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

Abstract

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.

Keywords: Chaperone; Protein disulfide isomerase; Reductase activity; Tempol.