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.     

Aedes aegypti ferritin.

Diseases transmitted by hematophagous (blood-feeding) insects are responsible for millions of human deaths worldwide. In hematophagous insects, the blood meal is important for regulating egg maturation. Although a high concentration of iron is toxic for most organisms, hematophagous insects seem unaffected by the iron load in a blood meal. One means by which hematophagous insects handle this iron load is, perhaps, by the expression of iron-binding proteins, specifically the iron storage protein ferritin. In vertebrates, ferritin is an oligomer composed of two types of subunits called heavy and light chains, and is part of the constitutive antioxidant response. Previously, we found that the insect midgut, a main site of iron load, is also a primary site of ferritin expression and that, in the yellow fever mosquito, Aedes aegypti, the expression of the ferritin heavy-chain homologue (HCH) is induced following blood feeding. We now show that the expression of the Aedes ferritin light-chain homologue (LCH) is also induced with blood-feeding, and that the genes of the LCH and HCH are tightly clustered. mRNA levels for both LCH- and HCH-genes increase with iron, H2O2 and hemin treatment, and the temporal expression of the genes is very similar. These results confirm that ferritin could serve as the cytotoxic protector in mosquitoes against the oxidative challenge of the bloodmeal. Finally, although the Aedes LCH has no iron responsive element (IRE) at its 5'-untranslated region (UTR), the 5'-UTR contains several introns that are alternatively spliced, and this alternative splicing event is different from any ferritin message seen to date.     

Drosophila melanogaster ferritin: cDNA encoding a light chain homologue, temporal and tissue specific expression of both subunit types.

Drosophila melanogaster secreted ferritin like the cytosolic ferritins of other organisms is composed of two subunits, a heavy chain homologue (HCH) and a light chain homologue (LCH). We report the cloning of a cDNA encoding the ferritin LCH of this insect. As predicted from the gene sequence, it contains no iron responsive element (IRE). Northern blot analysis reveals two mRNAs that differ in length due to the choice of polyadenylation signals. Message levels vary through the life cycle of the fly and are markedly increased by high levels of dietary iron. The gut is the main site of increased message synthesis and iron preferentially increases the amount of shorter messages. Western blotting reveals that LCH is the predominant ferritin subunit in all life stages. The amount of LCH protein corresponds well with the message levels in control animals, while in iron-fed animals LCH does not increase proportionally with the message levels. In contrast, the amount of HCH is less than that would be predicted from message levels in control animals, but corresponds well in iron-fed animals. Ferritin is abundant in gut and hemolymph of larvae and adults and in ovaries of adult flies. At pupariation, ferritin becomes more abundant in hemolymph than in other tissues.     

Organization of the ferritin genes in Drosophila melanogaster

The organization of two closely clustered genes, Fer1HCH and Fer2LCH, encoding the heavy-chain homolog (HCH) and the light-chain homolog (LCH) subunits of Drosophila melanogaster ferritin are reported here. The 5019-bp sequence of the cluster was assembled from genomic fragments obtained by polymerase chain reaction (PCR) amplification of genomic DNA and from sequences obtained from the Berkeley Drosophila Genome Project (BDGP) (http://www.fruitfly.org). These genes, located at position 99F1, have different exon-intron structures (Fer1HCH has three introns and Fer2LCH has two introns) and are divergently transcribed. Computer analysis of the possibly shared promoter regions revealed the presence of putative metal regulatory elements (MREs), a finding consistent with the upregulation of these genes by iron, and putative NF-kappaB-like binding sites. The structure of two other invertebrate ferritin genes, from the nematode Caenorhabditis elegans (located on chromosomes I and V), was also analyzed. Both nematode genes have two introns, lack iron-responsive elements (IREs), and encode ferritin subunits similar to vertebrate H chains. These findings, along with comparisons of ferritin genes from invertebrates, vertebrates, and plants, suggest that the specialization of ferritin H and L type chains, the complex exon-intron organization of plant and vertebrate genes, and the use of the IRE/iron regulatory protein (IRP) mechanism for regulation of ferritin synthesis are recent evolutionary acquisitions.     

