Crystallization and preliminary X-ray diffraction data for the aconitase form of human iron-regulatory protein 1.

Iron-regulatory proteins (IRPs) 1 and 2 are closely related molecules involved in animal iron metabolism. Both proteins can bind to specific mRNA regions called iron-responsive elements and thereby control the expression of proteins involved in the uptake, storage and utilization of iron. In iron-replete cells, IRP1, but not IRP2, binds a [4Fe-4S] cluster and functions as a cytoplasmic aconitase, with simultaneous loss of its RNA-binding ability. Whereas IRP2 is known to be involved in Fe homeostasis, the role of IRP1 is less clear; it may provide a link between citrate and iron metabolisms and be involved in oxidative stress response. Here, two crystal forms of the aconitase version of recombinant human IRP1 are reported. An X-ray fluorescence measurement performed on a gold-derivative crystal showed the unexpected presence of zinc, in addition to gold and iron. Both native and MAD X-ray data at the Au, Fe and Zn absorption edges have been collected from these crystals.

Peroxisomal targeting of mammalian hydroxyacid oxidase 1 requires the C-terminal tripeptide SKI.

Peroxisomal proteins are post-translationally imported into peroxisomes after recognition by specific receptors. The best-defined peroxisomal targeting signal is a C-terminal tripeptide SKL. Different functional variants of this tripeptide have been defined, but mutants with a SKI sequence were recognized as being inefficiently targeted to peroxisomes. Recently, we have cloned a cDNA for the mouse hydroxyacid oxidase 1 (Hao1), a protein that seems to be localized in peroxisomes. Interestingly, the mouse Hao1 sequence comprises a C-terminal SKI tripeptide. We have analyzed the subcellular localization of Hao1 and tested whether its SKI sequence acts as a targeting signal. Ltk(-) and Cos-7 cells were transfected with vectors expressing a fusion protein of green fluorescence protein and Hao1, as well as mutants thereof. Targeting to peroxisomes of the fusion protein with the wild-type SKI sequence was highly selective and as complete as with the peroxisome-specific SKL sequence. By contrast, targeting was lost in a mutant with the sequence CKM. The data show that mammalian Hao1 is a peroxisomal protein and that the C-terminal sequence SKI acts as the targeting signal.

Glycoprotein-associated amino acid exchangers: broadening the range of transport specificity.

Members of the newly discovered glycoprotein-associated amino acid transporter family (gpaAT-family) share a similar primary structure with >40% identity, a predicted 12-transmembrane segment topology and the requirement for association with a glycoprotein (heavy chain) for functional surface expression. Five of the six identified gpaATs (light chains) associate with the surface antigen 4F2 heavy chain (4F2hc = CD98), a ubiquitous plasma membrane protein induced in cell proliferation, and which is also highly expressed at the basolateral surface of amino acid transporting epithelia. The differing tissue localizations of the 4F2hc-associated gpaATs appear to complement each other. As yet, a single gpaAT (b(0,+)AT) has been shown to associate with rBAT, a 4F2hc-related glycoprotein mainly localized in intestine and kidney luminal brush-border membranes. The transport characteristics of gpaATs have been shown, by expression in heterologous systems, to correspond to the previously described transport systems L, y+L, xc- and b(o,+). These (obligatory) exchangers of broad substrate specificity (with the exception of xCT) are expected to equilibrate the concentrations of their substrate amino acids across membranes. Thus, the driving force provided by a transmembrane gradient of one substrate amino acid, such as that generated by a parallel functioning unidirectional transporter, can be used by a gpaAT to fuel the secondary active vectorial transport of other exchangeable species. Vectorial transport of specific amino acids is also promoted by the intrinsic asymmetry of these exchangers. The fact that genetic defects of the epithelial gpaATs b(0,+)AT and y+LAT1 cause non-type I cystinuria and lysinuric protein intolerance, respectively, demonstrates that these gpaATs perform vectorial secondary and/or tertiary active transport of specific amino acids in vivo.

The Pit-1 homeodomain and beta-domain interact with Ets-1 and modulate synergistic activation of the rat prolactin promoter.

