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PubMed2004

Identification of a heme-sensing domain in iron regulatory protein 2.

Iron regulatory protein 2 coordinates the cellular regulation of iron metabolism by binding to iron-responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. The mechanisms that prevent degradation during iron deficiency or promote degradation during iron sufficiency are not delineated. Iron regulatory protein 2 contains a domain not present in the closely related iron regulatory protein 1, and we found that this domain binds heme with high affinity. A cysteine within the domain is axially liganded to the heme, as occurs in cytochrome P450. The protein-bound heme reacts with molecular oxygen to mediate the oxidation of cysteine, including beta-elimination of the sulfur to yield alanine. This covalent modification may thus mark the protein molecule for degradation by the proteasome system, providing another mechanism by which heme can regulate the level of iron regulatory protein 2.

Binding Sites↗

PubMed2003

Oxygen and iron regulation of iron regulatory protein 2.

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 CoCl2 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 alpha (HIF-1 alpha), 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.

Cell Hypoxia↗

PubMed1995

Requirements for iron-regulated degradation of the RNA binding protein, iron regulatory protein 2.

Iron regulatory proteins (IRPs) regulate the expression of genes involved in iron metabolism whose transcripts contain RNA stem-loop motifs known as iron-responsive elements (IREs). When iron concentrations are low, IRPs bind to IREs in the 5' untranslated region (UTR) of transcripts where they repress translation, or the 3' UTR of transcripts where they inhibit degradation. The RNA binding activities of the homologous proteins IRP1 and IRP2 are both regulated post-translationally. The binding activity of IRP2 is regulated by the degradation of the protein when cells are iron-replete. Here, we demonstrate that a 73 amino acid sequence that corresponds to a unique exon in IRP2 contains a sequence required for rapid degradation in iron-replete cells. The deletion of this sequence eliminates the rapid turnover of IRP2, whereas the transfer of this sequence to the corresponding position in the homologous protein IRP1 confers the capacity for iron-dependent degradation upon IRP1. Site-directed mutagenesis has demonstrated that specific cysteines within the IRP2 exon are required for iron-dependent degradation. The degradation of IRP2 appears to be mediated by the proteasome in iron-replete cells. When degradation is prevented, the RNA binding activity of IRP2 is not regulated by iron concentration. Thus, degradation is required for the regulation of the RNA binding activity of IRP2.

Amino Acid Sequence↗

PubMed2000

Effects of interferon-gamma and lipopolysaccharide on macrophage iron metabolism are mediated by nitric oxide-induced degradation of iron regulatory protein 2.

Iron regulatory proteins (IRP-1 and IRP-2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements, which are located in the 3'-untranslated region and the 5'-untranslated region of their respective mRNAs. Cellular iron levels affect binding of IRPs to iron-responsive elements and consequently expression of TfR and ferritin. Moreover, NO(*), a redox species of nitric oxide that interacts primarily with iron, can activate IRP-1 RNA binding activity resulting in an increase in TfR mRNA levels. Recently we found that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO(+) (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA binding of IRP-2 followed by IRP-2 degradation, and these changes were associated with a decrease in TfR mRNA levels (Kim, S., and Ponka, P. (1999) J. Biol. Chem. 274, 33035-33042). In this study, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) increased IRP-1 binding activity, whereas RNA binding of IRP-2 decreased and was followed by a degradation of this protein. Moreover, the decrease of IRP-2 binding/protein levels was associated with a decrease in TfR mRNA levels in LPS/IFN-gamma-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. Furthermore, LPS/IFN-gamma-stimulated RAW 264.7 cells showed increased rates of ferritin synthesis. These results suggest that NO(+)-mediated degradation of IRP-2 plays a major role in iron metabolism during inflammation.

Animals↗

PubMed1999

Hypoxia post-translationally activates iron-regulatory protein 2.

Iron-regulatory proteins 1 and 2 (IRP1 and IRP2) are RNA-binding proteins that post-transcriptionally regulate the expression of mRNAs that code for proteins involved in the maintenance of iron and energy homeostasis. Here we show that hypoxia differentially regulates the RNA binding activities of IRP1 and IRP2 in human 293 and in mouse Hepa-1 cells. In contrast to IRP1, where hypoxic exposure decreases IRP1 RNA binding activity, hypoxia increases IRP2 RNA binding activity. The hypoxic increase in IRP2 RNA binding activity results from increased IRP2 protein levels. Cobalt, which mimics hypoxia by activation of hypoxia-inducible factor 1 (HIF-1), also increases IRP2 protein levels; however, cobalt-induced IRP2 lacks RNA binding activity. Addition of a reductant to cobalt-treated extracts restored IRP2 RNA binding activity. Hypoxic activation of IRP2 is not because of an increase in transcriptional activation by HIF-1, because IRP2 accumulates in Hepa-1 cells lacking a functional HIF-1beta subunit, nor is it because of an increase in IRP2 mRNA stability. Rather, our data indicate that hypoxia increases IRP2 levels by a post-translational mechanism involving protein stability. Differential regulation of IRP1 and IRP2 during hypoxia may regulate specific IRP target mRNAs whose expression is required for hypoxic adaptation. Furthermore, these data imply mechanistic parallels between the hypoxia-induced post-transcriptional regulation of IRP2 and HIF-1alpha.

