Decreased protein nitration in macrophages that overexpress indoleamine 2, 3-dioxygenase.
The activity of indoleamine 2, 3-dioxygenase (IDO; E.C. 1.13.11.42) catalyzes the oxidative cleavage of tryptophan to form kynurenine. IDO activity consumes superoxide anions; therefore, we postulated that over-expression of IDO might mitigate superoxide-anion dependent, oxidative modification of cellular proteins in vitro. We prepared and characterized RAW 264.7 macrophages that were stably transfected with either an IDO expression vector or the control (empty) vector. We detected IDO mRNA, protein, and enzyme activity in the IDO-transfected macrophages, but not in the macrophages transfected with the empty vector. To generate superoxide anions in situ, we treated the IDO-and control-transfected cultures with xanthine or hypoxanthine, and then used ELISA methods to quantitate the relative levels of oxidatively modified proteins in total cell lysates. The levels of protein carbonyls were similar in IDO-transfected and vector-transfected macrophages; however, protein nitration was significantly less in IDO-transfected cells compared to control transfectants. In addition, steady-state levels of superoxide anions were significantly lower in the IDO-transfected cultures compared with control transfectants. Our results are consistent with the concept that, besides degrading tryptophan, IDO activity may protect cells from oxidative damage.
Keskin
Derin B
DB
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, 30912, USA, Derin_keskin@dfci.harvard.edu.
Marshall
Brendan
B
Munn
David
D
Mellor
Andrew L
AL
Gearhart
Debra A
DA
D000818:Animals; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009566:Nitrates / Q000378:metabolism*; D050050:Protein Carbonylation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014162:Transfection
D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D009566:Nitrates; D012333:RNA, Messenger; D013481:Superoxides