Alterations in the interaction between iron regulatory proteins and their iron responsive element in normal and Alzheimer's diseased brains.

Iron regulatory proteins (IRPs) are cytoplasmic mRNA binding proteins involved in intracellular regulation of iron homeostasis. IRPs regulate expression of ferritin and transferrin receptor at the mRNA level by interacting with a conserved RNA structure termed the iron-responsive element (IRE). This concordant regulation of transferrin receptors and ferritin is designed so a cell can obtain iron when it is needed, and sequester iron when it is in excess. However, we have reported that iron accumulates in the brain in Alzheimer's disease without a concomitant increase in ferritin. An increase in iron without proper sequestration can increase the vulnerability of cells to oxidative stress. Oxidative stress is a component of many neurological diseases including Alzheimer's. We hypothesized that alterations in the IRP/IRE interaction could be the site at which iron mismanagement occurs in the Alzheimer's brains. In this report we demonstrate that in normal human brain extracts, the IRP is detected as a double IRE/IRP complex by RNA band shift assay, but in 2 of 6 Alzheimer's brain (AD) extracts examined a single IRE/IRP complex was obtained. Furthermore, the mobility of the single IRE/IRP complex in Alzheimer's brain extracts is decreased relative to the double IRE/IRP complex. Western blot and RNA band super shift assay demonstrate that IRP1 is involved in the formation of the single IRE/IRP complex. In vitro analyses suggest that the stability of the doublet complex and single AD complex are different. The single complex from the AD brain are more stable. A more stable IRE/IRP complex in the AD brain could increase stability of the transferrin receptor mRNA and inhibit ferritin synthesis. At the cellular level, the outcome of this alteration in the molecular regulatory mechanism would be increased iron accumulation without an increase in ferritin; identical to the observation we reported in AD brains. The appearance of the single IRE/IRP complex in Alzheimer's brain extracts is associated with relatively high endogenous ribonuclease activity. We propose that elevated RNase activity is one mechanism by which the iron regulatory system becomes dysfunctional.     

Role of RNA secondary structure of the iron-responsive element in translational regulation of ferritin synthesis.

Iron regulates synthesis of the iron storage protein ferritin at the translational level through interaction between a stem-loop structure, the iron-responsive element (IRE), located in the 5'-untranslated region (5'-UTR) of ferritin mRNAs, and a protein, the iron regulatory protein (IRP). The role of IRE secondary structure in translational regulation of ferritin synthesis was explored by introducing ferritin constructs containing mutations in the IRE into Rat-2 fibroblasts. Our in vivo studies demonstrate that size and sequence of the loop within the IRE and the distance and/or spatial relationship of this loop to the bulged nucleotide region closest to the loop must be preserved in order to observe iron-dependent translation of ferritin mRNA. In contrast, changes in nucleotide sequence of the upper stem can be introduced without affecting translational regulation in vivo, as long as a stem can be formed. Our in vivo results suggest that only a very small variation in the affinity of interaction of IRP with IRE can be tolerated in order to maintain iron-dependent regulation of translation.     

Excess capacity of the iron regulatory protein system

Iron regulatory proteins (IRP1 and IRP2) are master regulators of cellular iron metabolism. IRPs bind to iron-responsive elements (IREs) present in the untranslated regions of mRNAs encoding proteins of iron storage, uptake, transport, and export. Because simultaneous knockout of IRP1 and IRP2 is embryonically lethal, it has not been possible to use dual knockouts to explore the consequences of loss of both IRP1 and IRP2 in mammalian cells. In this report, we describe the use of small interfering RNA to assess the relative contributions of IRP1 and IRP2 in epithelial cells. Stable cell lines were created in which either IRP1, IRP2, or both were knocked down. Knockdown of IRP1 decreased IRE binding activity but did not affect ferritin H and transferrin receptor 1 (TfR1) expression, whereas knockdown of IRP2 marginally affected IRE binding activity but caused an increase in ferritin H and a decrease in TfR1. Knockdown of both IRPs resulted in a greater reduction of IRE binding activity and more severe perturbation of ferritin H and TfR1 expression compared with single IRP knockdown. Even though the knockdown of IRP-1, IRP-2, or both was efficient, resulting in nondetectable protein and under 5% of wild type levels of mRNA, all stable knockdowns retained an ability to modulate ferritin H and TfR1 appropriately in response to iron challenge. However, further knockdown of IRPs accomplished by transient transfection of small interfering RNA in stable knockdown cells completely abolished the response of ferritin H and TfR1 to iron challenge, demonstrating an extensive excess capacity of the IRP system.     