Animals↗

PubMed2005

Altered body iron distribution and microcytosis in mice deficient in iron regulatory protein 2 (IRP2).

Iron regulatory protein 2 (IRP2)-deficient mice have been reported to suffer from late-onset neurodegeneration by an unknown mechanism. We report that young adult Irp2-/- mice display signs of iron mismanagement within the central iron recycling pathway in the mammalian body, the liver-bone marrow-spleen axis, with altered body iron distribution and compromised hematopoiesis. In comparison with wild-type littermates, Irp2-/- mice are mildly microcytic with reduced serum hemoglobin levels and hematocrit. Serum iron and transferrin saturation are unchanged, and hence microcytosis is not due to an overt decrease in systemic iron availability. The liver and duodenum are iron loaded, while the spleen is iron deficient, associated with a reduced expression of the iron exporter ferroportin. A reduction in transferrin receptor 1 (TfR1) mRNA levels in the bone marrow of Irp2-/- mice can plausibly explain the microcytosis by an intrinsic defect in erythropoiesis due to a failure to adequately protect TfR1 mRNA against degradation. This study links a classic regulator of cellular iron metabolism to systemic iron homeostasis and erythropoietic TfR1 expression. Furthermore, this work uncovers aspects of mammalian iron metabolism that can or cannot be compensated for by the expression of IRP1.

Animals↗

PubMed1998

Involvement of heme in the degradation of iron-regulatory protein 2.

Iron-regulatory proteins (IRPs) recognize and bind to specific RNA structures called iron-responsive elements. Mediation of these binding interactions by iron and iron-containing compounds regulates several post-transcriptional events relevant to iron metabolism. There are two known IRPs, IRP1 and IRP2, both of which can respond to iron fluxes in the cell. There is ample evidence that IRP1 is converted by iron to cytoplasmic aconitase in vivo. It has also been shown that, under certain conditions, a significant fraction of IRP1 is degraded in cells exposed to iron or heme. Studies have shown that the degradation of IRP1 that is induced by iron can be inhibited by either desferrioxamine mesylate (an iron chelator) or succinyl acetone (an inhibitor of heme synthesis), whereas the degradation induced by heme cannot. This suggests that heme rather than iron is responsible for this degradation. Several laboratories have shown that IRP2 is also degraded in cells treated with iron salts. We now show evidence suggesting that this IRP2 degradation may be mediated by heme. Thus, in experiments analogous to those used previously to study IRP1, we find that IRP2 is degraded in rabbit fibroblast cells exposed to heme or iron salts. However, as shown earlier with IRP1, both desferrioxamine mesylate and succinyl acetone will inhibit the degradation of IRP2 induced by iron but not that induced by heme.

Animals↗

PubMed2005

Microcytic anemia, erythropoietic protoporphyria, and neurodegeneration in mice with targeted deletion of iron-regulatory protein 2.

Iron-regulatory proteins (IRPs) 1 and 2 posttranscriptionally regulate expression of transferrin receptor (TfR), ferritin, and other iron metabolism proteins. Mice with targeted deletion of IRP2 overexpress ferritin and express abnormally low TfR levels in multiple tissues. Despite this misregulation, there are no apparent pathologic consequences in tissues such as the liver and kidney. However, in the central nervous system, evidence of abnormal iron metabolism in IRP2-/- mice precedes the development of adult-onset progressive neurodegeneration, characterized by widespread axonal degeneration and neuronal loss. Here, we report that ablation of IRP2 results in iron-limited erythropoiesis. TfR expression in erythroid precursors of IRP2-/- mice is reduced, and bone marrow iron stores are absent, even though transferrin saturation levels are normal. Marked overexpression of 5-aminolevulinic acid synthase 2 (Alas2) results from loss of IRP-dependent translational repression, and markedly increased levels of free protoporphyrin IX and zinc protoporphyrin are generated in IRP2-/- erythroid cells. IRP2-/- mice represent a new paradigm of genetic microcytic anemia. We postulate that IRP2 mutations or deletions may be a cause of refractory microcytic anemia and bone marrow iron depletion in patients with normal transferrin saturations, elevated serum ferritins, elevated red cell protoporphyrin IX levels, and adult-onset neurodegeneration.

