Oxygen and Iron Regulation of Iron Regulatory Protein 2

Abstract

Iron regulatory protein 2 (IRP2) is a central regulator of cellular iron homeostasis due to its regulation of specific mRNAs encoding proteins of iron uptake and storage. Iron regulates IRP2 by mediating its rapid proteasomal degradation, where hypoxia and the hypoxia mimetics CoCl 2 and desferrioxamine (DFO) stabilize it. Previous studies showed that iron-mediated degradation of IRP2 requires the presence of critical cysteines that reside within a 73-amino acid unique region. Here we show that a mutant IRP2 protein lacking this 73-amino acid region degraded at a rate similar to that of wild-type IRP2. In addition, DFO and hypoxia blocked the degradation of both the wild-type and mutant IRP2 proteins. Recently, members of the 2-oxoglutarate (2-OG)-dependent dioxygenase family have been shown to hydroxylate hypoxia-inducible factor-1␣ (HIF-1␣), a modification required for its ubiquitination and proteasomal degradation. Since 2-OG-dependent dioxygenases require iron and oxygen, in addition to 2-OG, for substrate hydroxylation, we hypothesized that this activity may be involved in the regulation of IRP2 stability. To test this we used the 2-OG-dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG) and showed that it blocked iron-mediated IRP2 degradation. In addition, hypoxia, DFO and DMOG blocked IRP2 ubiquitination. These data indicate that the region of IRP2 that is involved in IRP2 iron-mediated degradation lies outside of the 73-amino acid unique region and suggest a model whereby 2-OG-dependent dioxygenase activity may be involved in the oxygen and iron regulation of IRP2 protein stability.Iron regulatory proteins 1 and 2 (IRP1 and IRP2) 1 are cytosolic RNA-binding proteins that have a central role in iron homeostasis (reviewed in Refs. 1 and 2). IRP1 and IRP2 bind to stem-loop structures, known as iron-responsive elements, that are found in the untranslated regions of mRNAs encoding proteins important in iron homeostasis. The interaction of IRPs with iron-responsive elements represses translation of ferritin mRNA while increasing the stability of the transferrin receptor mRNA. Since IRP1 and IRP2 RNA binding activities are regulated by intracellular iron concentration, these proteins couple cellular iron with the regulation of expression of proteins important in iron homeostasis. In iron-replete cells, IRP1 RNA binding activity decreases as the protein assembles a [4Fe-4S] cluster having cytosolic aconitase activity, whereas in irondepleted cells apo-IRP1 binds RNA (3). In the case of IRP2, iron controls its RNA binding activity through the regulation of protein stability. This is achieved through iron stimulation of its ubiquitination and proteasomal degradation (4 -7). Thus, the amount of IRP2 is inversely related to cellular iron concentration. The mechanism by which iron accelerates IRP2 degradation has been reported to involve iron-catalyzed oxidation of the protein within a unique 73-amino acid region termed the "degradation domain" (6, 7). It has been recently shown that in vitro iron can oxidiz...

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.…”

“…The reducing activity of ascorbate is considered essential for the replenishment of ferrous iron and activation of prolyl-hydroxylases. Prompted by earlier data showing at least partial stabilization of IRP2 by pharmacological inhibitors of prolyl-hydroxylases [15,16], we hypothesized that oxidative stress may also protect IRP2 against degradation, by analogy to HIFa. Indeed, treatments of cells with H 2 O 2 or menadione stabilized IRP2 ( Figs.…”

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

“…More recent results have proposed that the 73aa-Domain plays a role in IRP2 degradation in response to haem and that the cysteine residue of a Cys-Pro-Phe-His, haem regulatory-like, motif in the domain binds oxidized haem [17][18][19]. However, studies by independent groups showed that an IRP2 deletion mutant lacking the full-length domain remains sensitive to iron-dependent degradation, similar to wild-type IRP2 [20,21].…”

Biology of ferritin in mammals: an update on iron storage, oxidative damage and neurodegeneration

“…Under reducing condition there is no urgent need to sequester iron and ferritin synthesis can be repressed, while the opposite happens in the presence of an oxidative environment. No definitive explanation exist yet for the existence of two IRPs with different type of regulation, even though IRP2 seems to be the only functional IRE ligand at low oxygen tension (Hanson et al 2003).…”