Selective Translational Control of the Alzheimer Amyloid Precursor Protein Transcript by Iron Regulatory Protein-1

Iron influx increases the translation of the Alzheimer amyloid precursor protein (APP) via an iron-responsive element (IRE) RNA stem loop in its 5′-untranslated region. Equal modulated interaction of the iron regulatory proteins (IRP1 and IRP2) with canonical IREs controls iron-dependent translation of the ferritin subunits. However, our immunoprecipitation RT-PCR and RNA binding experiments demonstrated that IRP1, but not IRP2, selectively bound the APP IRE in human neural cells. This selective IRP1 interaction pattern was evident in human brain and blood tissue from normal and Alzheimer disease patients. We computer-predicted an optimal novel RNA stem loop structure for the human, rhesus monkey, and mouse APP IREs with reference to the canonical ferritin IREs but also the IREs encoded by erythroid heme biosynthetic aminolevulinate synthase and Hif-2α mRNAs, which preferentially bind IRP1. Selective 2′-hydroxyl acylation analyzed by primer extension analysis was consistent with a 13-base single-stranded terminal loop and a conserved GC-rich stem. Biotinylated RNA probes deleted of the conserved CAGA motif in the terminal loop did not bind to IRP1 relative to wild type probes and could no longer base pair to form a predicted AGA triloop. An AGU pseudo-triloop is key for IRP1 binding to the canonical ferritin IREs. RNA probes encoding the APP IRE stem loop exhibited the same high affinity binding to rhIRP1 as occurs for the H-ferritin IRE (35 pm). Intracellular iron chelation increased binding of IRP1 to the APP IRE, decreasing intracellular APP expression in SH-SY5Y cells. Functionally, shRNA knockdown of IRP1 caused increased expression of neural APP consistent with IRP1-APP IRE-driven translation.     

Controlled cortical impact injury affects dopaminergic transmission in the rat striatum

The therapeutic benefits of dopamine (DA) agonists after traumatic brain injury (TBI) imply a role for DA systems in mediating functional deficits post‐TBI. We investigated how experimental TBI affects striatal dopamine systems using fast scan cyclic voltammetry (FSCV), western blot, and d‐amphetamine‐induced rotational behavior. Adult male Sprague–Dawley rats were injured by a controlled cortical impact (CCI) delivered unilaterally to the parietal cortex, or were naïve controls. Amphetamine‐induced rotational behavior was assessed 10 days post‐CCI. Fourteen days post‐CCI, animals were anesthetized and underwent FSCV with bilateral striatal carbon fiber microelectrode placement and stimulating electrode placement in the medial forebrain bundle (MFB). Evoked DA overflow was assessed in the striatum as the MFB was electrically stimulated at 60 Hz for 10 s. In 23% of injured animals, but no naïve animals, rotation was observed with amphetamine administration. Compared with naïves, striatal evoked DA overflow was lower for injured animals in the striatum ipsilateral to injury (p < 0.05). Injured animals exhibited a decrease in V_max (52% of naïve, p < 0.05) for DA clearance in the hemisphere ipsilateral to injury compared with naïves. Dopamine transporter (DAT) expression was proportionally decreased in the striatum ipsilateral to injury compared with naïve animals (60% of naïve, p < 0.05), despite no injury‐related changes in vesicular monoamine transporter or D₂ receptor expression (DRD₂) in this region. Collectively, these data appear to confirm that the clinical efficacy of dopamine agonists in the treatment of TBI may be related to disruptions in the activity of subcortical dopamine systems.     