Anemia↗

PubMed2003

Iron regulatory protein 2 as iron sensor. Iron-dependent oxidative modification of cysteine.

Iron regulatory protein 2 coordinates cellular regulation of iron metabolism by binding to iron responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. This iron-dependent degradation requires the presence of a 73-residue degradation domain, but its functions have not yet been established. We now show that the domain can act as an iron sensor, mediating its own covalent modification. The domain forms an iron-binding site with three cysteine residues located in the middle of the domain. It then reacts with molecular oxygen to generate a reactive oxidizing species at the iron-binding site. One cysteine residue is oxidized to dehydrocysteine and other products. This covalent modification may thus mark the protein molecule for degradation by the proteasome system.

Amino Acids↗

PubMed1999

Control of transferrin receptor expression via nitric oxide-mediated modulation of iron-regulatory protein 2.

Cellular iron storage and uptake are coordinately regulated post-transcriptionally by cytoplasmic factors, iron-regulatory proteins 1 and 2 (IRP-1 and IRP-2). When iron in the intracellular transit pool is scarce, IRPs bind to iron-responsive elements (IREs) in the 5'-untranslated region of the ferritin mRNA and 3'-untranslated region of the transferrin receptor (TfR) mRNA. Such binding inhibits translation of ferritin mRNA and stabilizes the mRNA for TfR, whereas the opposite scenario develops when iron in the transit pool is plentiful. However, we (Richardson, D. R., Neumannova, V., Nagy, E., and Ponka, P. (1995) Blood 86, 3211-3219) and others reported that the binding of IRPs to IREs can also be modulated by nitric oxide (NO). In this study, we showed that a short exposure of RAW 264.7 cells (a murine macrophage cell line) to the NO(+) donor, sodium nitroprusside (SNP), caused a significant decrease in IRP-2 binding to the IREs followed by IRP-2 degradation and that these changes occurred without affecting IRP-1 binding. The SNP-mediated degradation of IRP-2 in RAW 264.7 cells could be prevented by MG-132 or lactacystin, known inhibitors of proteasome-dependent protein degradation. A SNP-mediated decrease in IRP-2 binding and levels was associated with a dramatic decrease in TfR mRNA levels and an increase in ferritin synthesis. Importantly, the proteasome inhibitor MG-132 prevented the SNP-mediated decrease in TfR mRNA levels. These observations suggest that IRP-2 can play an important role in controlling transferrin receptor expression.

Animals↗

PubMed2004

Genetic ablations of iron regulatory proteins 1 and 2 reveal why iron regulatory protein 2 dominates iron homeostasis.

The two iron regulatory proteins IRP1 and IRP2 bind to transcripts of ferritin, transferrin receptor and other target genes to control the expression of iron metabolism proteins at the post-transcriptional level. Here we compare the effects of genetic ablation of IRP1 to IRP2 in mice. IRP1-/- mice misregulate iron metabolism only in the kidney and brown fat, two tissues in which the endogenous expression level of IRP1 greatly exceeds that of IRP2, whereas IRP2-/- mice misregulate the expression of target proteins in all tissues. Surprisingly, the RNA-binding activity of IRP1 does not increase in animals on a low-iron diet that is sufficient to activate IRP2. In animal tissues, most of the bifunctional IRP1 is in the form of cytosolic aconitase rather than an RNA-binding protein. Our findings indicate that the small RNA-binding fraction of IRP1, which is insensitive to cellular iron status, contributes to basal mammalian iron homeostasis, whereas IRP2 is sensitive to iron status and can compensate for the loss of IRP1 by increasing its binding activity. Thus, IRP2 dominates post-transcriptional regulation of iron metabolism in mammals.

Animals↗

PubMed1999

Stabilization of iron regulatory protein 2, IRP2, by aluminum.

Iron regulatory protein 2 (IRP2) is one of the central regulators of iron homeostasis. IRP2 regulates expression of molecules involved in iron metabolism by binding to iron responsive elements (IREs) in the transcripts of those molecules in iron depletion. IRP2 is regulated by the accelerated degradation initiated by the iron-catalyzed oxidation. Here we report that aluminum antagonizes the iron-induced decrease in IRE binding activity of IRP2. Aluminum also inhibits iron-induced oxidation of IRP2 in vitro. These results suggest that aluminum stabilizes IRP2 by interfering with the iron-catalyzed oxidation, which results in perturbation of iron metabolism.

