Multiple determinants within iron-responsive elements dictate iron regulatory protein binding and regulatory hierarchy

Iron regulatory proteins (IRPs) are iron-regulated RNA binding proteins that, along with iron-responsive elements (IREs), control the translation of a diverse set of mRNA with 5′ IRE. Dysregulation of IRP action causes disease with etiology that may reflect differential control of IRE-containing mRNA. IREs are defined by a conserved stem–loop structure including a midstem bulge at C8 and a terminal CAGUGH sequence that forms an AGU pseudo-triloop and N19 bulge. C8 and the pseudo-triloop nucleotides make the majority of the 22 identified bonds with IRP1. We show that IRP1 binds 5′ IREs in a hierarchy extending over a ninefold range of affinities that encompasses changes in IRE binding affinity observed with human L-ferritin IRE mutants. The limits of this IRE binding hierarchy are predicted to arise due to small differences in binding energy (e.g., equivalent to one H-bond). We demonstrate that multiple regions of the IRE stem not predicted to contact IRP1 help establish the binding hierarchy with the sequence and structure of the C8 region displaying a major role. In contrast, base-pairing and stacking in the upper stem region proximal to the terminal loop had a minor role. Unexpectedly, an N20 bulge compensated for the lack of an N19 bulge, suggesting the existence of novel IREs. Taken together, we suggest that a regulatory binding hierarchy is established through the impact of the IRE stem on the strength, not the number, of bonds between C8 or pseudo-triloop nucleotides and IRP1 or through their impact on an induced fit mechanism of binding.     

Nucleotide-Specific Recognition of Iron-Responsive Elements by Iron Regulatory Protein 1

IRP1 [iron regulatory protein (IRP) 1] is a bifunctional protein with mutually exclusive end-states. In one mode of operation, IRP1 binds iron-responsive element (IRE) stem–loops in messenger RNAs encoding proteins of iron metabolism to control their rate of translation. In its other mode, IRP1 serves as cytoplasmic aconitase to correlate iron availability with the energy and oxidative stress status of the cell. IRP1/IRE binding occurs through two separate interfaces, which together contribute about two-dozen hydrogen bonds. Five amino acids make base-specific contacts and are expected to contribute significantly to binding affinity and specificity of this protein:RNA interaction. In this mutagenesis study, each of the five base-specific amino acids was changed to alter binding at each site. Analysis of IRE binding affinity and translational repression activity of the resulting IRP1 mutants showed that four of the five contact points contribute uniquely to the overall binding affinity of the IRP1:IRE interaction, while one site was found to be unimportant. The stronger-than-expected effect on binding affinity of mutations at Lys379 and Ser681, residues that make contact with the conserved nucleotides G16 and C8, respectively, identified them as particularly critical for providing specificity and stability to IRP1:IRE complex formation. We also show that even though the base-specific RNA-binding residues are not part of the aconitase active site, their substitutions can affect the aconitase activity of holo-IRP1, positively or negatively.     

Modulation of iron regulatory protein-1 by various metals.

Iron regulatory protein-1 (IRP-1) is known as a cytosolic aconitase and a central regulator of iron (Fe) homeostasis. IRP-1 regulates the expression of Fe metabolism-related proteins by interacting with the Fe-responsive element (IRE) in the untranslated regions of mRNAs of these proteins. However, it is less known whether IRP-1 modulates various non-Fe metals. In the present study, we showed that treatment of homogenously purified IRP-1 with non-Fe metals decreased the affinity to IRE in RNA band shift assays and increased aconitase activity. Non-Fe metals also inhibited (55)Fe incorporation into the fourth labile position of the Fe-S cluster of IRP-1. In PLC hepatoma cells, metal loading inactivated binding activity and activated enzyme activity. It also suppressed transferrin receptor mRNA expression in the cells. These results suggest that various non-Fe metals modulate IRP-1 by conversion of the 3Fe-4S apo-form to a [1 non-Fe metal + 3Fe]-4Fe holo-form.     

