Cysteine Oxidation Regulates the RNA-Binding Activity of Iron Regulatory Protein 2

Abstract

Iron regulatory protein 2 (IRP2) is an RNA-binding protein that regulates the posttranscriptional expression of proteins required for iron homeostasis such as ferritin and transferrin receptor 1. IRP2 RNA-binding activity is primarily regulated by iron-mediated proteasomal degradation, but studies have suggested that IRP2 RNA binding is also regulated by thiol oxidation. We generated a model of IRP2 bound to RNA and found that two cysteines (C512 and C516) are predicted to lie in the RNA-binding cleft. Site-directed mutagenesis and thiol modification show that, while IRP2 C512 and C516 do not directly interact with RNA, both cysteines are located within the RNA-binding cleft and must be unmodified/reduced for IRP2-RNA interactions. Oxidative stress induced by cellular glucose deprivation reduces the RNA-binding activity of IRP2 but not IRP2-C512S or IRP2-C516S, consistent with the formation of a disulfide bond between IRP2 C512 and C516 during oxidative stress. Decreased IRP2 RNA binding is correlated with reduced transferrin receptor 1 mRNA abundance. These studies provide insight into the structural basis for IRP2-RNA interactions and reveal an iron-independent mechanism for regulating iron homeostasis through the redox regulation of IRP2 cysteines.Iron is an essential nutrient required for a variety of cellular processes, including DNA synthesis, respiration, and heme biosynthesis. However, ferrous iron readily reacts with hydroperoxides to produce hydroxyl radicals that can cause cellular damage. As both iron excess and deficiency are deleterious, cells have developed mechanisms to ensure that iron levels are sufficient for cellular need but at the same time limit iron toxicity.Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are the key iron sensors in mammalian cells (32,48). IRPs are cytosolic proteins that bind RNA stem-loops known as iron-responsive elements (IREs) located in the 5Ј or 3Ј untranslated regions of mRNAs encoding proteins involved in iron homeostasis. IRP binding to a 5Ј IRE present in ferritin (iron storage) or ferroportin (iron exporter) mRNAs represses protein translation. IRP binding to 3Ј IREs, such as those in transferrin receptor 1 (TfR-1) and divalent metal transporter 1 (DMT1) (iron importers), stabilizes the mRNA and increases protein expression. While both IRPs function as RNA-binding proteins when iron content is low, increased cellular iron regulates IRP1 and IRP2 by different mechanisms. Increased cellular iron results in the assembly of an [4Fe-4S] cluster in the RNA-binding cleft of IRP1, which allows IRP1 to function as a cytosolic aconitase. Unlike IRP1, IRP2 does not coordinate an [4Fe-4S] cluster or function as an aconitase. Instead, the RNA-binding activity of IRP2 is reduced by iron-dependent polyubiquitylation and proteasomal degradation. The role of IRP2 as the predominant RNA-binding protein in vivo has been established in murine knockout models in which Irp1 Ϫ/Ϫ mice display no overt phenotype, whereas Irp2 Ϫ/Ϫ mice develop microcytic anemia and locomotor deficits...

PGRL1 Participates in Iron-induced Remodeling of the Photosynthetic Apparatus and in Energy Metabolism in Chlamydomonas reinhardtii

“…Obviously, the two hypotheses are not mutually exclusive, and a model where the iron-loading and redox state of cysteines would be synergistic to impact CEF and iron-sensing is also conceivable. In this context it is of note that for IRP2 also, cysteine oxidation besides iron availability regulates the RNA binding activity of the protein, thereby demonstrating that iron-and redox-dependent regulation may occur in conjunction (58). In general oxidoreduction of protein thiols in redox regulation is a common (59) and in particular a well known mechanism in chloroplasts (60,61).…”

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

“…The binding is modulated by cellular iron and redox status: when iron levels are low, both IRP1 and IRP2 sit onto the IREs of H and L (figure 4) mRNAs, thus blocking their translation; when iron is available IRP2 is degraded by the proteasome (Guo et al 1995), while IRP1 binds to a 4Fe-4S cluster and covert to the cytosolic aconitase enzyme (Rouault and Tong 2005). Under oxidative condition the iron sulfur cluster of aconitase can be destroyed, thus inducing the switch to IRP1 (Pantopoulos et al 1997), while the oxidation of cysteines in IRP2 promotes its degradation (Zumbrennen et al 2009). 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.…”

Fumarate Mediates a Chronic Proliferative Signal in Fumarate Hydratase-Inactivated Cancer Cells by Increasing Transcription and Translation of Ferritin Genes

“…3C and Table S2). C512 was previously shown to be critical in maintaining the translational repression function of IRP2 (11). Thus, succination of IRP2 may partly contribute to the increased ferritin protein levels in FH-inactivated cells.…”