Pit-1/GHF-1 is a pituitary-specific, POU homeodomain transcription factor required for development of somatotroph, lactotroph, and thyrotroph cell lineages and regulation of the temporal and spatial expression of the growth hormone, prolactin (PRL), and thyrotropin-beta genes. Synergistic interaction of Pit-1 with a member of the Ets family of transcription factors, Ets-1, has been shown to be an important mechanism regulating basal and Ras-induced lactotroph-specific rat (r) PRL promoter activity. Pit-1beta/GHF-2, an alternatively spliced isoform containing a 26-amino acid insert (beta-domain) within its transcription-activation domain, physically interacts with Ets-1 but fails to synergize. By using a series of Pit-1 internal-deletion constructs in a transient transfection protocol to reconstitute rPRL promoter activity in HeLa cells, we have determined that the functional and physical interaction of Pit-1 and Ets-1 is mediated via the POU homeodomain, which is common to both Pit-1 and Pit-1beta. Although the Pit-1 homeodomain is both necessary and sufficient for direct binding to Ets-1 in a DNA-independent manner, an additional interaction surface was mapped to the beta-domain, specific to the Pit-1beta isoform. Thus, the unique transcriptional properties of Pit-1 and Pit-1beta on the rPRL promoter may be due to the formation of functionally distinct complexes of these two Pit-1 isoforms with Ets-1.

Luminal heterodimeric amino acid transporter defective in cystinuria.

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b(0,+) type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b(0,+)AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b(0,+) amino acid transporter complex. We demonstrate its b(0,+)-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b(0,+)AT protein is the product of the gene defective in non-type I cystinuria.

Identification of RNA-binding surfaces in iron regulatory protein-1.

Post-transcriptional regulation of mRNA translation and stability in iron metabolism involves the interaction between the trans-acting cytoplasmic iron regulatory proteins (IRP-1 and IRP-2) and cis-acting iron-responsive elements (IREs) in mRNA 5'- or 3'-untranslated regions. IRP-1 can adopt two conformations: one with a [4Fe-4S]-cluster, unable to bind IREs, which functions as a cytoplasmic aconitase; one lacking this cluster, which accumulates in iron-deprived cells and binds mRNA firmly. We investigated which surfaces of IRP-1 interact with IREs. Surface areas were predicted on the basis of the crystallized porcine mitochondrial aconitase structure. We selected nine sequences absent or different in mitochondrial and Escherichia coli aconitases, both being devoid of RNA-binding properties. Mutations in two regions of domain 4 of IRP-1 lowered the affinity for a wild-type IRE up to 7-fold in vitro, whereas the aconitase activity, a control for structural integrity, was not affected. Scatchard plot analysis with mutant IREs indicated that domain 4 is involved in the binding specificity. This conclusion was confirmed with hybrid proteins in which IRP-1 surface loops were grafted into IRP-2. The results indicate that arginines 728 and 732 contact the IRE bulge, whereas region 685-689 is necessary for recognition of the IRE loop.

Experimental hemochromatosis due to MHC class I HFE deficiency: immune status and iron metabolism.

The puzzling linkage between genetic hemochromatosis and histocompatibility loci became even more so when the gene involved, HFE, was identified. Indeed, within the well defined, mainly peptide-binding, MHC class I family of molecules, HFE seems to perform an unusual yet essential function. As yet, our understanding of HFE function in iron homeostasis is only partial; an even more open question is its possible role in the immune system. To advance on both of these avenues, we report the deletion of HFE alpha1 and alpha2 putative ligand binding domains in vivo. HFE-deficient animals were analyzed for a comprehensive set of metabolic and immune parameters. Faithfully mimicking human hemochromatosis, mice homozygous for this deletion develop iron overload, characterized by a higher plasma iron content and a raised transferrin saturation as well as an elevated hepatic iron load. The primary defect could, indeed, be traced to an augmented duodenal iron absorption. In parallel, measurement of the gut mucosal iron content as well as iron regulatory proteins allows a more informed evaluation of various hypotheses regarding the precise role of HFE in iron homeostasis. Finally, an extensive phenotyping of primary and secondary lymphoid organs including the gut provides no compelling evidence for an obvious immune-linked function for HFE.

LAT2, a new basolateral 4F2hc/CD98-associated amino acid transporter of kidney and intestine.

Glycoprotein-associated amino acid transporters (gpaAT) are permease-related proteins that require heterodimerization to express their function. So far, four vertebrate gpaATs have been shown to associate with 4F2hc/CD98 for functional expression, whereas one gpaAT specifically associates with rBAT. In this study, we characterized a novel gpaAT, LAT2, for which mouse and human cDNAs were identified by expressed sequence tag data base searches. The encoded ortholog proteins are 531 and 535 amino acids long and 92% identical. They share 52 and 48% residues with the gpaATs LAT1 and y(+)LAT1, respectively. When mouse LAT2 and human 4F2hc cRNAs were co-injected into Xenopus oocytes, disulfide-linked heterodimers were formed, and an L-type amino acid uptake was induced, which differed slightly from that produced by LAT1-4F2hc: the apparent affinity for L-phenylalanine was higher, and L-alanine was transported at physiological concentrations. In the presence of an external amino acid substrate, LAT2-4F2hc also mediated amino acid efflux. LAT2 mRNA is expressed mainly in kidney and intestine, whereas LAT1 mRNA is expressed widely. Immunofluorescence experiments showed colocalization of 4F2hc and LAT2 at the basolateral membrane of kidney proximal tubules and small intestine epithelia. In conclusion, LAT2 forms with LAT1 a subfamily of L-type gpaATs. We propose that LAT1 is involved in cellular amino acid uptake, whereas LAT2 plays a role in epithelial amino acid (re)absorption.

Iron regulatory protein as an endogenous sensor of iron in rat intestinal mucosa. Possible implications for the regulation of iron absorption.

Duodenal enterocytes adjust intestinal iron absorption to the body's state of iron repletion. Here we tested how iron supply from the blood modulates the RNA-binding activity of iron regulatory proteins (IRP-1 and IRP-2) in immature duodenal rat enterocytes, and whether the modulation is compatible with the hypothesis that IRPs, in turn, may regulate the expression of iron transport proteins in maturating enterocytes during migration to the villus tips. Tissue uptake of parenterally applied 59Fe along the duodenal crypt-villus axis was compared to local IRP-1 and IRP-2 activity and to duodenal 59Fe transport capacity 12 h, 48 h, and 72 h after intravenous iron administration to iron-deficient rats. IRP-1 and IRP-2 activity was significantly increased in iron-deficiency. 59Fe administrated from the blood side was almost exclusively taken up by crypt enterocytes. Accordingly, the activity of IRP-1 decreased at this site 12 h after parenteral iron administration, but remained high at the villus tips. After 48 h the bulk of 59Fe containing enterocytes had migrated to the villus tips. Correspondingly, IRP-1 activity was decreased at duodenal villus tips after 48 h. IRP-2 activity also tended to decrease, though the change was statistically not significant. IRP-2 activity remained significantly higher at duodenal villus tips than in crypts, even after 72 h. Intestinal iron absorption capacity decreased with the same delay as IRP-1 activity after intravenous iron administration. In the ileum 59Fe uptake from the blood and IRP activity showed no significant difference between crypt and villus region. Luminal administration of iron decreased duodenal IRP-1 and IRP-2 activity at tips and crypts within 2 h. Thus, recently absorbed iron becomes available to cytosolic IRP during its passage through the enterocyte. Our results are compatible with a role of IRPs in gearing the expression of intestinal iron transporters in the duodenal brushborder to the body's state of iron repletion.

Molecular cloning of mouse glycolate oxidase. High evolutionary conservation and presence of an iron-responsive element-like sequence in the mRNA.

Iron regulatory proteins (IRPs) control the synthesis of several proteins in iron metabolism by binding to iron-responsive elements (IREs), a hairpin structure in the untranslated region (UTR) of corresponding mRNAs. Binding of IRPs to IREs in the 5' UTR inhibits translation of ferritin heavy and light chain, erythroid aminolevulinic acid synthase, mitochondrial aconitase, and Drosophila succinate dehydrogenase b, whereas IRP binding to IREs in the 3' UTR of transferrin receptor mRNA prolongs mRNA half-life. To identify new targets of IRPs, we devised a method to enrich IRE-containing mRNAs by using recombinant IRP-1 as an affinity matrix. A cDNA library established from enriched mRNA was screened by an RNA-protein band shift assay. This revealed a novel IRE-like sequence in the 3' UTR of a liver-specific mouse mRNA. The newly identified cDNA codes for a protein with high homology to plant glycolate oxidase (GOX). Recombinant protein expressed in bacteria displayed enzymatic GOX activity. Therefore, this cDNA represents the first vertebrate GOX homologue. The IRE-like sequence in mouse GOX exhibited strong binding to IRPs at room temperature. However, it differs from functional IREs by a mismatch in the middle of its upper stem and did not confer iron-dependent regulation in cells.

Iron and gene expression: molecular mechanisms regulating cellular iron homeostasis.

In recent years, specific post-transcriptional mechanisms in the cytoplasm of vertebrate cells have been elucidated that directly affect the stability and translation of mRNAs coding for central proteins in iron metabolism. This review shall focus primarily on these mechanisms. Other levels of control, either affecting gene transcription and/ or related to the function of iron-capturing substances and transmembrane transport, are also likely to exist and to influence the iron balance and utilization. They are, however, much less clear.

Translational regulation of mRNAs with distinct IRE sequences by iron regulatory proteins 1 and 2.

Iron regulatory proteins 1 and 2 (IRP-1, IRP-2) interact with iron-responsive elements (IREs) present in the 5'- or 3'-untranslated regions (UTR) of several mRNAs coding for proteins in iron metabolism. Whereas binding of IRP-1 and -2 to an IRE in the 5'-UTR inhibits mRNA translation in vitro, it has remained unknown whether either endogenous protein is sufficient to control translation in mammalian cells. We analyzed this question by taking advantage of published mutant IREs that are exclusively recognized by either IRP-1 or IRP-2 in vitro. These IREs were inserted into the 5'-UTR of a human growth hormone reporter mRNA, and translational regulation was measured in stably transfected mouse L cells. Cells cultured in iron-rich or -depleted medium were labeled with [35S]methionine, and secreted growth hormone was immunoprecipitated. IREs with loop sequence specific for IRP-1 (UAGUAC), IRP-2 (CCGAGC), or both proteins (GAGUCG and the wild-type CAGUGC sequence) all mediated translational regulation, in contrast to a control sequence (GCUCCG) that binds neither IRP-1 nor IRP-2. Control experiments excluded IRP-1 binding to the IRP-2-specific sequence in vivo. The present data demonstrate that IRP-1 and IRP-2 can independently function as translational repressors in living cells.

Transferrin receptor expression is controlled differently by transferrin-bound and non-transferrin iron in human cells.

We studied the effects of iron supplied as transferrin-bound iron and iron supplied as non-transferrin iron on transferrin receptor expression by human cell lines. Defined conditions of iron supply were represented by (i) 5 microg/ml of iron-saturated transferrin (transferrin medium) and by (ii) 500 microM ferric citrate (ferric citrate medium). Transferrin receptor expression of studied cell lines (HeLa, K562, Jiyoye) grown as long-term cultures in transferrin medium was somewhat higher (up to 137% of the mean fluorescence intensity) than in ferric citrate medium. The receptor expression corresponded with cellular iron regulatory protein (IRP) activity (ratio activated/total), which was also higher in transferrin medium (0.69-0.84) than in ferric citrate medium (0.33-0.60). However, unexpectedly much higher (about 65-135-fold) cellular iron levels were found in ferric citrate medium (13.9-14.9 nmol/10(6) cells) than in transferrin medium (0.11-0.21 nmol/10(6) cells). In contrast to the iron levels, cellular ferritin levels of the cells in ferric citrate medium (38.3-130 ng/10(6) cells) were only about 2-7-fold higher than in transferrin medium (6.8-61.5 ng/10(6) cells). We suggest that iron supplied as non-transferrin iron (ferric citrate) is apparently less available for the control of transferrin receptor expression via IRP activity than iron supplied as transferrin.

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

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 micrograms/ml) medium into low-iron (5 microM 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 10(6) cells) were found for LIT cells HeLa and HID cells K562. Furthermore, the intracellular iron level (4.64 nmol/10(6) cells) of HeLa cells grown in low-iron medium was much higher than iron levels (0.15 or 0.20 nmol/10(6) 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 microM, 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.

Interaction between iron-regulatory proteins and their RNA target sequences, iron-responsive elements.

In this chapter, we have focused on the biochemistry of IRP-1 and the features which distinguish it from the related RNA-binding protein, IRP-2. IRP-1 is the cytoplasmic isoform of the enzyme aconitase, and, depending on iron status, may switch between enzymatic and RNA-binding activities. IRP-1 and IRP-2 are trans-acting regulators of mRNAs involved in iron uptake, storage and utilisation. The finding of an IRE in the citric acid cycle enzymes, mitochondrial aconitase and succinate dehydrogenase, suggests that the IRPs may also influence cellular energy production. These two proteins appear to bind RNAs with different but overlapping specificity, suggesting that they may regulate the stability or translation of as yet undefined mRNA targets, possibly extending their regulatory function beyond that of iron homeostasis. The interaction between the IRPs and the IRE represents one of the best characterised model systems for posttranscriptional gene control, and given that each IRP can also recognise its own unique set of RNAs, the search for new in vivo mRNA targets is expected to provide yet more surprises and insights into the fate of cytoplasmic mRNAs.

Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress.

As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. At the cellular level, the basic molecular framework for the regulation of iron uptake, storage, and utilization has been defined. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites, iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches.

Effects of overexpression of the transferrin receptor on the rates of transferrin recycling and uptake of non-transferrin-bound iron.

The possibilities that the recycling of the transferrin receptor is a rate-limiting step in the efflux of endocytosed transferrin, and that the receptor functions as a trans-membrane Fe transporter were investigated in untransfected Ltk- cells and in cells transfected with different levels of DNA for wild-type, mutant and chimeric human transferrin receptors. The uptake of transferrin-bound Fe and non-transferrin-bound Fe(II), and the surface binding, endocytosis and recycling of transferrin were measured. In cells that expressed increasing numbers of surface transferrin receptors, the rate of Fe uptake increased at a slower rate than the number of receptors. By measurement of the rates of endocytosis and recycling of transferrin it was shown that this effect was not due to a deficiency of endocytosis, but to a slower rate of recycling as the receptor numbers increased. Hence, a restricted recycling rate of the transferrin receptor appeared to be responsible for the slower rate of Fe uptake by cells with high receptor numbers, presumably because one or more cytosolic components required for recycling were in limited supply. The rate of uptake of non-transferrin-bound Fe(II) was not influenced by the number of transferrin receptors present on the surface of the cells even though this varied more than 20-fold between the different cell lines. Hence, this investigation does not support the hypothesis that the receptors play a direct role in the transport of Fe(II) across cell membranes, as has been proposed previously [Singer, S. J. (1989) Biol. Cell 65, 1-5].

Induction of ferritin synthesis in cells infected with Mengo virus.

We have recently identified ferritin as a cellular protein particle whose synthesis is stimulated in mouse or human cells infected by the picornavirus Mengo. Immunoprecipitation of the particle from infected murine L929 cells showed a 4- and 6-fold increase in the intracellular concentrations of H and L apoferritin subunits, respectively. This differential expression altered the H/L subunit ratio from 3.0 in uninfected cells to 2.2 in Mengo virus-infected cells. The induction is not due to an increase in transcription of the apoferritin L and H genes, nor is it due to an increase in stability of the apoferritin mRNAs. At the level of translation, the iron regulatory protein (IRP) remained intact, with similar amounts being detected in uninfected and infected cells. The Mengo virus RNA genome does not compete with the iron regulatory element (IRE) for the binding of IRP, and sequence analysis confirmed that there are no IREs in the virus RNA. The IRE binding activity of IRP in infected cells decreased approximately 30% compared with uninfected cells. The decrease in binding activity could be overcome by the addition of Desferal (deferoxamine mesylate; CIBA) an intracellular iron chelator, which suggests that virus infection causes an increase in intracellular free iron. Electron paramagnetic resonance (EPR) studies have confirmed the increase in free iron in Mengo virus infected cells. The permeability of cells for iron does not change in virus infected cells, suggesting that the induction of ferritin by Mengo virus is due to a change in the form of intracellular iron from a bound to a free state.

Iron regulatory proteins 1 and 2 bind distinct sets of RNA target sequences.

Iron regulatory proteins (IRPs) 1 and 2 bind with equally high affinity to iron-responsive element (IRE) RNA stem-loops located in mRNA untranslated regions and, thereby, post-transcriptionally regulate several genes of iron metabolism. In this study we define the RNA-binding specificities of mouse IRP-1 and IRP-2. By screening loop mutations of the ferritin H-chain IRE, we show that both IRPs bind well to a large number of IRE-like sequences. More significantly, each IRP was found to recognize a unique subset of IRE-like targets. These IRP-specific groups of IREs are distinct from one another and are characterized by changes in certain paired (IRP-1) or unpaired (IRP-2) loop nucleotides. We further demonstrate the application of such sequences as unique probes to detect and distinguish IRP-1 from IRP-2 in human cells, and observe that the IRPs are regulated similarly by iron and reducing agents in human and rodent cells. Importantly, the ability of each IRP to recognize an exclusive subset of IREs was conserved between species. These findings suggest that IRP-1 and IRP-2 may each regulate unique mRNA targets in vivo, possibly extending their function beyond the regulation of intracellular iron homeostasis.