Binding Of Iron Regulatory Proteins Research Articles

A temporal difference in the stabilization of two mRNAs with a 3' iron-responsive element during iron deficiency.

The interactions of iron regulatory proteins (IRPs) with mRNAs containing an iron-responsive element (IRE) maintain cellular iron homeostasis and coordinate it with metabolism and possibly cellular behavior. The mRNA encoding transferrin receptor-1 (TFRC, TfR1), which is a major means of iron importation, has five IREs within its 3' UTR, and IRP interactions help maintain cytosolic iron through the protection of the TfR1 mRNA from degradation. An IRE within the 3' UTR of an mRNA splice variant encoding human cell division cycle 14A (CDC14A) has the potential to coordinate the cellular iron status with cellular behavior through a similar IRP-mediated mechanism. However, the stability of the CDC14A splice variant was reported earlier to be unaffected by the cellular iron status, which suggested that the IRE is not functional. We labeled newly synthesized mRNA in HEK293 cells with 5-ethynyl uridine and found that the stability of the CDC14A variant is responsive to iron deprivation, but there are two major differences from the regulation of TfR1 mRNA stability. First, the decay of the CDC14A mRNA does not utilize the Roquin-mediated reaction that acts on the TfR1 mRNA, indicating that there is flexibility in the degradative machinery antagonized by the IRE-IRP interactions. Second, the stabilization of the CDC14A mRNA is delayed relative to the TfR1 mRNA and does not occur until IRP binding activity has been induced. The result is consistent with a hierarchy of IRP interactions in which the maintenance of cellular iron through the stabilization of the TfR1 mRNA is initially prioritized.

Iron-dependent post transcriptional control of mitochondrial aconitase expression.

Iron regulatory proteins (IRPs) control the translation of animal cell mRNAs encoding proteins with diverse roles. This includes the iron storage protein ferritin and the tricarboxylic cycle (TCA) enzyme mitochondrial aconitase (ACO2) through iron-dependent binding of IRP to the iron responsive element (IRE) in the 5' untranslated region (UTR). To further elucidate the mechanisms allowing IRPs to control translation of 5' IRE-containing mRNA differentially, we focused on Aco2 mRNA, which is weakly controlled versus the ferritins. Rat liver contains two classes of Aco2 mRNAs, with and without an IRE, due to alterations in the transcription start site. Structural analysis showed that the Aco2 IRE adopts the canonical IRE structure but lacks the dynamic internal loop/bulge five base pairs 5' of the CAGUG(U/C) terminal loop in the ferritin IREs. Unlike ferritin mRNAs, the Aco2 IRE lacks an extensive base-paired flanking region. Using a full-length Aco2 mRNA expression construct, iron controlled ACO2 expression in an IRE-dependent and IRE-independent manner, the latter of which was eliminated with the ACO23C3S mutant that cannot bind the FeS cluster. Iron regulation of ACO23C3S encoded by the full-length mRNA was completely IRE-dependent. Replacement of the Aco23C3S 5' UTR with the Fth1 IRE with base-paired flanking sequences substantially improved iron responsiveness, as did fusing of the Fth1 base-paired flanking sequences to the native IRE in the Aco3C3S construct. Our studies further define the mechanisms underlying the IRP-dependent translational regulatory hierarchy and reveal that Aco2 mRNA species lacking the IRE contribute to the expression of this TCA cycle enzyme.

IRE-dependent Regulation of Intestinal Dmt1 Prevails During Chronic Dietary Iron Deficiency but is Dispensable in Conditions of Acute Erythropoietic Stress.

IRE-dependent Regulation of Intestinal Dmt1 Prevails During Chronic Dietary Iron Deficiency but is Dispensable in Conditions of Acute Erythropoietic Stress.

Artemisinin compounds sensitize cancer cells to ferroptosis by regulating iron homeostasis.

