Nitration and Inactivation of IDO by Peroxynitrite

Abstract

IDO induction can deplete l-tryptophan in target cells, an effect partially responsible for the antimicrobial activities and antiallogeneic T cell responses of IFN-γ in human macrophages, dendritic cells, and bone marrow cells. l-Tryptophan depletion and NO production are both known to have an antimicrobial effect in macrophages, and the interaction of these two mechanisms is unclear. In this study we found that IDO activity was inhibited by the peroxynitrite generator, 3-(4-morpholinyl)sydnonimine, in PMA-differentiated cytokine-induced THP-1 (acute monocytic leukemia) cells and IFN-γ-stimulated PBMCs, whereas IDO protein expression was unaffected compared with that in untreated cells. Nitrotyrosine was detected in immunoprecipitated (IP)-IDO from PMA-differentiated cytokine-induced THP-1 cells treated with 3-(4-morpholinyl)sydnonimine, but not from untreated cells. Treatment of IP-IDO and recombinant IDO (rIDO) with peroxynitrite significantly decreased enzyme activity. Nitrotyrosine was detected in both peroxynitrite-treated IP-IDO and rIDO, but not in either untreated IP-IDO or rIDO. Peptide analysis by liquid chromatography/electrospray ionization and tandem mass spectrometry demonstrated that Tyr15, Tyr345, and Tyr353 in rIDO were nitrated by peroxynitrite. The levels of Tyr nitration and the inhibitory effect of peroxynitrite on IDO activity were significantly reduced in the Tyr15-to-Phe mutant. These results indicate that IDO is nitrated and inactivated by peroxynitrite and that nitration of Tyr15 in IDO protein is the most important factor in the inactivation of IDO.

Dynamic interplay between nitration and phosphorylation of tubulin cofactor B in the control of microtubule dynamics

“…4D). Previous studies have shown that protein nitration can have an inhibitory effect on enzymatic activity (21). To test whether the decrease in TCoB phosphorylation in NO donor-treated cells was not caused by nitration of Pak1 itself, we tested the nitration status of Pak1.…”

Mouse Mesenchymal Stem Cells Suppress Antigen-Specific TH Cell Immunity Independent of Indoleamine 2,3-Dioxygenase 1 (IDO1)

“…The mechanisms involved in post-transcriptional and post-translational regulation of IDO1 enzyme activity remain elusive but may involve nitration of the enzyme by peroxynitrite [34].…”

Astrocyte Indoleamine 2,3-Dioxygenase Is Induced by the TLR3 Ligand Poly(I:C): Mechanism of Induction and Role in Antiviral Response

“…In several cell systems, IDO expression and activity have been shown to be inhibited by NO. For example, NO enhances proteosomal degradation of IDO, and NO donors inactivate IDO by inducing nitration of critical tyrosine residues (25,33). Therefore, we investigated the relationship between IDO and iNOS expression in astrocytes.…”

“…IDO biosynthesis is regulated at several steps. In particular, IDO expression and/or activity have been shown to be downregulated by NO in several cell culture and cell-free systems (25,33,52). We therefore asked whether NO generated by astrocyte iNOS can affect IDO expression or activity.…”