title: 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-gamma 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-gamma-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.
authors.affiliation: Department of Informative Clinical Medicine, Gifu University Graduate School of Medicine, Gifu City, Japan.
authors.forename: Hidetsugu
authors.initials: H
authors.lastname: Fujigaki
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Saito
authors.forename: Felix
authors.initials: F
authors.lastname: Lin
authors.forename: Suwako
authors.initials: S
authors.lastname: Fujigaki
authors.forename: Kanako
authors.initials: K
authors.lastname: Takahashi
authors.forename: Brian M
authors.initials: BM
authors.lastname: Martin
authors.forename: Cai Y
authors.initials: CY
authors.lastname: Chen
authors.forename: Junichi
authors.initials: J
authors.lastname: Masuda
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Kowalak
authors.forename: Osamu
authors.initials: O
authors.lastname: Takikawa
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Seishima
authors.forename: Sanford P
authors.initials: SP
authors.lastname: Markey
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization; D014443:Tyrosine / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects
chemical_list: D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine