Distribution of divalent metal transporter 1 and metal transport protein 1 in the normal and Belgrade rat.

Iron accumulation in the brain occurs in a number of neurodegenerative diseases. Two new iron transport proteins have been identified that may help elucidate the mechanism of abnormal iron accumulation. The Divalent Metal Transporter 1 (DMT1), is responsible for iron uptake from the gut and transport from endosomes. The Metal Transport Protein 1 (MTP1) promotes iron export. In this study we determined the cellular and regional expression of these two transporters in the brains of normal adult and Belgrade rats. Belgrade rats have a defect in DMT1 that is associated with lower levels of iron in the brain. In the normal rat, DMT1 expression is highest in neurons in the striatum, cerebellum, thalamus, ependymal cells lining the third ventricle, and vascular cells throughout the brain. The staining in the ependymal cells and endothelial cells suggests that DMT1 has an important role in iron transport into the brain. In Belgrade rats, there is generalized decrease in immunodetectable DMT1 compared to normal rats except in the ependymal cells. This decrease in immunoreactivity, however, was absent on immunoblots. The immunoblot analysis indicates that this protein did not upregulate to compensate for the chronic defect in iron transport. MTP1 staining is found in most brain regions. MTP1 expression in the brain is robust in pyramidal neurons of the cerebral cortex but is not detected in the vascular endothelial cells and ependymal cells. MTP1 staining in Belgrade rats was decreased compared to normal, but similar to DMT1 this decrease was not corroborated by immunoblotting. These results indicate that DMT1 and MTP1 are involved in brain iron transport and this involvement is regionally and cellularly specific.

Osteoclast-stimulating factor interacts with the spinal muscular atrophy gene product to stimulate osteoclast formation.

We have recently identified and cloned an intracellular peptide termed osteoclast-stimulating factor (OSF) that increases osteoclast (OCL) formation and bone resorption through a cellular signal transduction cascade, possibly through its interaction with c-Src or related family members. To further identify participants in the OSF signaling cascade, we used yeast two-hybrid screening with Saccharomyces cerevisiae, and we found that the 40-kDa spinal muscular atrophy disease-determining gene product, survival motor neuron (SMN), interacts with the OSF-Src homology 3 domain. Reverse transcription-polymerase chain reaction analysis of SMN mRNA expression in cells of the OCL lineage demonstrates that expression of the exon 7 splice variant of SMN is restricted to mature OCLs, whereas the unspliced transcript was expressed in OCL precursors as well as mature OCLs. Treatment of murine bone marrow cultures with conditioned media (5% (v/v)) from 293 cells transiently expressing the SMN cDNA significantly increased OCL formation, compared with treatment with conditioned media from mock-transfected cells. Furthermore, OCL-stimulatory activity by OSF or SMN was abolished by antisense constructs to SMN or OSF, respectively. These data confirm the participation of SMN in the OSF-enhanced expression of an OCL stimulator. OSF-SMN interaction may provide more insights into novel cellular signaling mechanisms that may play an important role in congenital bone fractures associated with type I spinal muscular atrophy disease.

Expression of the duodenal iron transporters divalent-metal transporter 1 and ferroportin 1 in iron deficiency and iron overload.

BACKGROUND & AIMS: Imbalances of iron homeostasis are accompanied by alterations of intestinal iron absorption. The identification of divalent-metal transporter 1 (DMT1) and ferroportin 1 (FP1) has improved our understanding of transmembrane iron trafficking. To gain insight into the regulatory properties of these transporters in the duodenum, we studied their expression in patients with hereditary hemochromatosis (HFE-associated and non-HFE-associated), secondary iron overload, and iron deficiency. METHODS: DMT1, FP1 messenger RNA (mRNA), and protein expression were analyzed in duodenal biopsy specimens from patients by means of TaqMan real-time polymerase chain reaction, Western blotting technique, and immunohistochemistry. RESULTS: DMT1 and FP1 mRNA levels are positively correlated with each other in all patient groups (P < 0.001). Moreover, DMT1 and FP1 mRNA levels were significantly increased in patients with iron deficiency, HFE and non-HFE hemochromatosis, whereas they were unchanged in patients with secondary iron overload. Alterations in DMT1 and FP1 mRNA levels were paralleled by comparable changes in the duodenal expression of these proteins. In patients with normal iron status or iron deficiency, significant negative correlations between DMT1, FP1 mRNA, and serum iron parameters were found, which were absent in subjects with primary hemochromatosis. CONCLUSIONS: DMT1 and FP1 are centrally involved in iron uptake/transfer in the duodenum and in the adaptive changes of iron homeostasis to iron deficiency and overload.

Haptoglobin in lung defence.

Haptoglobin (Hp) has been known to be associated with the host defence response to infection and inflammation. The biological functions of Hp can be related to its ability to bind haemoglobin or to modulate immune response. Hp is expressed at a high level in lung cells, yet its protective role(s) in the lung is not known. Using transgenic mice overexpressing Hp, we demonstrated that Hp can reduce blood-induced lung injury. Hp-mediated haemoglobin catabolism in lung cells appears to be linked to iron mobilization, and may be an efficient mechanism to reduce oxidative damage associated with haemolysis.

A novel mammalian iron-regulated protein involved in intracellular iron metabolism.

We have isolated and characterized a novel iron-regulated gene that is homologous to the divalent metal transporter 1 family of metal transporters. This gene, termed metal transporter protein (mtp1), is expressed in tissues involved in body iron homeostasis including the developing and mature reticuloendothelial system, the duodenum, and the pregnant uterus. MTP1 is also expressed in muscle and central nervous system cells in the embryo. At the subcellular level, MTP1 is localized to the basolateral membrane of the duodenal epithelial cell and a cytoplasmic compartment of reticuloendothelial system cells. Overexpression of MTP1 in tissue culture cells results in intracellular iron depletion. In the adult mouse, MTP1 expression in the liver and duodenum are reciprocally regulated. Iron deficiency induces MTP1 expression in the duodenum but down-regulates expression in the liver. These data indicate that MTP1 is an iron-regulated membrane-spanning protein that is involved in intracellular iron metabolism.

Regulation of genes of iron metabolism by the iron-response proteins.

Iron is an essential nutrient, yet excess iron can be toxic to cells. The uptake of iron by mammalian cells is post-transcriptionally regulated by the interaction of iron-response proteins (IRP1 and IRP2) with iron-response elements (IREs) found in the mRNAs of genes of iron metabolism, such as ferritin, the transferrin receptor, erythroid aminolevulinic acid synthase, and mitochondrial aconitase. The IRPs are RNA binding proteins that bind to the IRE (found in the mRNAs of the regulated genes) in an iron- dependent manner. Binding of IRPs to the IREs leads to changes in the expression of the regulated genes and subsequent changes in the uptake, utilization, or storage of intracellular iron. Recent work has demonstrated that the binding of the IRPs to the IREs can also be modulated by changes in the redox state or oxidative stress level of the cell. These findings provide an important link between iron metabolism and states of oxidative stress.

The interaction between the iron-responsive element binding protein and its cognate RNA is highly dependent upon both RNA sequence and structure.

To assess the influence of RNA sequence/structure on the interaction RNAs with the iron-responsive element binding protein (IRE-BP), twenty eight altered RNAs were tested as competitors for an RNA corresponding to the ferritin H chain IRE. All changes in the loop of the predicted IRE hairpin and in the unpaired cytosine residue characteristically found in IRE stems significantly decreased the apparent affinity of the RNA for the IRE-BP. Similarly, alteration in the spacing and/or orientation of the loop and the unpaired cytosine of the stem by either increasing or decreasing the number of base pairs separating them significantly reduced efficacy as a competitor. It is inferred that the IRE-BP forms multiple contacts with its cognate RNA, and that these contacts, acting in concert, provide the basis for the high affinity of this interaction.

Cellular regulation of the iron-responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding.

The translation of ferritin mRNA and degradation of transferrin receptor mRNA are regulated by the interaction of an RNA-binding protein, the iron-responsive element binding protein (IRE-BP), with RNA stem-loop structures known as iron-responsive elements (IREs) contained within these transcripts. IRE-BP produced in iron-replete cells has aconitase (EC 4.2.1.3) activity. The protein shows extensive sequence homology with mitochondrial aconitase, and sequences of peptides prepared from cytosolic aconitase are identical with peptides of IRE-BP. As an active aconitase, IRE-BP is expected to have an Fe-S cluster, in analogy to other aconitases. This Fe-S cluster has been implicated as the region of the protein that senses intracellular iron levels and accordingly modifies the ability of the IRE-BP to interact with IREs. Expression of the IRE-BP in cultured cells has revealed that the IRE-BP functions either as an active aconitase, when the cells are iron-replete, or as an active RNA-binding protein, when the cells are iron-depleted. We compare properties of purified authentic cytosolic aconitase from beef liver with those of IRE-BP from tissue culture cells and establish that characteristics of the physiologically relevant form of the protein from iron-depleted cells resemble those of cytosolic aconitase apoprotein. We demonstrate that loss of the labile fourth iron atom of the Fe-S cluster results in loss of aconitase activity, but that more extensive cluster alteration is required before the IRE-BP acquires the capacity to bind RNA with the affinity seen in vivo. These results are consistent with a model in which the cubane Fe-S cluster is disassembled when intracellular iron is depleted.

Reciprocal control of RNA-binding and aconitase activity in the regulation of the iron-responsive element binding protein: role of the iron-sulfur cluster.

Several mechanisms of posttranscriptional gene regulation are involved in regulation of the expression of essential proteins of iron metabolism. Coordinate regulation of ferritin and transferrin receptor expression is produced by binding of a cytosolic protein, the iron-responsive element binding protein (IRE-BP) to specific stem-loop structures present in target RNAs. The affinity of this protein for its cognate RNA is regulated by the cell in response to changes in iron availability. The IRE-BP demonstrates a striking level of amino acid sequence identity to the iron-sulfur (Fe-S) protein mitochondrial aconitase. Moreover, the recombinant IRE-BP has aconitase function. The lability of the Fe-S cluster in mitochondrial aconitase has led us to propose that the mechanism by which iron levels are sensed by the IRE-BP involves changes in an Fe-S cluster in the IRE-BP. In this study, we demonstrate that procedures aimed at altering the IRE-BP Fe-S cluster in vitro reciprocally alter the RNA binding and aconitase activity of the IRE-BP. The changes in the RNA binding of the protein produced in vitro appear to match the previously described alterations of the protein in response to iron availability in the cell. Furthermore, iron manipulation of cells correlates with the activation or inactivation of the IRE-BP aconitase activity. The results are consistent with a model for the posttranslational regulation of the IRE-BP in which the Fe-S cluster is altered in response to the availability of intracellular iron and this, in turn, regulates the RNA-binding activity.

An iron-sulfur cluster plays a novel regulatory role in the iron-responsive element binding protein.

Post-transcriptional regulation of genes important in iron metabolism, ferritin and the transferrin receptor (TfR), is achieved through regulated binding of a cytosolic protein, the iron-responsive element binding protein (IRE-BP), to RNA stem-loop motifs known as iron-responsive elements (IREs). Binding of the IRE-BP represses ferritin translation and represses degradation of the TfR mRNA. The IRE-BP senses iron levels and accordingly modifies binding to IREs through a novel sensing mechanism. An iron-sulfur cluster of the IRE-BP reversibly binds iron; when cytosolic iron levels are depleted, the cluster becomes depleted of iron and the IRE-BP acquires the capacity to bind IREs. When cytosolic iron levels are replete, the IRE-BP loses RNA binding capacity, but acquires enzymatic activity as a functional aconitase. RNA binding and aconitase activity are mutually exclusive activities of the IRE-BP, and the state of the iron-sulfur cluster determines how the IRE-BP will function.

Cloning of the cDNA encoding an RNA regulatory protein--the human iron-responsive element-binding protein.

Iron-responsive elements (IREs) are stemloop structures found in the mRNAs encoding ferritin and the transferrin receptor. These elements participate in the iron-induced regulation of the translation of ferritin and the stability of the transferrin receptor mRNA. Regulation in both instances is mediated by binding of a cytosolic protein to the IREs. High-affinity binding is seen when cells are starved of iron and results in repression of ferritin translation and inhibition of transferrin receptor mRNA degradation. The IRE-binding protein (IRE-BP) has been identified as an approximately 90-kDa protein that has been purified by both affinity and conventional chromatography. In this report we use RNA affinity chromatography and two-dimensional gel electrophoresis to isolate the IRE-BP for protein sequencing. A degenerate oligonucleotide probe derived from a single peptide sequence was used to isolate a cDNA clone that encodes a protein containing 13 other sequenced peptides obtained from the IRE-BP. Consistent with previous characterization of the IRE-BP, the cDNA encodes a protein of 87 kDa with a slightly acidic pI, and the corresponding mRNA of approximately 3.6 kilobases is found in a variety of cell types. The encoded protein contains a nucleotide-binding consensus sequence and regions of cysteine and histidine clusters. This mRNA is encoded by a single gene on human chromosome 9, a finding consistent with previous localization by functional mapping. The protein contains no previously defined consensus motifs for either RNA or DNA binding. The simultaneous cloning of a different, but highly homologous, cDNA suggests that the IRE-BP is a member of a distinct gene family.

The inhibition of the iron responsive element RNA-protein interaction by heme does not mimic in vivo iron regulation.

Hemin at greater than 1 microM concentrations inhibits the interaction of the iron responsive element (IRE) and the iron responsive element binding protein (IRE-BP) as measured by gel retardation and UV cross-linking. Heme has recently been proposed to inhibit the repression of translation of an IRE-containing mRNA (Lin, J. J., Daniels-McQueen, S., Patino, M. M., Gaffield, L., Walden, W. E., and Thach, R. E., (1990) Science 247, 74-76). Our binding inhibition provides structural support for these observations. The action of hemin, however, does not mimic the physiologically demonstrated inhibition of high affinity binding of the IRE to IRE-BP by the oxidation of a sulfhydryl of the IRE-BP. In addition to this effect, hemin also inhibits a wide variety of RNA and DNA binding proteins, restriction endonucleases, and nucleases. Therefore, in vitro, the inhibitory effects of hemin are not limited to the interaction of the IRE-BP and the IRE, but are nonspecific and affect a wide variety of nucleic acid-protein interactions. Any hypothesis on the effects on protein-nucleic acid interactions employing greater than 1 microM concentrations of hemin should be interpreted with caution.

Specific high-affinity binding of host cell proteins to the 3' region of rubella virus RNA.

Replication of rubella virus is initiated at the 3' end of the genomic RNA. An inverted repeat sequence of 12 nucleotides that is capable of forming a stem-loop structure is located at the 3' end of the RNA, 59 nucleotides upstream from the poly (A) tail. We screened the 158-bp region of the 3' end of the virus, including the stem-loop structure, for its ability to bind to host-cell proteins. Specific high-affinity binding of three cytosolic proteins with relative molecular masses (Mr) of 61, 63 and 68 kD to the stem-loop structure was observed by UV-induced covalent crosslinking. Altering the stem structure by removal of specific bases abolished the binding interactions. The binding of the host proteins is greatly increased after infection and coincides with the appearance of negative strand RNA synthesis. The increase in binding is dependent on new protein synthesis. The amount of the 61-kD protein that binds varies in uninfected cells and is maximal in cells that are in the stationary phase of growth. All binding activity could be abrogated by alkaline phosphatase treatment of cell lysates. A possible role of these host proteins in the replication of rubella virus is discussed.

Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements.

The 5' untranslated region of the ferritin heavy-chain mRNA contains a stem-loop structure called an iron-responsive element (IRE), that is solely responsible for the iron-mediated control of ferritin translation. A 90-kilodalton protein, called the IRE binding protein (IRE-BP), binds to the IRE and acts as a translational repressor. IREs also explain the iron-dependent control of the degradation of the mRNA encoding the transferrin receptor. Scatchard analysis reveals that the IRE-BP exists in two states, each of which is able to specifically interact with the IRE. The higher-affinity state has a Kd of 10 to 30 pM, and the lower affinity state has a Kd of 2 to 5 nM. The reversible oxidation or reduction of a sulfhydryl is critical to this switching, and the reduced form is of the higher affinity while the oxidized form is of lower affinity. The in vivo rate of ferritin synthesis is correlated with the abundance of the high-affinity form of the IRE-BP. In lysates of cells treated with iron chelators, which decrease ferritin biosynthesis, a four- to fivefold increase in the binding activity is seen and this increase is entirely caused by an increase in high-affinity binding sites. In desferrioxamine-treated cells, the high-affinity form makes up about 50% of the total IRE-BP, whereas in hemin-treated cells, the high-affinity form makes up less than 1%. The total amount of IRE-BP in the cytosol of cells is the same regardless of the prior iron treatment of the cell. Furthermore, a mutated IRE is not able to interact with the IRE-BP in a high-affinity form but only at a single lower affinity Kd of 0.7 nM. Its interaction with the IRE-BP is insensitive to the sulfhydryl status of the protein.

The iron-responsive element binding protein: a method for the affinity purification of a regulatory RNA-binding protein.

A method of affinity purification of a regulatory protein that binds specific RNA sequences is described. RNAs containing the regulatory sequences are transcribed in vitro from oligonucleotide templates, biotinylated, and incubated with unfractionated cytosol. Specific RNA-protein complexes are bound in solution to avidin, and the resulting complex is bound to biotin-agarose beads. The cytosolic binding protein is released from the RNA in high salt, and a second round of purification yields an essentially homogeneous protein. Using this method, we have identified the protein in human liver that binds iron-responsive RNA regulatory sequences. Iron-responsive elements (IREs) are RNA stem-loops present in the mRNAs encoding ferritin and the transferrin receptor. IREs form the basis for the translational regulation of ferritin gene expression and the regulation of transferrin receptor mRNA degradation rates. The IRE binding protein purified by this technique migrates as a 90-kDa polypeptide on SDS/PAGE. The interaction of the purified protein with IRE-containing RNAs can be detected by gel-mobility shift assays or by covalent crosslinking induced by UV irradiation.