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.     

Hyphantria cunea ferritin heavy chain homologue: cDNA sequence and mRNA expression

We have sequenced a cDNA clone encoding a 26-kDa ferritin subunit, which was heavy chain homologue (HCH), in fall webworm, Hyphantria cunea. The HCH cDNA was obtained from the screening of a cDNA library using a PCR product. H. cunea ferritin is composed of 221 amino acid residues and their calculated mass is 26,160 Da. The protein contains the conserved motifs for the ferroxidase center typical for heavy chains of vertebrate ferritin. The iron-responsive element sequence with a predicted stem-loop structure is present in the 5'-untranslated region of ferritin HCH mRNA. The sequence alignment of ferritin HCH shows 68.9 and 68.7% identity with Galleria mellonella HCH (26 kDa ferritin) and Manduca sexta HCH, respectively. While G type insect ferritin vertebrate light chain homologue (LCH) is distantly related to H. cunea ferritin HCH (17.2-20.8%), the Northern blot analysis revealed that H. cunea ferritin HCH was ubiquitously expressed in various tissues and all developmental stages. The ferritin expression of midgut is more responsive to iron-fed, compared to fat body in H. cunea.     

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.     

Iron loaded ferritin secretion and inhibition by CI-976 in Aedes aegypti larval cells

Ferritin is a multimer of 24 subunits of heavy and light chains. In mammals, iron taken into cells is stored in ferritin or incorporated into iron-containing proteins. Very little ferritin is found circulating in mammalian serum; most is retained in the cytoplasm. Female mosquitoes, such as Aedes aegypti (yellow fever mosquito, Diptera), require a blood meal for oogenesis. Mosquitoes receive a potentially toxic level of iron in the blood meal which must be processed and stored. We demonstrate by ⁵⁹Fe pulse-chase experiments that cultured A. aegypti larval CCL-125 cells take up iron from culture media and store it in ferritin found mainly in the membrane fraction and secrete iron-loaded ferritin. We observe that in these larval cells ferritin co-localizes with ceramide-containing membranes in the absence of iron. With iron treatment, ferritin is found associated with ceramide-containing membranes as well as in cytoplasmic non-ceramide vesicles. Treatment of CCL-125 cells with iron and CI-976, an inhibitor of lysophospholipid acyl transferases, disrupts ferritin secretion with a concomitant decrease in cell viability. Interfering with ferritin secretion may limit the ability of mosquitoes to adjust to the high iron load of the blood meal and decrease iron delivery to the ovaries reducing egg numbers.     

Dopaminergic neuronal differentiation from rat embryonic neural precursors by Nurr1 overexpression

In vitro expanded CNS precursors could provide a renewable source of dopamine (DA) neurons for cell therapy in Parkinson's disease. Functional DA neurons have been derived previously from early midbrain precursors. Here we demonstrate the ability of Nurr1, a nuclear orphan receptor essential for midbrain DA neuron development in vivo, to induce dopaminergic differentiation in naïve CNS precursors in vitro. Independent of gestational age or brain region of origin, Nurr1‐induced precursors expressed dopaminergic markers and exhibited depolarization‐evoked DA release in vitro. However, these cells were less mature and secreted lower levels of DA than those derived from mesencephalic precursors. Transplantation of Nurr1‐induced DA neuron precursors resulted in limited survival and in vivo differentiation. No behavioral improvement in apomorphine‐induced rotation scores was observed. These results demonstrate that Nurr1 induces dopaminergic features in naïve CNS precursors in vitro. However, additional factors will be required to achieve in vivo function and to unravel the full potential of neural precursors for cell therapy in Parkinson's disease.     

Secreted ferritin: Mosquito defense against iron overload?