Aluminum↗

PubMed1995

Iron regulates the intracellular degradation of iron regulatory protein 2 by the proteasome.

Iron regulatory proteins (IRP1 and IRP2) are RNA-binding proteins that bind to specific structures, termed iron-responsive elements (IREs), that are located in the 5'- or 3'-untranslated regions of mRNAs that encode proteins involved in iron homeostasis. IRP1 and IRP2 RNA binding activities are regulated by iron; IRP1 and IRP2 bind IREs with high affinity in iron-depleted cells and with low affinity in iron-repleted cells. The decrease in IRP1 RNA binding activity occurs by a switch between apoprotein and 4Fe-4S forms, without changes in IRP1 levels, whereas the decrease in IRP2 RNA binding activity reflects a reduction in IRP2 levels. To determine the mechanism by which iron decreases IRP2 levels, we studied IRP2 regulation by iron in rat hepatoma and human HeLa cells. The iron-dependent decrease in IRP2 levels was not due to a decrease in the amount of IRP2 mRNA or to a decrease in the rate of IRP2 synthesis. Pulse-chase experiments demonstrated that iron resulted in a 3-fold increase in the degradation rate of IRP2. IRP2 degradation depends on protein synthesis, but not transcription, suggesting a requirement for a labile protein. IRP2 degradation is not prevented by lysosomal inhibitors or calpain II inhibitors, but is prevented by inhibitors that block proteasome function. These data suggest the involvement of the proteasome in iron-mediated IRP2 proteolysis.

Ammonium Chloride↗

PubMed1998

Iron-dependent oxidation, ubiquitination, and degradation of iron regulatory protein 2: implications for degradation of oxidized proteins.

The ability of iron to catalyze formation of reactive oxygen species significantly contributes to its toxicity in cells and animals. Iron uptake and distribution is regulated tightly in mammalian cells, in part by iron regulatory protein 2 (IRP2), a protein that is degraded efficiently by the proteasome in iron-replete cells. Here, we demonstrate that IRP2 is oxidized and ubiquitinated in cells before degradation. Moreover, iron-dependent oxidation converts IRP2 into a substrate for ubiquitination in vitro. A regulatory pathway is described in which excess iron is sensed by its ability to catalyze site-specific oxidations in IRP2, oxidized IRP2 is ubiquitinated, and ubiquitinated IRP2 subsequently is degraded by the proteasome. Selective targeting and removal of oxidatively modified proteins may contribute to the turnover of many proteins that are degraded by the proteasome.

Animals↗

PubMed1995

Characterization and expression of iron regulatory protein 2 (IRP2). Presence of multiple IRP2 transcripts regulated by intracellular iron levels.

Iron regulatory proteins (IRP1 and IRP2) are RNA-binding proteins that bind to stem-loop structures, termed iron-responsive elements (IREs), present in either the 5'- or 3'-untranslated regions of specific mRNAs. The binding of IRPs to 5'-IREs inhibits translation of mRNA, whereas the binding of IRPs to 3'-IREs stabilizes mRNA. To study the structure and regulation of IRP2, we isolated cDNAs for rat and human IRP2. The derived amino acid sequence of rat IPR2 is 93% identical with that of human IRP2 and is present in lower eukaryotes, indicating that IRP2 is highly conserved. IRP1 and IRP2 share 61% overall amino acid identity. IRP2 is ubiquitously expressed in rat tissues, the highest amounts present in skeletal muscle and heart. IRP2 is encoded by multiple mRNAs of 6.4, 4.0, and 3.7 kilobases. The 3'-untranslated region of rat IRP2 contains multiple polyadenylation signals, two of which could account for the 4.0-kb and 3.7-kb mRNAs. The 3.7-kb mRNA is increased in iron-depleted cells and occurs with a reciprocal decrease in the 6.4-kb transcript. These data suggest that the 3.7-kb mRNA is produced by alternative poly(A) site utilization in iron-depleted cells.

Amino Acid Sequence↗

PubMed2000

Internal loop/bulge and hairpin loop of the iron-responsive element of ferritin mRNA contribute to maximal iron regulatory protein 2 binding and translational regulation in the iso-iron-responsive element/iso-iron regulatory protein family.

