Involvement of the Iron Regulatory Protein from Eisenia andrei Earthworms in the Regulation of Cellular Iron Homeostasis

Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5′- or 3′-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5′-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.     

Binding of cytosolic aconitase to the iron responsive element of porcine mitochondrial aconitase mRNA.

The 5' end of porcine mitochondrial aconitase mRNA contains an iron responsive element (IRE)-like secondary structure (T. Dandekar, R. Stripecke, N. K. Gray, B. Goosen, A. Constable, H. E. Johansson, and M. W. Hentze (1991) EMBO J. 10, 1903-1909). A protein from a liver extract binds to a mitochondrial aconitase RNA probe and supports the identification of this sequence as an IRE. Purified cytosolic aconitase but not the mitochondrial enzyme binds to this IRE as well as to a ferritin IRE. All forms of cytosolic aconitase, [4Fe-4S] enzyme, [3Fe-4S] enzyme and apoenzyme bind with similar affinity. A Kd of 0.25 nM was calculated for the apoaconitase-IRE interaction from Scatchard analysis. These results support the conclusion that cytosolic aconitase is an IRE-binding protein which may regulate translation of mitochondrial aconitase mRNA.     

MicroRNA-1291-mediated silencing of IRE1α enhances Glypican-3 expression

MicroRNAs (miRNA) are generally described as negative regulators of gene expression. However, some evidence suggests that they may also play positive roles. As such, we reported that miR-1291 leads to a GPC3 mRNA expression increase in hepatoma cells through a 3′ untranslated region (UTR)-dependent mechanism. In the absence of any direct interaction between miR-1291 and GPC3 mRNA, we hypothesized that miR-1291 could act by silencing a negative regulator of GPC3 mRNA expression. Based on in silico predictions and experimental validation, we demonstrate herein that miR-1291 represses the expression of the mRNA encoding the endoplasmic reticulum (ER)-resident stress sensor IRE1α by interacting with a specific site located in the 5′ UTR. Moreover, we show, in vitro and in cultured cells, that IRE1α cleaves GPC3 mRNA at a 3′ UTR consensus site independently of ER stress, thereby prompting GPC3 mRNA degradation. Finally, we show that the expression of a miR-1291-resistant form of IRE1α abrogates the positive effects of miR-1291 on GPC3 mRNA expression. Collectively, our data demonstrate that miR-1291 is a biologically relevant regulator of GPC3 expression in hepatoma cells and acts through silencing of the ER stress sensor IRE1α.     

An atypical iron-responsive element (IRE) within crayfish ferritin mRNA and an iron regulatory protein 1 (IRP1)-like protein from crayfish hepatopancreas

A putative crayfish iron-responsive element (IRE) is present in the 5'-untranslated region of the crayfish ferritin mRNA. The putative crayfish IRE is in a cap-proximal position and shares most of the structural features of the consensus IRE, but the RNA stem-loop structure contains a bulge of a guanine instead of a cytosine at the expected position, so far thought to be a hallmark of IREs. By using an electromobility shift assay this IRE was shown to specifically bind purified recombinant human iron regulatory protein 1 (IRP1) as well as a factor(s) present in a homogenate of crayfish hepatopancreas, likely to be a crayfish IRP1 homologue. With mutations in the crayfish IRE, the affinity of IRP to IRE was drastically decreased. A cDNA encoding an IRP1-like protein was cloned from the hepatopancreas of crayfish. This protein has sequence similarities to IRP, and contains all the active-site residues of aconitase, two putative RNA-binding regions and a putative contact site between RNA and IRP. These results show that a crayfish IRE, lacking the bulged C, can bind IRP1 in vitro and that an IRP1-like protein present in crayfish hepatopancreas may have both aconitase and RNA-binding activities.     

Identification and characterization of the iron regulatory element in the ferritin gene of a plant (soybean)

Iron increases ferritin synthesis, targeting plant DNA and animal mRNA. The ferritin promoter in plants has not been identified, in contrast to the ferritin promoter and mRNA iron-responsive element (IRE) in animals. The soybean leaf, a natural tissue for ferritin expression, and DNA, with promoter deletions and luciferase or glucuronidase reporters, delivered with particle bombardment, were used to show that an 86-base pair fragment (iron regulatory element (FRE)) controlled iron-mediated derepression of the ferritin gene. Mutagenesis with linkers of random sequence detected two subdomains separated by 21 base pairs. FRE has no detectable homology to the animal IRE or to known promoters in DNA and bound a trans-acting factor in leaf cell extracts. FRE/factor binding was abrogated by increased tissue iron, in analogy to mRNA (IRE)/iron regulatory protein in animals. Maximum ferritin derepression was obtained with 50 microm iron citrate (1:10) or 500 microm iron citrate (1:1) but Fe-EDTA was ineffective, although the leaf iron concentration was increased; manganese, zinc, and copper had no effect. The basis for different responses in ferritin expression to different iron complexes, as well as the significance of using DNA but not mRNA as an iron regulatory target in plants, remain unknown.     