The antimalarial drug artemisinin and its derivatives have been explored as potential anticancer agents, but their underlying mechanisms are controversial. In this study, we found that artemisinin compounds can sensitize cancer cells to ferroptosis, a new form of programmed cell death driven by iron-dependent lipid peroxidation. Mechanistically, dihydroartemisinin (DAT) can induce lysosomal degradation of ferritin in an autophagy-independent manner, increasing the cellular free iron level and causing cells to become more sensitive to ferroptosis. Further, by associating with cellular free iron and thus stimulating the binding of iron-regulatory proteins (IRPs) with mRNA molecules containing iron-responsive element (IRE) sequences, DAT impinges on IRP/IRE-controlled iron homeostasis to further increase cellular free iron. Importantly, in both in vitro and a mouse xenograft model in which ferroptosis was triggered in cancer cells by the inducible knockout of GPX4, we found that DAT can augment GPX4 inhibition-induced ferroptosis in a cohort of cancer cells that are otherwise highly resistant to ferroptosis. Collectively, artemisinin compounds can sensitize cells to ferroptosis by regulating cellular iron homeostasis. Our findings can be exploited clinically to enhance the effect of future ferroptosis-inducing cancer therapies.

Leishmania donovani inhibits ferroportin translation by modulating FBXL5-IRP2 axis for its growth within host macrophages.

Hepcidin mediated ferroportin (Fpn) degradation in macrophages is a well adopted strategy to limit iron availability towards invading pathogens. Leishmania donovani (LD), a protozoan parasite, resides within macrophage and competes with host for availing iron. Using in vitro and in vivo model of infection, we reveal that LD decreases Fpn abundance in host macrophages by hepcidin independent mechanism. Unaffected level of Fpn-FLAG in LD infected J774 macrophage confirms that Fpn down-regulation is not due its degradation. While increased Fpn mRNA but decreased protein expression in macrophages suggests blocking of Fpn translation by LD infection that is confirmed by 35 S-methionine labelling assay. We further reveal that LD blocks Fpn translation by induced binding of iron regulatory proteins (IRPs) to the iron responsive element present in its 5'UTR. Supershift analysis provides evidence of involvement of IRP2 particularly during in vivo infection. Accordingly, a significant increase in IRP2 protein expression with simultaneous decrease in its stability regulator F-box and leucine-rich repeat Protein 5 (FBXL5) is detected in splenocytes of LD-infected mice. Increased intracellular growth due to compromised expressions of Fpn and FBXL5 by specific siRNAs reveals that LD uses a novel strategy of manipulating IRP2-FBXL5 axis to inhibit host Fpn expression.

Functional characterization of a novel non-coding mutation \u201cGhent +49A\u2009>\u2009G\u201d in the iron-responsive element of L-ferritin causing hereditary hyperferritinaemia-cataract syndrome

Hereditary hyperferritinaemia-cataract syndrome (HHCS) is a rare disorder usually caused by heterozygous mutations in the iron-responsive element (IRE) in the 5′ untranslated region (5′UTR) of the L-ferritin gene (FTL), disturbing the binding of iron regulatory proteins (IRPs) and the post-transcriptional regulation of ferritin expression. Here, the proband of a consanguineous family displayed moderate bilateral cataracts and elevated serum ferritin in the absence of iron overload. The parents and siblings showed variable degrees of mild bilateral cataracts combined with elevated levels of circulating ferritin. Sequencing of FTL identified a novel 5′UTR mutation c.-151A > G, also named “Ghent +49A > G”. The zygosity of the mutation, occurring in homozygous and heterozygous state in the proband and other affected family members respectively, correlated well with severity of ophthalmological and hematological manifestations. The substitution is expected to impair the secondary structure of the upper IRE stem. Functional characterization of +49A > G by electrophoretic mobility shift assays demonstrated a reduced binding affinity for IRP1 compared to the wild-type IRE of FTL. Overall, we have expanded the repertoire of deleterious biallelic FTL IRE mutations in HHCS with this novel +49A > G mutation, the zygosity of which correlated well with the disease expression.