Rg1 protects iron‐induced neurotoxicity through antioxidant and iron regulatory proteins in 6‐OHDA‐treated MES23.5 cells

Ginsenoside‐Rg1 is one of the pharmacologically active components isolated from ginseng. It was reported that Rg1 protected dopamine (DA) neurons in 6‐hydroxydopamine (6‐OHDA)‐induced Parkinson's disease (PD) models in vivo and in vitro. Our previous study also demonstrated that iron accumulation was involved in the toxicity of 6‐OHDA. However, whether Rg1 could protect DA neurons against 6‐OHDA toxicity by modulating iron accumulation and iron‐induced oxidative stress is not clear. Therefore, the present study was carried out to elucidate this effect in 6‐OHDA‐treated MES23.5 cells and the possible mechanisms were also conducted. Findings showed Rg1 restored iron‐induced decrease in mitochondrial transmembrane potential in MES23.5 cells, and increased ferrous iron influx was found in 6‐OHDA‐treated cells. Rg1 pretreatment could decrease this iron influx by inhibiting 6‐OHDA‐induced up‐regulation of an iron importer protein divalent metal transporter 1 with iron responsive element (DMT1 + IRE). Furthermore, findings also showed that the effect of Rg1 on DMT1 + IRE expression was due to its inhibition of iron regulatory proteins (IRPs) by its antioxidant effect. These results suggested that the neuroprotective effect of Rg1 against iron toxicity in 6‐OHDA‐treated cells was to decrease the cellular iron accumulation and attenuate the improper up‐regulation of DMT1 + IRE via IRE/IRP system. This provides new insight to understand the pharmacological effects of Rg1 on iron‐induced degeneration of DA neurons. J. Cell. Biochem. 111: 1537–1545, 2010. © 2010 Wiley‐Liss, Inc.     

Cellular iron transport

Iron has a split personality as an essential nutrient that also has the potential to generate reactive oxygen species. We discuss how different cell types within specific tissues manage this schizophrenia. The emphasis in enterocytes is on regulating the body's supply of iron by regulating transport into the blood stream. In developing red blood cells, adaptations in transport manage the body's highest flux of iron. Hepatocytes buffer the body's stock of iron. Macrophage recycle the iron from effete red cells among other iron management tasks. Pneumocytes provide a barrier to prevent illicit entry that, when at risk of breaching, leads to a need to handle the dangers in a fashion essentially shared with macrophage. We also discuss or introduce cell types including renal cells, neurons, other brain cells, and more where our ignorance, currently still vast, needs to be removed by future research.     

Human iron transporters

Human iron transporters manage iron carefully because tissues need iron for critical functions, but too much iron increases the risk of reactive oxygen species. Iron acquisition occurs in the duodenum via divalent metal transporter (DMT1) and ferroportin. Iron trafficking depends largely on the transferrin cycle. Nevertheless, non-digestive tissues have a variety of other iron transporters that may render DMT1 modestly redundant, and DMT1 levels exceed those needed for the just-mentioned tasks. This review begins to consider why and also describes advances after 2008 that begin to address this challenge.     

Extracellular H2O2 and not superoxide determines the compartment-specific activation of transferrin receptor by iron regulatory protein 1

Iron regulatory protein 1 (IRP1) functions as translational regulator that plays a central role in coordinating the cellular iron metabolism by binding to the mRNA of target genes such as the transferrin receptor (TfR)—the major iron uptake protein. Reactive oxygen species such as H₂O₂ and that are both co-released by inflammatory cells modulate IRP1 in opposing directions. While H₂O₂—similar to iron depletion—strongly induces IRP1 via a signalling cascade, inactivates the mRNA binding activity by a direct chemical attack. These findings have raised the question of whether compartmentalization may be an important mechanism for isolating these biological reactants when released from inflammatory cells during the oxygen burst cascade. To address this question, we studied cytosolic IRP1 and its downstream target TfR in conjunction with a tightly controlled biochemical modulation of extracellular and H₂O₂ levels mimicking the oxygen burst cascade of inflammatory cells. We here demonstrate that IRP1 activity and expression of TfR are solely dependent on H₂O₂ when co-released with from xanthine oxidase. Our findings confirm that extracellular H₂O₂ determines the functionality of the IRP1 cluster and its downstream targets while the reactivity of is limited to its compartment of origin.     