The yellow fever mosquito, Aedes aegypti, must blood feed in order to complete her life cycle. The blood meal provides a high level of iron that is required for egg development. We are interested in developing control strategies that interfere with this process. We show that A. aegypti larval cells synthesize and secrete ferritin in response to iron exposure. Cytoplasmic ferritin is maximal at low levels of iron, consists of both the light chain (LCH) and heavy chain (HCH) subunits and reflects cytoplasmic iron levels. Secreted ferritin increases in direct linear relationship to iron dose and consists primarily of HCH subunits. Although the messages for both subunits increase with iron treatment, our data indicate that mosquito HCH synthesis could be partially controlled at the translational level as well. Importantly, we show that exposure of mosquito cells to iron at low concentrations increases cytoplasmic iron, while higher iron levels results in a decline in cytoplasmic iron levels indicating that excess iron is removed from mosquito cells. Our work indicates that HCH synthesis and ferritin secretion are key factors in the response of mosquito cells to iron exposure and could be the primary mechanisms that allow these insects to defend against an intracellular iron overload.     

Thyrotropin-releasing hormone and epidermal growth factor regulate iron-regulatory protein binding in pituitary cells via protein kinase C-dependent and -independent signaling pathways

Intracellular iron homeostasis is regulated, in part, by interactions between iron-regulatory proteins (IRP1 and IRP2) and iron-responsive elements (IREs) in ferritin and transferrin receptor mRNAs. In addition to iron, cellular oxidative stress induced by H(2)O(2), nitric oxide, and hypoxia, and hormonal activation by thyroid hormone and erythropoeitin have each been shown to regulate IRP binding to IREs. Hormonal signals, in particular mediated through protein kinase C (PKC), play a central role in the modulation of IRP/IRE interactions since phorbol esters were shown to activate IRP binding (Eisenstein, R. S., Tuazon, P. T., Schalinske, K. L., Anderson, S. A., and Traugh, J. A. (1993) J. Biol. Chem. 268, 27363-27370). In pituitary thyrotrophs (TtT97), we found that thyrotropin releasing hormone (TRH) and epidermal growth factor (EGF) increased IRP binding to a ferritin IRE, dependent on PKC and mitogen-activated protein kinase (MAPK) activity. In contrast, TRH and EGF decreased IRP binding in pituitary lactotrophs (GH3), despite activation of PKC and MAPK. IRP1 and IRP2 levels remained constant and IRP2 binding was predominant throughout. TRH and EGF markedly decreased IRP binding in MAPK kinase inhibitor-treated GH3 cells, whereas, they increased IRP binding in phosphatase inhibitor-treated GH3 cells. IRE-dependent CAT reporter translational expression closely reflected IRP binding to the ferritin IRE in both GH3 and TtT97 cells. Interestingly, ferritin protein levels were regulated similarly by TRH in both cell lines. These data link two different cell receptor systems to common signaling pathways that regulate IRP binding and ferritin expression. Remarkably, for TRH and EGF, these effects may be PKC-dependent or -independent determined by the cell type.     

Orientation behavior of Propylaea japonica toward visual and olfactory cues from its prey–host plant combination

The lady beetle Propylaea japonica (Thunberg) (Coleoptera: Coccinellidae) is an important predator of aphids in agroecosystems. The inundative release of coccinellid beetles can be an effective biological control strategy. An understanding of how biological control agents perceive and use stimuli from host plants is the key to successfully implement commercially produced predators. Here, we studied the relative role of visual and volatile cues. Dual‐choice assays using foraging‐naïve and foraging‐experienced P. japonica adults were conducted using cotton plants [Gossypium hirsutum L. (Malvaceae)] with or without infestation by the cotton aphid, Aphis gossypii (Glover) (Hemiptera: Aphididae). Overall, experienced beetles were more attracted than naïve beetles toward cues associated with aphid‐infested plants. Experienced beetles were also more responsive to olfactory cues compared with naïve beetles. Both foraging‐naïve and ‐experienced lady beetles integrate olfactory and visual cues from plants infested with aphids, with an apparently greater reliance on olfactory cues. The results suggest that foraging experience may increase prey location in P. japonica.