Thermodynamic and Kinetic Analyses of Iron Response Element (IRE)-mRNA Binding to Iron Regulatory Protein, IRP1

Comparison of kinetic and thermodynamic properties of IRP1 (iron regulatory protein1) binding to FRT (ferritin) and ACO2 (aconitase2) IRE-RNAs, with or without Mn2+, revealed differences specific to each IRE-RNA. Conserved among animal mRNAs, IRE-RNA structures are noncoding and bind Fe2+ to regulate biosynthesis rates of the encoded, iron homeostatic proteins. IRP1 protein binds IRE-RNA, inhibiting mRNA activity; Fe2+ decreases IRE-mRNA/IRP1 binding, increasing encoded protein synthesis. Here, we observed heat, 5 °C to 30 °C, increased IRP1 binding to IRE-RNA 4-fold (FRT IRE-RNA) or 3-fold (ACO2 IRE-RNA), which was enthalpy driven and entropy favorable. Mn2+ (50 µM, 25 °C) increased IRE-RNA/IRP1 binding (Kd) 12-fold (FRT IRE-RNA) or 6-fold (ACO2 IRE-RNA); enthalpic contributions decreased ~61% (FRT) or ~32% (ACO2), and entropic contributions increased ~39% (FRT) or ~68% (ACO2). IRE-RNA/IRP1 binding changed activation energies: FRT IRE-RNA 47.0 ± 2.5 kJ/mol, ACO2 IRE-RNA 35.0 ± 2.0 kJ/mol. Mn2+ (50 µM) decreased the activation energy of RNA-IRP1 binding for both IRE-RNAs. The observations suggest decreased RNA hydrogen bonding and changed RNA conformation upon IRP1 binding and illustrate how small, conserved, sequence differences among IRE-mRNAs selectively influence thermodynamic and kinetic selectivity of the protein/RNA interactions.

A role for amyloid precursor protein translation to restore iron homeostasis and ameliorate lead (Pb) neurotoxicity.

Iron supplementation ameliorates the neurotoxicity of the environmental contaminant lead (Pb); however, the mechanism remains undefined. Iron is an essential nutrient but high levels are toxic due to the catalytic generation of destructive hydroxyl radicals. Using human neuroblastoma SH-SY5Y cells to model human neurons, we investigated the effect of Pb on proteins of iron homeostasis: the Alzheimer's amyloid precursor protein (APP), which stabilizes the iron exporter ferroportin 1; and, the heavy subunit of the iron-storage protein, ferritin (FTH). Lead (Pb(II) and Pb(IV) inhibited APP translation and raised cytosolic iron(II). Lead also increased iron regulatory protein-1 binding to the cognate 5'untranslated region-specific iron-responsive element (IRE) of APP and FTH mRNAs. Concurrent iron treatment rescued cells from Pb toxicity by specifically restoring APP synthesis, i.e. levels of the APP-related protein, APLP-2, were unchanged. Significantly, iron/IRE-independent over-expression of APP695 protected SH-SY5Y cells from Pb toxicity, demonstrating that APP plays a key role in maintaining safe levels of intracellular iron. Overall, our data support a model of neurotoxicity where Pb enhances iron regulatory protein/IRE-mediated repression of APP and FTH translation. We propose novel treatment options for Pb poisoning to include chelators and the use of small molecules to maintain APP and FTH translation. We propose the following cascade for Lead (Pb) toxicity to neurons; by targeting the interaction between Iron regulatory protein-1 and Iron-responsive elements, Pb caused translational repression of proteins that control intracellular iron homeostasis, including the Alzheimer's amyloid precursor protein (APP) that stabilizes the iron exporter ferroportin, and the ferroxidase heavy subunit of the iron-storage protein, ferritin. When unregulated, IRE-independent over-expression of APP695 protected SH-SY5Y neurons from Pb toxicity. There is a novel and key role for APP in maintaining safe levels of intracellular iron pertinent to lead toxicity.

