Nitroarachidonic Acid (NO2AA) Inhibits Protein Disulfide Isomerase (PDI) Through Reversible Covalent Adduct Formation with Critical Cysteine Residues

Nitroarachidonic acid (NO 2 AA) is a nitroalkene exhibiting pleiotropic anti-inflammatory actions in many cell types. We have recently shown that NO 2 AA inhibits phagocytic NADPH oxidase 2 (NOX2) by preventing formation of the active complex in the membrane, without affecting the phosphorylation of cytosolic subunits. Recent work indicates the participation of Protein disulfide isomerase (PDI) activity on NOX2 active complex formation. Thus, the nitroalkene could affect PDI activity which might help to explain our previous observations on NOX2 inhibition. We determined that both the reductase and chaperone activities of PDI were inhibited by NO 2 AA in a dose and time- dependent manner, being a reversible effect. The mechanism of inhibition did not involve the release of nitric oxide ( ● NO). Since nitroalkenes are potent electrophiles and PDI has critical cysteine residues for its activity, the formation of a reversible covalent adduct between NO 2 AA and PDI may occur. We demonstrated the reversible covalent modification of PDI by NO 2 AA using the modified PEG switch assay as well as LC-MS/MS. Trypsinization of modified PDI confirmed that Cys residues present in the active site of PDI were key targets accounting for nitroalkene modification. Future work will be directed to determine if these modifications participates in the reported NO 2 AA modulation of NOX2.