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.     

Molecular cloning, expression and characterization of cDNAs encoding the ferritin subunits from the beetle, Apriona germari

Insect secreted ferritins are composed of subunits, which resemble heavy and light chains of vertebrate cytosolic ferritins. We describe here the cloning, expression and characterization of cDNAs encoding the ferritin heavy-chain homologue (HCH) and light-chain homologue (LCH) from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae). The A. germari ferritin LCH and HCH cDNA sequences were comprised of 672 and 636 bp encoding 224 and 212 amino acid residues, respectively. The A. germari ferritin HCH subunit contained the conserved motifs for the ferroxidase center typical of vertebrate ferritin heavy chains and the iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5′-untranslated region (UTR) of ferritin HCH mRNA. However, the A. germari ferritin LCH subunit had no IRE at its 5′-UTR and ferroxidase center residues. Phylogenetic analysis confirmed the deduced protein sequences of A. germari ferritin HCH and LCH being divided into two types, G type (LCH) and S type (HCH). Southern blot analysis suggested the possible presence of each A. germari ferritin subunit gene as a single copy and Northern blot analysis confirmed a higher expression pattern in midgut than fat body. The cDNAs encoding the A. germari ferritin subunits were expressed as approximately 30 kDa (LCH) and 26 kDa (HCH) polypeptides in baculovirus-infected insect cells. Western blot analysis and iron staining assay confirmed that A. germari ferritin has a native molecular mass of approximately 680 kDa.     

Evolution of the iron-responsive element

An RNA hairpin structure referred to as the iron-responsive element (IRE) and iron regulatory proteins (IRPs) are key players in the control of iron metabolism in animal cells. They regulate translation initiation or mRNA stability, and the IRE is found in a variety of mRNAs, such as those encoding ferritin, transferrin receptor (Tfr), erythroid aminolevulinic acid synthase (eALAS), mitochondrial aconitase (mACO), ferroportin, and divalent metal transporter 1 (DMT1). We have studied the evolution of the IRE by considering all mRNAs previously known to be associated with this structure and by computationally examining its occurrence in a large variety of eukaryotic organisms. More than 100 novel sequences together with approximately 50 IREs that were previously reported resulted in a comprehensive view of the phylogenetic distribution of this element. A comparison of the different mRNAs shows that the IREs of eALAS and mACO are found in chordates, those of ferroportin and Tfr1 are found in vertebrates, and the IRE of DMT1 is confined to mammals. In contrast, the IRE of ferritin occurs in a majority of metazoa including lower metazoa such as sponges and Nematostella (sea anemone). These findings suggest that the ferritin IRE represents the ancestral version of this type of translational control and that during the evolution of higher animals the IRE structure was adopted by other genes. On the basis of primary sequence comparison between different organisms, we suggest that some of these IREs developed by "convergent evolution" through stepwise changes in sequence, rather than by recombination events.     

Regulation of ferritin and transferrin receptor mRNAs

Iron regulates the synthesis of two proteins critical for iron metabolism, ferritin and the transferrin receptor, through novel mRNA/protein interactions. The mRNA regulatory sequence (iron-responsive element (IRE)) occurs in the 5'-untranslated region of all ferritin mRNAs and is repeated as five variations in the 3'-untranslated region of transferrin receptor mRNA. When iron is in excess, ferritin synthesis and iron storage increase. At the same time, transferrin receptor synthesis and iron uptake decrease. Location of the common IRE regulatory sequence in different noncoding regions of the two mRNAs may explain how iron can have opposite metabolic effects; when the IRE is in the 5'-untranslated region of ferritin mRNA, translation is enhanced by excess iron whereas the presence of the IREs in the 3'-untranslated region of the transferrin receptor mRNA leads to iron-dependent degradation. How and where iron actually acts is not yet known. A soluble 90-kDa regulatory protein which has been recently purified to homogeneity from liver and red cells specifically blocks translation of ferritin mRNA and binds IRE sequences but does not appear to be an iron-binding protein. The protein is the first specific eukaryotic mRNA regulator identified and confirms predictions 20 years old. Concerted regulation by iron of ferritin and transferrin receptor mRNAs may also define a more general strategy for using common mRNA sequences to coordinate the synthesis of metabolically related proteins.     