Transferrin receptor mRNA interactions contributing to iron homeostasis

The transferrin receptor is the primary means of iron importation for most mammalian cells and understanding its regulatory mechanisms is relevant to hematologic, oncologic, and other disorders in which iron homeostasis is perturbed. The 3′ UTR of the transferrin receptor mRNA contains an instability element that is protected from degradation during iron depletion through interactions of iron regulatory proteins (IRPs) with five iron-responsive elements (IREs). The structural features required for degradation and the site of IRP binding required for in situ protection remain unclear. An RNA-CLIP strategy is described here that identifies the predominant site of IRP-1 interaction within a nontransformed primary cell line. This approach avoided complications associated with the use of elevated concentrations of protein and/or mRNA and detected interactions within the native environment of the mRNA. A compensatory mutagenesis strategy indicates that the instability element at minimum consists of three non-IRE stem–loops that can function additively, suggesting that they are not forming one highly interdependent structure. Although the IREs are not essential for instability, they enhance instability when IRP interactions are inhibited. These results are supportive of a mechanism for a graded response to the intracellular iron resulting from a progressive loss of IRP protection.

Regulation of iron homeostasis by the p53-ISCU pathway.

Accumulation of iron in tissues increases the risk of cancer, but iron regulatory mechanisms in cancer tissues are largely unknown. Here, we report that p53 regulates iron metabolism through the transcriptional regulation of ISCU (iron-sulfur cluster assembly enzyme), which encodes a scaffold protein that plays a critical role in Fe-S cluster biogenesis. p53 activation induced ISCU expression through binding to an intronic p53-binding site. Knockdown of ISCU enhanced the binding of iron regulatory protein 1 (IRP1), a cytosolic Fe-S protein, to an iron-responsive element in the 5′ UTR of ferritin heavy polypeptide 1 (FTH1) mRNA and subsequently reduced the translation of FTH1, a major iron storage protein. In addition, in response to DNA damage, p53 induced FTH1 and suppressed transferrin receptor, which regulates iron entry into cells. HCT116 p53+/+ cells were resistant to iron accumulation, but HCT116 p53−/− cells accumulated intracellular iron after DNA damage. Moreover, excess dietary iron caused significant elevation of serum iron levels in p53−/− mice. ISCU expression was decreased in the majority of human liver cancer tissues, and its reduced expression was significantly associated with p53 mutation. Our finding revealed a novel role of the p53-ISCU pathway in the maintenance of iron homeostasis in hepatocellular carcinogenesis.

Iron Regulatory Protein 1 Suppresses Hypoxia-Induced Iron Uptake Proteins Expression and Decreases Iron Levels in HepG2 Cells.

Transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1) are important proteins for cellular iron uptake, and both are regulated transcriptionally through the binding of hypoxia-inducible factor 1 (HIF-1) to hypoxia-responsive elements (HREs) under hypoxic conditions. These proteins are also regulated post-transcriptionally through the binding of iron regulatory protein 1 (IRP1) to iron-responsive elements (IREs) located in the mRNA untranslated region (UTR) to control cellular iron homeostasis. In iron-deficient cells, IRP1-IRE interactions stabilize TfR1 and DMT1 mRNAs, enhancing iron uptake. However, little is known about the impact of IRP1 on the regulation of cellular iron homeostasis under hypoxia. Thus, to investigate the role of IRP1 in hypoxic condition, overexpression and knockdown assays were performed using HepG2 cells. The overexpression of IRP1 suppressed the hypoxia-induced increase in TfR1 and DMT1 (+IRE) expression and reduced the stability of TfR1 and DMT1 (+IRE) mRNAs under hypoxia, whereas IRP1 knockdown further increased the hypoxia-induced expression of both proteins, preventing the decrease in IRE-dependent luciferase activity induced by hypoxia. Under hypoxic conditions, ferrous iron uptake, the labile iron pool (LIP), and total intracellular iron reduced when IRP1 was overexpressed and further increased when IRP1 was knocked down. IRP1 expression declined and TfR1/DMT1 (+IRE) expression increased with the time of hypoxia prolonged, whereas the binding of IRP1 to the IRE of TfR1/DMT1 mRNA maintained. In summary, IRP1 suppressed TfR1/DMT1 (+IRE) expression, limited the cellular iron content and decreased lactate dehydrogenase (LDH) release induced by hypoxia.

