yvette Cedex, France.
Matthieu
M
Réfrégiers
Laboratoire de Chimie Physique UMR 8000, Univ. Paris-Sud, CNRS, Université Paris Saclay, Orsay Cedex, France.
Chantal
C
Houée-Levin
Laboratoire de Chimie Physique UMR 8000, Univ. Paris-Sud, CNRS, Université Paris Saclay, Orsay Cedex, France.
Laura
L
Baciou
D016718:Arachidonic Acid / Q000737:chemistry*; D002942:Circular Dichroism; D006801:Humans; D019255:NADPH Oxidases / Q000737:chemistry* / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D010750:Phosphoproteins; D013438:Sulfhydryl Compounds; C079789:neutrophil cytosol factor 67K; D016718:Arachidonic Acid; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1
NADPH oxidase; arachidonic acid; circular dichroism; superoxide production; thiol groups