Characteristics of the interaction of the ferritin repressor protein with the iron-responsive element

Summary The iron-responsive regulation of ferritin mRNA translation is mediated by the specific interaction of the ferritin repressor protein (FRP) with the iron-responsive element (IRE), a highly conserved 28-nucleotide sequence located in the 5 untranslated region of ferritin mRNAs. The IRE alone is necessary and sufficient to confer repression of translation by FRP upon a heterologous message, chloramphenicol acetyltransferase, in an in vitro translation system. The activity of FRP is sensitive to iron in vivo. Cytoplasmic extracts of rabbit kidney cells show reduction of FRP activity when grown in the presence of iron, as detected by RNA band shift assay. Using a nitrocellulose filter binding assay to examine the interaction of FRP with the IRE in more detail, we find that purified FRP has a single high-affinity binding site for the IRE with aK _d of 20–50 pM. Hemin pretreatment decreases the total amount of FRP which can bind to the IRE. This effect is dependent on hemin concentration. Interestingly, the FRP which remains active at a given hemin concentration binds to the IRE with the same high affinity as untreated FRP. A variety of hemin concentrations were examined for their effect on preformed FRP/IRE complexes. All hemin concentrations tested resulted in rapid complex breakdown. The final amount of complex breakdown corresponds to the concentration of hemin present in the reaction. The effect of hemin on FRP activity suggests that a specific hemin binding site exists on FRP.Abbreviations IRE iron-responsive element - FRP ferritin repressor protein - CAT chloramphenicol acetyltransferase - ORF open reading frame     

Biochemical and pathogenic properties of the natural isolate of Shewanella algae from Peter the Great Bay, Sea of Japan

Pathogenic properties of the natural isolate of Shewanella algae from the coelomic fluid of the sea cucumber Apostichopus japonicus (Peter the Great Bay, Sea of Japan) were investigated. The isolate had oxydative metabolism, was positive for ornithine decarboxylase, cytochrome oxidase, catalase, DNase and gelatinase, hemolytically active, did not produce acid from carbohydrates, and did not hydrolyze urea and esculin. The strain was resistant to penicillin, amoxicillin, and ampicillin and susceptible to tetracycline and carbenicillin. Among cellular fatty acids, 13:0-i, 15:0-i, 16:0, 16:1(n-7), 17:0-i, and 17:0-ai dominated. These biochemical properties made it possible to attribute the isolated bacteria to the genus Shewanella and identified as S. algae. The cells of this bacterium were introduced into the coelomic cavity of another echinoderm, the sea urchin Strongylocentrotus nudus. As a result, in about 24 h the animals became slow and 3-8 days after the inoculation died. Dividing bacteria were being found during the experiment in the coelomic fluid as well as in the phagosomes of amoebocytes, i.e. cells acting as phagocytes in the coelomic fluid. The studies of the invasive properties of strain 156 showed that bacterial cells entered the subcuticular space of S. nudus and A. japonicus through the cuticle and stayed there for a long time without penetrating epithelium and exerting toxic effect upon the organisms of the laboratory animals. Pathogenic effect of S. algae can be manifested only if the cutaneous epithelium is destroyed permitting it to penetrate the lower tissue layers. The toxicity of S. algae is confirmed by in vitro experiments. The inoculation of the embryonic cells of S. nudus with samples of this bacterium caused the death of 10% of cells within an hour and 100% of cells within 12 h after inoculation. The results of the investigations demonstrate that S. algae could produce opportunistic infection in the sea cucumber A. japonicus and the sea urchin S. nudus, which may be natural reservoirs of this human pathogen.     

Three ferritin subunits involved in immune defense from bay scallop Argopecten irradians

Ferritin is a ubiquitous protein that plays an important role in iron storage and iron-withholding strategy of innate immunity. In this study, three genes encoding different ferritin subunits were cloned from bay scallop Argopecten irradians (AiFer1, AiFer2 and AiFer3) by rapid amplification of cDNA ends (RACE) approaches based on the known ESTs. The open reading frames of the three ferritins are of 516 bp, 522 bp and 519 bp, encoding 171,173 and 172 amino acids, respectively. All the AiFers contain a putative Iron Regulatory Element (IRE) in their 5′-untranslated regions. The deduced amino acid sequences of AiFers possess both the ferroxidase center of mammalian H ferritin and the iron nucleation site of mammalian L ferritin. Gene structure study revealed two distinct structured genes encoding a ferritin subunit (AiFer3). Quantitative real-time PCR analysis indicated the significant up-regulation of AiFers in hemocytes after challenged with Listonella anguillarum, though the magnitudes of AiFer1 and AiFer2 were much higher than that of AiFer3. Taken together, these results suggest that AiFers are likely to play roles in both iron storage and innate immune defense against microbial infections.