Catecholamine Stress Hormones Regulate Cellular Iron Homeostasis by a Posttranscriptional Mechanism Mediated by Iron Regulatory Protein: IMPLICATION IN ENERGY HOMEOSTASIS

Adequate availability of iron is important for cellular energy metabolism. Catecholamines such as epinephrine and norepinephrine promote energy expenditure to adapt to conditions that arose due to stress. To restore the energy balance, epinephrine/norepinephrine-exposed cells may face higher iron demand. So far, no direct role of epinephrine/norepinephrine in cellular iron homeostasis has been reported. Here we show that epinephrine/norepinephrine regulates iron homeostasis components such as transferrin receptor-1 and ferritin-H in hepatic and skeletal muscle cells by promoting the binding of iron regulatory proteins to iron-responsive elements present in the UTRs of transferrin receptor-1 and ferritin-H transcripts. Increased transferrin receptor-1, decreased ferritin-H, and increased iron-responsive element-iron regulatory protein interaction are also observed in liver and muscle tissues of epinephrine/norepinephrine-injected mice. We demonstrate the role of epinephrine/norepinephrine-induced generation of reactive oxygen species in converting cytosolic aconitase (ACO1) into iron regulatory protein-1 to bind iron-responsive elements present in UTRs of transferrin receptor-1 and ferritin-H. Our study further reveals that mitochondrial iron content and mitochondrial aconitase (ACO2) activity are elevated by epinephrine/norepinephrine that are blocked by the antioxidant N-acetyl cysteine and iron regulatory protein-1 siRNA, suggesting involvement of reactive oxygen species and iron regulatory protein-1 in this mechanism. This study reveals epinephrine and norepinephrine as novel regulators of cellular iron homeostasis.

The physiological functions of iron regulatory proteins in iron homeostasis - an update.

Iron regulatory proteins (IRPs) regulate the expression of genes involved in iron metabolism by binding to RNA stem-loop structures known as iron responsive elements (IREs) in target mRNAs. IRP binding inhibits the translation of mRNAs that contain an IRE in the 5′untranslated region of the transcripts, and increases the stability of mRNAs that contain IREs in the 3′untranslated region of transcripts. By these mechanisms, IRPs increase cellular iron absorption and decrease storage and export of iron to maintain an optimal intracellular iron balance. There are two members of the mammalian IRP protein family, IRP1 and IRP2, and they have redundant functions as evidenced by the embryonic lethality of the mice that completely lack IRP expression (Irp1-/-/Irp2-/- mice), which contrasts with the fact that Irp1-/- and Irp2-/- mice are viable. In addition, Irp2-/- mice also display neurodegenerative symptoms and microcytic hypochromic anemia, suggesting that IRP2 function predominates in the nervous system and erythropoietic homeostasis. Though the physiological significance of IRP1 had been unclear since Irp1-/- animals were first assessed in the early 1990s, recent studies indicate that IRP1 plays an essential function in orchestrating the balance between erythropoiesis and bodily iron homeostasis. Additionally, Irp1-/- mice develop pulmonary hypertension, and they experience sudden death when maintained on an iron-deficient diet, indicating that IRP1 has a critical role in the pulmonary and cardiovascular systems. This review summarizes recent progress that has been made in understanding the physiological roles of IRP1 and IRP2, and further discusses the implications for clinical research on patients with idiopathic polycythemia, pulmonary hypertension, and neurodegeneration.

