Endogenous nitration of iron regulatory protein-1 (IRP-1) in nitric oxide-producing murine macrophages: further insight into the mechanism of nitration in vivo and its impact on IRP-1 functions.
Iron regulatory protein-1 (IRP-1) is a bifunctional [4Fe-4S] protein that functions as a cytosolic aconitase or as a trans-regulatory factor controlling iron homeostasis at a post-transcriptional level. Because IRP-1 is a sensitive target protein for nitric oxide (NO), we investigated whether this protein is nitrated in inflammatory macrophages and whether this post-transcriptional modification changes its activities. RAW 264.7 macrophages were first stimulated with interferon-gamma and lipopolysaccharide (IFN-gamma/LPS) and then triggered by phorbol 12-myristate 13-acetate (PMA) in order to promote co-generation of NO* and O*2-.. IRP-1 was isolated by immunoprecipitation and analyzed for protein-bound nitrotyrosine by Western blotting. We show that nitration of endogenous IRP-1 in NO-producing macrophages boosted to produce O*2- was accompanied by aconitase inhibition and impairment of its capacity to bind the iron-responsive element (IRE) of ferritin mRNA. Lost IRE-binding activity was not recovered by exposure of IRP-1 to 2% 2-mercaptoethanol and was not due to protein degradation. Inclusion of cis-aconitate with cell extract to stabilize the [4Fe-4S] cluster of holo-IRP-1 rendered protein insensitive to nitration by peroxynitrite, suggesting that loss of [Fe-S] cluster and subsequent change of conformation are prerequisites for tyrosine nitration. IRP-1 nitration was strongly reduced when IFN-gamma/LPS/PMA-stimulated cells were incubated with myeloperoxidase inhibitors, which points to the contribution of the nitrite/H2O2/peroxidase pathway to IRP-1 nitration in vivo. Interestingly, under these conditions, IRP-1 recovered full IRE binding as assessed by treatment with 2% 2-mercaptoethanol. Peroxidase-mediated nitration of critical tyrosine residues, by holding IRP-1 in an inactive state, may constitute, in activated macrophages, a self-protecting mechanism against iron-induced toxicity.
Gonzalez
Deyarina
D
Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, Avenue de la Terrasse, 91190 Gif-sur-Yvette, France.
Drapier
Jean-Claude
JC
Bouton
Cécile
C
D000154:Aconitate Hydratase / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002392:Catechin / Q000737:chemistry; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005293:Ferritins / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D007371:Interferon-gamma / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D035941:Iron Regulatory Protein 1 / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008070:Lipopolysaccharides / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009584:Nitrogen / Q000737:chemistry*; D010544:Peroxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000494:pharmacology; D011485:Protein Binding; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012457:Salicylamides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
D007506:Iron-Sulfur Proteins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012457:Salicylamides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D008623:Mercaptoethanol; D007371:Interferon-gamma; C005703:salicylhydroxamic acid; D002392:Catechin; D005293:Ferritins; D006861:Hydrogen Peroxide; D007501:Iron; D010544:Peroxidases; D019001:Nitric Oxide Synthase; D000154:Aconitate Hydratase; D035941:Iron Regulatory Protein 1; D009584:Nitrogen; D013755:Tetradecanoylphorbol Acetate