Arachidonic Acid and Nitroarachidonic: Effects on NADPH Oxidase Activity.

Arachidonic acid (AA) is a polyunsaturated fatty acid that participates in the inflammatory response mainly through bioactive-lipids formation in macrophages and also in the phagocytic NADPH oxidase 2 (NOX2) activation. NOX2 is the enzyme responsible for a huge superoxide formation in macrophages, essential to eliminate pathogens inside the phagosome. The oxidase is an enzymatic complex comprised of a membrane-bound flavocytochrome b 558 (gp91phox/p22phox), three cytosolic subunits (p47phox, p40phox and p67phox) and a Rac-GTPase. The enzyme becomes active when macrophages are exposed to appropriate stimuli that trigger the phosphorylation of cytosolic subunits and its migration to plasmatic membrane to form the active complex. It is proposed that AA stimulates NOX2 activity through AA interaction with different components of the NADPH oxidase complex. In inflammatory conditions, there is an increase in reactive oxygen and nitrogen species that results in the production of nitrated derivatives of AA, such as nitroarachidonic acid (NO2-AA). NO2-AA is capable to inhibit NOX2 activity by interfering with p47phox migration to the membrane without affecting phosphorylation of cytosolic proteins. Also, NO2-AA is capable to interact with protein disulfide isomerase (PDI), which is involved on NOX2 active complex formation. It has been demonstrated that NO2-AA forms a covalent adduct with PDI that could prevent the interaction with NOX2 and it would explain the inhibitory effects of the fatty acid upon NOX2. Together, current data indicate that AA is an important activator of NOX2 formed in the early events of the inflammatory response, leading to a massive production of oxidants that may, in turn, promote NO2-AA formation and shutting down the oxidative burst. Hence, AA and its derivatives could have antagonistic roles on NOX2 activity regulation.

Nitroxide 4-hydroxy-2,2',6,6'-tetramethylpiperidine 1-oxyl (Tempol) inhibits the reductase activity of protein disulfide isomerase via covalent binding to the Cys400 residue on CXXC redox motif at the a'active site.

BACKGROUND AND AIM: Oxidative stress arising from inflammatory processes is a serious cause of cell and tissue damage. Tempol is an efficient antioxidant with superoxide dismutase-like activity. The purpose of this paper is to address the inhibition of protein disulfide isomerase (PDI), an essential redox chaperone whose active sites contain the Cys-Gly-His-Cys (CXXC) motif, by the nitroxide Tempol. RESULTS: In the presence of Tempol (5-120 µM), the reductase activity of PDI was reversibly affected both in vitro and in activated mice neutrophils, with an IC50 of 22.9 ± 10.8 µM. Inhibitory activity was confirmed by using both the insulin method and fluorescent formation of eosin-glutathione (E-GSH). The capacity of Tempol to bind the enzyme was determined by EPR and mass spectrometry. EPR Tempol signal decreased in the presence of PDI while remained unaffected when PDI thiols were previously blocked with NEM. When total protein was analyzed, 1 and 4 molecules of Tempol were bound to the protein. However, only one was found to be covalently bound to PDI at the a'active site. More specifically, Cys400 was modified by Tempol. CONCLUSION: We have shown that the nitroxide Tempol acts as an inhibitor of PDI through covalent binding to the Cys400 of the protein structure. Since PDI is coupled with the assembly of the NADPH oxidase complex of phagocytes, these findings reveal a novel action of Tempol that presents potential clinical applications for therapeutic intervention to target PDI knockdown in pathological processes in which this protein is engaged.

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

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.

Nitroarachidonic acid prevents NADPH oxidase assembly and superoxide radical production in activated macrophages.

Nitration of arachidonic acid (AA) to nitroarachidonic acid (AANO2) leads to anti-inflammatory intracellular activities during macrophage activation. However, less is known about the capacity of AANO2 to regulate the production of reactive oxygen species under proinflammatory conditions. One of the immediate responses upon macrophage activation involves the production of superoxide radical (O2(•-)) due to the NADPH-dependent univalent reduction of oxygen to O2(•-) by the phagocytic NADPH oxidase isoform (NOX2), the activity of NOX2 being the main source of O2(•-) in monocytes/macrophages. Because the NOX2 and AA pathways are connected, we propose that AANO2 can modulate macrophage activation by inhibiting O2(•-) formation by NOX2. When macrophages were activated in the presence of AANO2, a significant inhibition of NOX2 activity was observed as evaluated by cytochrome c reduction, luminol chemiluminescence, Amplex red fluorescence, and flow cytometry; this process also occurs under physiological mimic conditions within the phagosomes. AANO2 decreased O2(•-) production in a dose- (IC50=4.1±1.8 µM AANO2) and time-dependent manner. The observed inhibition was not due to a decreased phosphorylation of the cytosolic subunits (e.g., p40(phox) and p47(phox)), as analyzed by immunoprecipitation and Western blot. However, a reduction in the migration to the membrane of p47(phox) was obtained, suggesting that the protective actions involve the prevention of the correct assembly of the active enzyme in the membrane. Finally, the observed in vitro effects were confirmed in an in vivo inflammatory model, in which subcutaneous injection of AANO2 was able to decrease NOX2 activity in macrophages from thioglycolate-treated mice.

Nitro-fatty acids: formation, redox signaling, and therapeutic potential.

SIGNIFICANCE: Nitrated derivatives of unsaturated fatty acids (nitro-fatty acids) are being formed and detected in human plasma, cell membranes, and tissue, triggering signaling cascades via covalent and reversible post-translational modifications of susceptible nucleophilic amino acids in transcriptional regulatory proteins and enzymes. RECENT ADVANCES: Nitro-fatty acids modulate metabolic as well as inflammatory signaling pathways, including the p65 subunit of nuclear factor κB and the transcription factor peroxisome proliferator-activated receptor-γ. Moreover, nitro-fatty acids can activate heat shock as well as phase II antioxidant responses. As electrophiles, they also activate the Nuclear factor erythroid 2-related factor 2 pathway. CRITICAL ISSUES: We first discuss the mechanisms of nitro-fatty acid formation as well as their key chemical and biochemical properties, including their capacity to release nitric oxide and exert antioxidant actions. The electrophilic properties of nitro-fatty acids to activate anti-inflammatory signaling pathways are discussed in detail. A critical issue is the influence of nitroarachidonic acid on prostaglandin endoperoxide H synthases, modulating inflammatory processes through redirection of arachidonic acid metabolism and signaling. FUTURE DIRECTIONS: Based on this information, we analyze in vivo data supporting nitro-fatty acids as promising pharmacological tools to prevent inflammatory diseases associated with oxidative and nitrative stress conditions. A key future issue is to evaluate whether nitro-fatty acid supplementation would be useful for human diseases linked to inflammation as well as their potential toxicity when administered by long periods of time.