Hypoxia induced changes in expression of proteins involved in iron uptake and storage in cultured lens epithelial cells

Hypoxia inducible factor (HIF) regulates expression of over 60 genes by binding to hypoxia response elements (HRE) located upstream of the transcriptional start sites. Many genes encoding proteins involved in iron transport and homeostasis are regulated by HIF. Expression of iron handling proteins can also be translationally regulated by binding of iron regulatory protein (IRP) to iron responsive elements (IREs) on the mRNA of ferritin chains and transferrin receptor (TfR). Lens epithelial cells (LEC) function in a low oxygen environment. This increases the risk of iron catalyzed formation of reactive oxygen species (ROS) and oxidative cell damage. We examined changes in expression of ferritin (iron storage protein) and Tf/TfR1 (iron uptake proteins) in LEC cultured under hypoxic conditions. Ferritin consists of 24 subunits of two types, heavy (H-chain) and light (L-chain) assembled in a cell specific ratio. Real-time PCR showed that 24 h exposure to hypoxia lowered transcription of both ferritin chains by over 50% when compared with normoxic LEC. However it increased the level of ferritin chain proteins (20% average). We previously found that 6 h exposure of LEC to hypoxia increased the concentration of cytosolic iron which would stimulate translation of ferritin chains. This elevated ferritin concentration increased the iron storage capacity of LEC. Hypoxic LEC labeled with 59FeTf incorporated 70% more iron into ferritin after 6 h as compared to normoxic LEC. Exposure of LEC to hypoxia for 24 h reduced the concentration of TfR1 in cell lysates. As a result, hypoxic LEC internalized less Tf at this later time point. Incorporation of 59Fe into ferritin of hypoxic LEC after 24 h did not differ from that of normoxic LEC due to lower 59FeTf uptake. This study showed that hypoxia acutely increased iron storage capacity and lowered iron uptake due to changes in expression of iron handling proteins. These changes may better protect LEC against oxidative stress by limiting iron-catalyzed ROS formation in the low oxygen environment in which the lens resides.

Iron Regulatory Protein-1 Protects against Mitoferrin-1-deficient Porphyria

Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1(+/gt);Irp1(-/-) erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5'-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1(gt/gt) cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1(gt/gt) cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.

Abstract 1578: Small molecule HIF-2a inhibitors down regulate its activity in vivo and ameliorate the vhl zebrafish phenotype.