Induction of iron regulatory protein 1 RNA-binding activity by nitric oxide is associated with a concomitant increase in the labile iron pool: implications for DNA damage

Iron regulatory protein 1 (IRP1) is a bifunctional [4Fe-4S] protein that controls iron homeostasis. Switching off its function from an aconitase to an apo-IRP1 interacting with iron-responsive element-containing mRNAs depends on the reduced availability of iron in labile iron pool (LIP). Although the modulation of IRP1 by nitric oxide has been characterized, its impact on LIP remains unknown. Here, we show that inhibition of IRP1 aconitase activity and induction of its IRE-binding activity during exposure of L5178Y mouse lymphoma cells to NO are associated with an increase in LIP levels. Removal of NO resulted in a reverse regulation of IRP1 activities accompanied by a decrease of LIP. The increased iron burden in LIP caused by NO exacerbated hydrogen peroxide-induced genotoxicity in L5178Y cells. We demonstrate that the increase in LIP levels in response to chronic but not burst exposure of L5178Y cells to NO is associated with alterations in the expression of proteins involved in iron metabolism.     

Generation of conditional alleles of the murine Iron Regulatory Protein (IRP)-1 and -2 genes

Central aspects of cellular iron metabolism are controlled by IRP1 and IRP2, which are ubiquitously expressed in mouse organs and cells. Total and constitutive deficiency of both IRPs causes embryonic lethality in the mouse. To bypass the early lethality and to study organ-specific and/or temporal functions of IRP1 and/or IRP2 we generated Irp1 and Irp2 conditional alleles. We used mouse lines where a betaGeo gene trap construct was inserted into the second intron of the Irp1 and the Irp2 gene, generating hypomorphic alleles by interrupting the corresponding open reading frame near the amino-termini. The gene trap cassettes are flanked by Frt sites and were co-inserted with LoxP sites flanking exon 3. Flp-mediated removal of the gene trap construct generates floxed alleles with wildtype functions. For both Irp genes, Cre-assisted deletion of exon 3 generates complete null alleles that, in the case of IRP2, are associated with altered body iron distribution and compromised hematopoiesis. If not removed, the gene trap construct causes partially penetrant embryonic lethality unrelated to IRP deficiency when inserted within the Irp1 but not the Irp2 locus. We discuss the implications for functional genomics in the mouse.     

固醇调节元件结合蛋白1及其靶基因网络

固醇调节元件结合蛋白1(Sterol regulatory element-binding protein 1, SREBP-1)是重要的核转录因子之一, 能调控内源性胆固醇、脂肪酸、甘油三酯和磷脂合成所需酶的表达, 以维持血脂动态平衡。研究表明, SREBP-1及其靶基因网络的异常可引起胰岛素抵抗、Ⅱ型糖尿病、心功能紊乱、血管并发症和肝脂肪变等一系列代谢性疾病。近年高通量组学技术的发展极大扩展了对SREBP-1靶基因及其转录调控模式的了解。文章对SREBP-1蛋白结构、活化过程、DNA结合位点及其调控的靶基因等方面的研究进展进行了综述, 并着重介绍了基于组学数据的转录调控网络的构建, 这将有助于更好的认识SREBP-1在脂类代谢中的作用, 为深入探讨脂质代谢性疾病的治疗提供新线索。     

用IRE—FINDER算法在非翻译区搜索人和小鼠的铁反应元件

铁反应元件 (IRE)具有的高度保守的RNA茎环结构 ,是胞质中IRE结合铁调控蛋白 (IRP)的位点。IRP与IRE的结合受细胞内铁含量的调控 ,与铁代谢有着密切的关系。许多与铁代谢有关的基因在 5′UTR或 3′UTR都存在IRE。当铁离子缺乏时 ,IRE与IRP结合 ,使核糖体的翻译进程受阻 ,抑制mRNA的翻译 ;而当铁离子浓度较高时 ,IRP就释放IRE ,促进翻译。因此 ,IRE在 5′UTR调节着翻译的起始[1] 。如果IRE位于 3′UTR ,IRE与IRP的结合可以稳定mRNA并预防其被降解。所有已知的IRE均为C1G5型 ,即环的 1号和 5号碱基位置分别是保守的C和G ;另外还存在一类体外合成的U1A5型IRE ,即环的 1号和 5号碱基位置分别是保守的U和A ,能够在体外有效地结合IRP。由于IRE与铁代谢、氧化性损伤 (oxidativestress)和衰老等过程都有密切的关系 ,因此在临床及新药研制等方面都很受重视。通过改进搜索IRE的算法 ,用计算机程序对人与鼠的mRNA非编码区(UTR)序列数据库进行全面的搜索 ,期望找出可能存在的C1G5或U1A5类型的IRE ,或包含这两种类型IRE的基因。     

O-GlcNAc inhibits interaction between Sp1 and sterol regulatory element binding protein 2

