Nitroarachidonic acid (NO2AA) inhibits protein disulfide isomerase (PDI) through reversible covalent adduct formation with critical cysteines

Abstract

Background: Nitroarachidonic acid (NO₂AA) exhibits pleiotropic anti-inflammatory actions in a variety of cell types. We have recently shown that NO₂AA inhibits phagocytic NADPH oxidase 2 (NOX2) by preventing the formation of the active complex. Recent work indicates the participation of protein disulfide isomerase (PDI) activity in NOX2 activation. Cysteine (Cys) residues at PDI active sites could be targets for NO₂AA- nitroalkylation regulating PDI activity which could explain our previous observation.

Methods: PDI reductase and chaperone activities were assessed using the insulin and GFP renaturation methods in the presence or absence of NO₂AA. To determine the covalent reaction with PDI as well as the site of reaction, the PEG-switch assay and LC-MS/MS studies were performed.

Results and conclusions: We determined that both activities of PDI were inhibited by NO₂AA in a dose- and time- dependent manner and independent from release of nitric oxide. Since nitroalkenes are potent electrophiles and PDI has critical Cys residues for its activity, then formation of a covalent adduct between NO₂AA and PDI is feasible. To this end we demonstrated the reversible covalent modification of PDI by NO₂AA. Trypsinization of modified PDI confirmed that the Cys residues present in the active site a' of PDI were key targets accounting for nitroalkene modification.

General significance: PDI may contribute to NOX2 activation. As such, inhibition of PDI by NO₂AA might be involved in preventing NOX2 activation. Future work will be directed to determine if the covalent modifications observed play a role in the reported NO₂AA inhibition of NOX2 activity.

Keywords: Mass spectrometry; Nitroalkene; Nitroarachidonic acid; Protein disulfide isomerase.