Abstract Hypoxia Inducible Factor (HIF) target genes are involved in several cellular processes including angiogenesis, erythropoiesis, metabolism and stem cell proliferation. The von Hippel-Lindau (VHL) tumor suppressor protein targets HIF for degradation in conditions of normal oxygen tension. Loss-of-VHL leads to constitutive expression of HIF. We have discovered small molecules that repress HIF-2α translation by enhancing the binding of Iron-Regulatory Protein 1 (IRP1) to the Iron-Responsive Element (IRE) within the 5’-UTR of HIF-2α mRNA and showed that these small molecules specifically down-regulate HIF-2α expression in vitro. To validate the HIF-2α inhibitors in vivo, first we tested their efficacy to inhibit acute hif induction in wild-type (wt) zebrafish embryos challenged with the hypoxia mimetic DMOG. Treatment of wt embryos with 100μM of DMOG up-regulated the expression of zebrafish HIF-target genes, such as vascular endothelial growth factor (vegf), erythropoietin (epo) and prolyl-hydroxylase 3 (phd3). Pre-treatment of embryos with the small molecule HIF-2α inhibitor 76 (10nM) significantly decreased the DMOG-induced up-regulation of HIF target genes and resulted in decrease of DMOG-induced erythrocytosis and blood vessel sprouting. Furthermore, we tested the efficacy of this HIF-2α inhibitor in a vhl zebrafish model (homozygougous mutants harbouring inactivating vhl mutations), which recapitulates aspects of human VHL disease (erythrocytosis, abnormal vascular proliferation in sites such as retina and brain, early lethality). Treatment with inhibitor 76 (10nM, 100nM and 1μM) down regulated the expression of the HIF-target genes phd3, epo and vegf in a dose response manner and rescued the erythrocytosis and sprouting phenotype of vhl embryos. To image and quantify the blood vessel abnormalities of vhl embryos, we generated a Fli-GFP::vhl+/- line and showed that compound 76 improves the abnormal connections between the intersegmental vessels of the Fli-GFP::vhl embryo tail, as well as their human hemangioblastoma-resembling brain vessel proliferation. Finally, we showed that treatment of vhl embryos with compound 76 rescues early lethality. Our data demonstrate that HIF-2α inhibitor 76 is active in vivo at low concentrations and encourage further development and preclinical testing of this HIF-2α inhibitor for treatment of VHL-associated lesions, such as renal cell carcinoma, hemangioblastoma and neuroendocrine tumors. Citation Format: Ana Metelo, Li Xiang, Rania Baker, Lee Kamentsky, Anne Carpenter, Randall Peterson. Small molecule HIF-2a inhibitors down regulate its activity in vivo and ameliorate the vhl zebrafish phenotype. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1578. doi:10.1158/1538-7445.AM2013-1578

Fe 2+ binds iron responsive element-RNA, selectively changing protein-binding affinities and regulating mRNA repression and activation

Iron increases synthesis rates of proteins encoded in iron-responsive element (IRE)-mRNAs; metabolic iron ("free," "labile") is Fe(2+). The noncoding IRE-RNA structure, approximately 30nt, folds into a stem loop to control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA riboregulators bind specifically to iron-regulatory proteins (IRP) proteins, inhibiting ribosome binding. Deletion of the IRE-RNA from an mRNA decreases both IRP binding and IRP-independent protein synthesis, indicating effects of other "factors." Current models of IRE-mRNA regulation, emphasizing iron-dependent degradation/modification of IRP, lack answers about how iron increases IRE-RNA/IRP protein dissociation or how IRE-RNA, after IRP dissociation, influences protein synthesis rates. However, we observed Fe(2+) (anaerobic) or Mn(2+) selectively increase the IRE-RNA/IRP K(D). Here we show: (i) Fe(2+) binds to the IRE-RNA, altering its conformation (by 2-aminopurine fluorescence and ethidium bromide displacement); (ii) metal ions increase translation of IRE-mRNA in vitro; (iii) eukaryotic initiation factor (eIF)4F binds specifically with high affinity to IRE-RNA; (iv) Fe(2+) increased eIF4F/IRE-RNA binding, which outcompetes IRP binding; (v) exogenous eIF4F rescued metal-dependent IRE-RNA translation in eIF4F-depeleted extracts. The regulation by metabolic iron binding to IRE-RNA to decrease inhibitor protein (IRP) binding and increase activator protein (eIF4F) binding identifies IRE-RNA as a riboregulator.

