S-Nitrosylation of IRP2 Regulates Its Stability via the Ubiquitin-Proteasome Pathway

Abstract

Nitric oxide (NO) is an important signaling molecule that interacts with different targets depending on its redox state. NO can interact with thiol groups resulting in S-nitrosylation of proteins, but the functional implications of this modification are not yet fully understood. We have reported that treatment of RAW 264.7 cells with NO caused a decrease in levels of iron regulatory protein 2 (IRP2), which binds to iron-responsive elements present in untranslated regions of mRNAs for several proteins involved in iron metabolism. In this study, we show that NO causes S-nitrosylation of IRP2, both in vitro and in vivo, and this modification leads to IRP2 ubiquitination followed by its degradation in the proteasome. Moreover, mutation of one cysteine (C178S) prevents NO-mediated degradation of IRP2. Hence, S-nitrosylation is a novel signal for IRP2 degradation via the ubiquitin-proteasome pathway.Physiologically, the majority of cells in vertebrates acquire iron from a well-characterized plasma glycoprotein, transferrin (Tf). Iron uptake from Tf involves the binding of Tf to the Tf receptors (TfR), internalization of Tf within an endocytic vesicle by receptor-mediated endocytosis, and the release of iron from Tf by a decrease in endosomal pH (15,39,41). Following iron release from Tf within endosomes, Fe 2ϩ passes through the endosomal membrane by divalent metal transporter 1 (2, 8) and then enters the poorly characterized intracellular labile pool (LIP). Intracellular iron that exceeds the requirement for the synthesis of functional heme and nonheme iron-containing proteins is stored within ferritin (39, 41).In general, cellular iron homeostasis is regulated posttranscriptionally by the cytoplasmic factors iron regulatory proteins 1 and 2 (IRP1 and IRP2), which "sense" iron levels in the LIP (7,14,35,41). In the absence of iron in the LIP, IRPs bind to specific nucleotide sequences called iron-responsive elements (IREs), which are located in the 3Ј untranslated region of TfR mRNA (36, 41) and the 5Ј untranslated region of ferritin mRNA (14,30,41). The binding of IRPs to IREs stabilizes TfR mRNA and blocks ferritin mRNA translation. In ironreplete cells, IRP1 contains a [4Fe-4S] cluster and binds RNA with low affinity (13, 41). IRP2, however, does not have the iron-sulfur cluster and is degraded under iron-replete conditions. This degradation is dependent on a 73-amino-acid insertion, rich in cysteine, which is absent in IRP1 (11,20). Hence, the expansion of the LIP inactivates IRP1 binding to IREs and leads to a degradation of IRP2, resulting in a rapid degradation of TfR mRNA and an efficient translation of ferritin mRNA (7,35,41). Importantly, IRPs can also be affected by various forms of oxidative stress and nitric oxide (NO) (6,12,15,38,40,46).NO is an important signaling molecule (17, 18) that interacts with different targets depending on its redox state. The reduced form of NO (the notation NO is used here as a generic expression encompassing all nitrogen monoxide species), NO ⅐ , interacts mainly with iron ...

Redox control of iron regulatory protein 2 stability

“…We speculate that the Fe-O-Fe center of FBXL5 may be amenable to redox control and sensitized by oxidative stress. However, our data do not exclude the possibility for alternative pathways that may contribute to the redox control of IRP2 stability, for example involving prolyl-hydroxylases [15,16] or endogenous heme [37][38][39]. Dissection of the molecular pathway underlying the redox regulation of IRP2 awaits further experimentation.…”

Human iron regulatory protein 2 is easily cleaved in its specific domain: consequences for the haem binding properties of the protein

“…The results presented herein indicate that IRP2 may be degraded in cells by additional pathways to the one leading to the proteasome [14,[41][42][43]. The mechanism by which this protein senses intracellular iron awaits further investigation.…”

Iron Regulatory Protein 1 Outcompetes Iron Regulatory Protein 2 in Regulating Cellular Iron Homeostasis in Response to Nitric Oxide

“…Under these conditions, discrepancies mostly concern not only the intrinsic contribution of each IRP to regulate downstream IRP targets but also the mechanism of NO-dependent IRP2 down-regulation (12-14, 17, 35). An explanation for the effect of NO has been proposed, suggesting that S-nitrosylation at Cys-178 leads to proteasomedependent IRP2 degradation (17). However, this mechanism, once believed in, was not confirmed by other authors and is still a matter of controversy (35).…”

“…Most studies focusing on the regulation of IRPs in macrophages reported that NO, in contrast to iron, conversely regulates IRP1 and IRP2. Although the IRP1 IRE binding activity is strongly activated by NO, IRP2 is down-regulated in NO-treated (14,17) or IFN-␥ LPS-stimulated macrophages (12,13). Under these conditions, discrepancies mostly concern not only the intrinsic contribution of each IRP to regulate downstream IRP targets but also the mechanism of NO-dependent IRP2 down-regulation (12-14, 17, 35).…”