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J. Biol. Chem., Vol. 279, Issue 41, 43345-43351, October 8, 2004

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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^*

Deyarina Gonzalez, Jean-Claude Drapier, and Cécile Bouton

From the Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, Avenue de la Terrasse, 91190 Gif-sur-Yvette, France

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- and lipopolysaccharide (IFN-/LPS) and then triggered by phorbol 12-myristate 13-acetate (PMA) in order to promote co-generation of NO^. and . 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 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-/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.

Received for publication, February 20, 2004 , and in revised form, July 15, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 33-1-69-82-30-10; Fax: 33-1-69-07-72-47; E-mail: Cecile.Bouton{at}icsn.cnrs-gif.fr.

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