Abstract 4253: In vivo activity of small molecule HIF2α inhibitors

Abstract Hypoxia-inducible factor (HIF) is a central regulator of the cellular response to hypoxia. Hypoxia promotes HIF activity by increasing its stability, transcriptional activity and translation. HIF target genes involved in several cellular processes including proliferation, angiogenesis, metabolism and promotion of the “stem-cell” phenotype. In normoxic conditions, HIF-α is recognized for degradation by the von Hippel-Lindau tumor suppressor protein (VHL). Loss-of-VHL function leads to constitutive expression of HIF regulatory subunits and consequent over-expression of HIF-target genes. We and others have shown that HIF2a is necessary and sufficient for the development of VHL-related renal cell carcinoma. We have developed small molecules that repress HIF-2α translation, in vitro, by enhancing the binding of Iron-Regulatory Protein 1 (IRP1) to the Iron-Responsive Element (IRE) within the 5′-UTR of HIF-2α mRNA (Zimmer M. et al. Molecular Cell 2008;32: 828-848). We showed that these small molecules down-regulate HIF2a expression in vitro, in all renal cell carcinoma cell lines. To validate these small molecules in vivo, we tested their efficacy on wild-type zebrafish embryos. Treatment of 3 days post fertilization zebrafish embryos with 100microM of the hypoxic mimetic DMOG or hypoxia dramatically upregulated the expression of HIF-target genes, such as Vascular Endothelial Growth Factor (Vegf), erythropoietin (Epo) and prolyl-hydroxylase 3 (Phd3). Pre-treatment of embryos with the small molecule HIF2a inhibitor 76 potently decreased the DMOG-induced upregulation of HIF target genes. The IC50 values of these small molecules were significantly less in vivo than in vitro. We are currently testing the efficacy of HIF2a inhibitors in rescuing the VHL zebrafish phenotype and prolonging survival of VHL mutant zebrafish embryos. In addition, data on the efficacy of these inhibitors on the growth of human renal cell carcinoma cells as tumors in the nude mouse xenograft assay will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4253. doi:1538-7445.AM2012-4253

Cell density-dependent shift in activity of iron regulatory protein 1 (IRP-1)/cytosolic (c-)aconitase

Iron regulatory protein 1 (IRP-1) is a bifunctional protein involved in iron homeostasis and metabolism. In one state, it binds to specific sequences in the mRNA's of several proteins involved in iron and energy metabolism, thereby influencing their expression post-transcriptionally. In another state it contains a [4Fe-4S] iron-sulfur cofactor and displays aconitase activity in the cytosol. We have shown that this protein binds and hydrolyzes ATP, with kinetic and thermodynamic equilibrium constants that predict saturation with ATP, favouring a non-RNA-binding form at normal cellular ATP levels, and thus pointing to additional function(s) of the protein. Here we show for the first time that the RNA-binding and aconitase forms of IRP-1 can undergo interconversion dependent on the density of cells growing in culture. Thus, in high density confluent cultures, compared with low density, actively proliferating cultures, cytosolic aconitase activity is increased whereas RNA binding activity is diminished. This is accompanied by a decrease in transferrin receptor expression in confluent cells, possibly due to loss of the transcript-stabilizing activity of bound IRP-1. In high density HepG2 cultures, cytosolic glutamate and the ratio of reduced-to-oxidized glutathione were increased. We propose that increased cytosolic aconitase activity in confluent cultures may divert cytosolic citrate away from the fatty acid/membrane synthetic pathways required by dividing cells, into a glutamate-dependent maintenance of cellular macromolecular synthesis. In addition, this may confer additional protection from oxidative stress due to down-regulation of iron acquisition from transferrin and increased glutamate for glutathione synthesis.

Two covariance models for iron-responsive elements

Iron-responsive elements (IREs) function in the 5’ or 3’ untranslated regions (UTRs) of mRNAs as post-transcriptional structured cis-acting RNA regulatory elements. One known functional mechanism is the binding of Iron Regulatory Proteins (IRPs) to 5’ UTR IREs, reducing translation rates at low iron levels. Another known mechanism is IRPs binding to 3’ UTR IREs in other mRNAs, increasing RNA stability. Experimentally proven elements are quite small, have some diversity of sequence and structure, and functional genes have similar pseudogenes in the genome. This paper presents two new IRE covariance models, comprising a new IRE clan in the RFAM database to encompass this variation without over-generalisation. Two IRE models rather than a single model is consistent with experimentally proven structures and predictions. All of the IREs with experimental support are modelled. These two new models show a marked increase in the sensitivity and specificity in detection of known iron-responsive elements and ability to predict novel IREs.