O-linked N-acetylglucosamine (O-GlcNAc), a monosaccharide N-acetylglucosamine on the serine and threonine residues of nucleocytoplasmic proteins, is a novel protein modification that is ubiquitous among eukaryotes and implicated in cell regulation. Recent evidence indicates that O-GlcNAc regulates protein-protein interactions. Here we provide evidence that O-GlcNAc interrupts a known interaction between Sp1 and sterol regulatory element binding protein 2 (SREBP2), thereby inhibiting expression of the gene encoding acetyl-CoA synthetase 1, which is involved in lipid synthesis. This study suggests a novel mechanism in which lipid biosynthesis may be regulated by O-GlcNAc.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes.     

The inability of cells to grow in low iron correlates with increasing activity of their iron regulatory protein (IRP)

Summary We studied the factors that determine the differing growth requirements of low-iron-tolerant (LIT) versus high-iron-dependent (HID) cells for extracellular nontransferrin iron. The growth of LIT cells HeLa and THP-1, when transferred from transferrin (5 μg/ml) medium into low-iron (5 μM ferric citrate) medium, was not significantly affected while HID cells Jiyoye and K562 showed nearly no growth. HeLa and THP-1 cells, as well as Jiyoye and K562 cells, do not produce transferrin in sufficient amounts to support their growth in low-iron medium. Surprisingly, similar rates of iron uptake in low-iron medium (0.033 and 0.032 nmol Fe/min and 10⁶ cells) were found for LIT cells HeLa and HID cells K562. Furthermore, the intracellular iron level (4.64 nmol/10⁶ cells) of HeLa cells grown in low-iron medium was much higher than iron levels (0.15 or 0.20 nmol/10⁶ cells) of HeLa or K562 cells grown in transferrin medium. We demonstrated that the activity (ratio activated/total) of the iron regulatory protein (IRP) in HID cells Jiyoye and K562 increased more than twofold (from 0.32 to 0.79 and from 0.47 to 1.12, respectively) within 48 h after their transfer into low-iron medium. In the case of LIT cells HeLa and THP-1, IRP activity stayed at similar or slightly decreased levels (0.86–0.73 and 0.58–0.55, respectively). Addition of iron chelator deferoxamine (50 μM, i.e., about half-maximal growth-inhibitory dose) resulted in significantly increased activity of IRP also in HeLa and THP-1 cells. We hypothesize that the relatively higher bioavailability of nontransferrin iron in LIT cells, over that in HID cells, determines the differing responses observed under low-iron conditions.     

Effects of hypoxic preconditioning on the expression of iron influx and efflux proteins in primary neuron culture

The mechanisms of neuroprotection induced by hypoxic preconditioning (HP) and the effects of HP on iron metabolism proteins in the brain have not been fully elucidated. Based on the accumulated information, we hypothesized that HP would be able to affect the expression of iron metabolism proteins in the brain and that the changes in the expression of these proteins induced by HP might be partly associated with the HP-induced neuroprotection. Here, we demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (Fpn1) proteins and thus increase transferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release, and also a progressive increase in cellular iron content in the cultured neurons. We concluded that HP has the ability to speed iron transport rate and proposed that the increase in iron transport rate and cellular iron in neurons might be one of the mechanisms involved in neuroprotection in the HP neurons. We also demonstrated that Fpn1 expression was significantly affected by HIF-1alpha, implying that the gene encoding this iron efflux protein is hypoxia-inducible.     

The role of reactive oxygen and nitrogen species in cellular iron metabolism

The catalytic role of iron in the Haber-Weiss chemistry, which results in propagation of damaging reactive oxygen species (ROS), is well established. In this review, we attempt to summarize the recent evidence showing the reverse: That reactive oxygen and nitrogen species can significantly affect iron metabolism. Their interaction with iron-regulatory proteins (IRPs) seems to be one of the essential mechanisms of influencing iron homeostasis. Iron depletion is known to provoke normal iron uptake via IRPs, superoxide and hydrogen peroxide are supposed to cause unnecessary iron uptake by similar mechanism. Furthermore, ROS are able to release iron from iron-containing molecules. On the contrary, nitric oxide (NO) appears to be involved in cellular defense against the iron-mediated ROS generation probably mainly by inducing iron removal from cells. In addition, NO may attenuate the effect of superoxide by mutual reaction, although the reaction product—peroxynitrite—is capable to produce highly reactive hydroxyl radicals.