Iron-responsive elements (IREs), a natural group of mRNA-specific sequences, bind iron regulatory proteins (IRPs) differentially and fold into hairpins [with a hexaloop (HL) CAGUGX] with helical distortions: an internal loop/bulge (IL/B) (UGC/C) or C-bulge. C-bulge iso-IREs bind IRP2 more poorly, as oligomers (n = 28-30), and have a weaker signal response in vivo. Two trans-loop GC base pairs occur in the ferritin IRE (IL/B and HL) but only one in C-bulge iso-IREs (HL); metal ions and protons perturb the IL/B [Gdaniec et al. (1998) Biochemistry 37, 1505-1512]. IRE function (translation) and physical properties (T(m) and accessibility to nucleases) are now compared for IL/B and C-bulge IREs and for HL mutants. Conversion of the IL/B into a C-bulge by a single deletion in the IL/B or by substituting the HL CG base pair with UA both derepressed ferritin synthesis 4-fold in rabbit reticulocyte lysates (IRP1 + IRP2), confirming differences in IRP2 binding observed for the oligomers. Since the engineered C-bulge IRE was more helical near the IL/B [Cu(phen)(2) resistant] and more stable (T(m) increased) and the HL mutant was less helical near the IL/B (ribonuclease T1 sensitive) and less stable (T(m) decreased), both CG trans-loop base pairs contribute to maximum IRP2 binding and translational regulation. The (1)H NMR spectrum of the Mg-IRE complex revealed, in contrast to the localized IL/B effects of Co(III) hexaammine observed previously, perturbation of the IL/B plus HL and interloop helix. The lower stability and greater helix distortion in the ferritin IL/B-IRE compared to the C-bulge iso-IREs create a combinatorial set of RNA/protein interactions that control protein synthesis rates with a range of signal sensitivities.

Animals↗

PubMed1994

Molecular characterization of a second iron-responsive element binding protein, iron regulatory protein 2. Structure, function, and post-translational regulation.

Several genes critical to the uptake, sequestration, and utilization of iron are regulated at the post-transcriptional level. The mRNAs encoded by these genes contain highly conserved stem-loop structures called iron-responsive elements (IREs). IREs function as the nucleic acid-binding sites for a cytosolic RNA-binding protein called the IRE-binding protein or IRE-BP. Binding of the IRE-BP to IREs is reversibly regulated by the iron status of the cell. The IRE-BP is highly conserved among human, rat, mouse, and rabbit, and it is identical to the cytosolic form of aconitase. In this study, we demonstrate that a distinct human gene encoding a protein which is 57% identical to the initially described IRE-BP, now referred to as iron regulatory protein 1 or IRP1, is also capable of binding to IREs with the same in vitro affinity and specificity the originally identified protein. This second gene product, which we call IRP2, is expressed in many tissues, but its mRNA abundance and tissue distribution are different from IRP1. In most cell lines tested, levels of IRP2 are inversely regulated by iron levels due to iron-dependent regulation of the half-life of the protein. In addition to changes in total amounts of IRP2, we demonstrate that the IRE binding activity of IRP2 can also vary up to 4-fold in the absence of any change in IRP2 protein levels. The possible reasons for the existence of a second IRP are discussed.

Amino Acid Sequence↗

PubMed2002

Multiple, conserved iron-responsive elements in the 3'-untranslated region of transferrin receptor mRNA enhance binding of iron regulatory protein 2.

Synthesis of proteins for iron homeostasis is regulated by specific, combinatorial mRNA/protein interactions between RNA stem-loop structures (iron-responsive elements, IREs) and iron-regulatory proteins (IRP1 and IRP2), controlling either mRNA translation or stability. The transferrin receptor 3'-untranslated region (TfR-3'-UTR) mRNA is unique in having five IREs, linked by AU-rich elements. A C-bulge in the stem of each TfR-IRE folds into an IRE that has low IRP2 binding, whereas a loop/bulge in the stem of the ferritin-IRE allows equivalent IRP1 and IRP2 binding. Effects of multiple IRE interactions with IRP1 and IRP2 were compared between the native TfR-3'-UTR sequence (5xIRE) and RNA with only 3 or 2 IREs. We show 1) equivalent IRP1 and IRP2 binding to multiple TfR-IRE RNAs; 2) increased IRP-dependent nuclease resistance of 5xIRE compared with lower IRE copy-number RNAs; 3) distorted TfR-IRE helix structure within the context of 5xIRE, detected by Cu-(phen)(2) binding/cleavage, that coincides with ferritin-IRE conformation and enhanced IRP2 binding; and 4) variable IRP1 and IRP2 expression in human cells and during development (IRP2-mRNA predominated). Changes in TfR-IRE structure conferred by the full length TfR-3'-UTR mRNA explain in part evolutionary conservation of multiple IRE-RNA, which allows TfR mRNA stabilization and receptor synthesis when IRP activity varies, and ensures iron uptake for cell growth.

3' Untranslated Regions↗