title: Nitroarachidonic acid (NO2AA) inhibits protein disulfide isomerase (PDI) through reversible covalent adduct formation with critical cysteines.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina-Universidad de la República, Montevideo, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina-Universidad de la República, Montevideo, Uruguay.
authors.forename: Lucía
authors.initials: L
authors.lastname: González-Perilli
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina-Universidad de la República, Montevideo, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina-Universidad de la República, Montevideo, Uruguay.
authors.forename: Mauricio
authors.initials: M
authors.lastname: Mastrogiovanni
authors.affiliation: Vascular Biology Laboratory, Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo, Brazil.
authors.forename: Denise
authors.initials: D
authors.lastname: de Castro Fernandes
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina-Universidad de la República, Montevideo, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina-Universidad de la República, Montevideo, Uruguay.
authors.forename: Homero
authors.initials: H
authors.lastname: Rubbo
authors.affiliation: Vascular Biology Laboratory, Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo, Brazil.
authors.forename: Francisco
authors.initials: F
authors.lastname: Laurindo
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina-Universidad de la República, Montevideo, Uruguay; Center for Free Radical and Biomedical Research, Facultad de Medicina-Universidad de la República, Montevideo, Uruguay. Electronic address: trocha@fmed.edu.uy.
authors.forename: Andrés
authors.initials: A
authors.lastname: Trostchansky
mesh_terms: D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D016718:Arachidonic Acid / Q000494:pharmacology*; D020134:Catalytic Domain; D003545:Cysteine / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000037:antagonists & inhibitors*; D014357:Trypsin / Q000378:metabolism
chemical_list: D000893:Anti-Inflammatory Agents; D008562:Membrane Glycoproteins; D016718:Arachidonic Acid; D009569:Nitric Oxide; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D014357:Trypsin; D019704:Protein Disulfide-Isomerases; D003545:Cysteine
keywords: Mass spectrometry; Nitroalkene; Nitroarachidonic acid; Protein disulfide isomerase