No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Post-transcriptional regulation of genes of Fe metabolism in mammalian cells
Abstract
Iron metabolism is tightly regulated in mammalian cells. Here we describe several types of post-transcriptional mechanisms
that have been identified in regulation of genes of iron metabolism. Iron-dependent regulation of stability of the transcript
is the key to regulation of the expression of the transferrin receptor. Selective repression of translation in iron-depleted
cells is the key to regulation of the expression of ferritin and several other genes that require iron for function. Specific
regulatory proteins that directly sense iron levels in cells are needed to coordinate iron metabolism. These proteins, known
as iron regulatory proteins (IRPs), bind to specific RNA stem-loops in transcripts with high affinity when cells are depleted
of iron. The two IRPS, IRP1 and IRP2, are post-translationally modified by iron, but the nature of the post-translational
regulatory process differs. IRP1 assembles an iron-sulfur cluster which determines its function, whereas IRP2 is rapidly degraded
in the presence of iron. Direct binding of iron is likely to be involved in sensing of iron levels by IRP1 and IRP2. Although
each IRP binds to the RNA stem-loop motifs known as iron-responsive elements (IREs) with high affinity, it is possible that
each protein binds an additional, unique set of target-binding motifs. The list of potential genes that are regulated by IRPs
continues to grow, and much remains to be learned about the regulation of mammalian iron metabolism.
... However, the relative insolubility of uncomplexed iron at physiological pH and the propensity of iron to initiate formation of reactive oxygen species raises potential problems regarding its safe and efficient use. Mammals possess a network of proteins that promote the transport, use, and storage of iron (1)(2)(3)(4). Diferric transferrin transports iron between tissues and binds to cell surface transferrin receptors (TfR). The diferric transferrin-TfR complex is internalized, resulting in delivery of iron to the cytoplasm and recycling of apo-Tf and TfR to the cell surface. ...
... IRP1 and IRP2 respond to numerous effectors including iron, phorbol ester, NO, and oxidative stress, suggesting that the presence of multiple IRE-binding proteins provides the means to broaden the circumstances under which iron metabolism may be regulated (referenced in refs. [1][2][3][4]. IRP1 is the most abundant IRE binding protein in nearly all mammalian tissues examined to date (5-7). The relative abundance of the two proteins varies between different tissues, and, when examined by immunoblotting, both IRPs have appeared to be ubiquitously expressed (5,6). ...
... These two regulatory RNA binding proteins were identified based on their specific interaction with IREs as well as their capacity to modulate translation of IRE-containing mRNAs in vitro (referenced in refs. [1][2][3][4]. Their similar affinity for IREs in natural mRNAs has raised the question of why two IRPs exist. However, two general observations have provided potential explanations for the existence of multiple IRE binding proteins. ...
Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that are central components of a sensory and regulatory network that modulates vertebrate iron homeostasis. IRPs regulate iron metabolism by binding to iron responsive element(s) (IREs) in the 5' or 3' untranslated region of ferritin or transferrin receptor (TfR) mRNAs. Two IRPs, IRP1 and IRP2, have been identified previously. IRP1 exhibits two mutually exclusive functions as an RNA binding protein or as the cytosolic isoform of aconitase. We demonstrate that the Ba/F3 family of murine pro-B lymphocytes represents the first example of a mammalian cell line that fails to express IRP1 protein or mRNA. First, all of the IRE binding activity in Ba/F3-gp55 cells is attributable to IRP2. Second, synthesis of IRP2, but not of IRP1, is detectable in Ba/F3-gp55 cells. Third, the Ba/F3 family of cells express IRP2 mRNA at a level similar to other murine cell lines, but IRP1 mRNA is not detectable. In the Ba/F3 family of cells, alterations in iron status modulated ferritin biosynthesis and TfR mRNA level over as much as a 20- and 14-fold range, respectively. We conclude that IRP1 is not essential for regulation of ferritin or TfR expression by iron and that IRP2 can act as the sole IRE-dependent mediator of cellular iron homeostasis.
... Conversely, the binding of IRPs to 3Ј-IREs stabilizes TfR mRNA. [3][4][5] IRP1 is a bifunctional protein: in iron-replete cells, the presence of a [4Fe-4S] cluster in the IRP1 molecule prevents its binding to IRE and determines the aconitase activity of the protein. When iron is scarce, IRP1 loses its cluster, thus enabling its interaction with IRE. ...
... Similarly, there was no difference in basal IRE binding activity of IRP2 ( Figure 3C). Keeping in mind that trans-regulatory activities of IRP1 or IRP2 are directly influenced by the fluctuations in intracellular iron concentration [3][4][5], it is noteworthy that, despite a marked difference in the LIP levels, the RNA binding activities of IRP1 and IRP2 were similar in the 2 cell lines. To clarify this phenomenon, we attempted to establish whether there is a quantitative relationship in LY cells between LIP and IRP levels. ...
... These results led us to conclude that the physiologically closely related LY cell sublines constitute-in mammalian cells-an exception to the classically accepted converse correlation between the cellular ''free'' iron content and the IRE binding activity of IRPs. 3,5 With regard to IRP1, the similar RNA binding activity observed in the 2 cell lines, despite a marked difference in their LIP content, could be explained by our finding that IRP1 levels in iron-deficient LY-S cells are half those in iron-rich LY-R maternal cells. The proportion of IRP1 molecules active as IRE binding protein is therefore higher in low LIP-containing LY-S mutant cells. ...
The redox properties of iron make this metal a key participant in oxygen-mediated toxicity. Accordingly, L5178Y (LY) mouse lymphoma cell lines, which display a unique inverse cross-sensitivity to ionizing radiation (IR) and hydrogen peroxide (H(2)O(2)), are a suitable model for the study of possible differences in the constitutive control of intracellular iron availability. We report here that the level of iron in the cytosolic labile iron pool (LIP), ie, potentially active in the Fenton reaction, is more than 3-fold higher in IR-resistant, H(2)O(2)-sensitive (LY-R) cells than in IR-sensitive, H(2)O(2)-resistant (LY-S) cells. This difference is associated with markedly greater content of ferritin H-subunits (H-Ft) in LY-S than in LY-R cells. Our results show that different expression of H-Ft in LY cells is a consequence of an up-regulation of H-Ft mRNA in the LY-S mutant cell line. In contrast, posttranscriptional control of iron metabolism mediated by iron-responsive element-iron regulatory proteins (IRPs) interaction is similar in the 2 cell lines, although IRP1 protein levels in iron-rich LY-R cells are twice those in iron-deficient LY-S cells. In showing that LY cell lines exhibit 2 different patterns of intracellular iron regulation, our results highlight both the role of high LIP in the establishment of pro-oxidant status in mammalian cells and the antioxidant role of ferritin. (Blood. 2000;95:2960-2966)
... In crystals of mt-aconitase the Fe-S cluster is in a solventfilled cleft (25). By analogy, assuming the IRP-specific insertions are only in the surface loops that do not affect folding (4,5,26), the IRE binding site has been suggested to be close to or the same as the binding cleft of the Fe-S cluster. Some residues predicted to reside in the putative cleft of iso-IRP1/c-aconitase are required for both aconitase function and iron regulation of mRNA function/RNA binding, based on site-directed mutagenesis and cross-linking studies (4,5,26). ...
... By analogy, assuming the IRP-specific insertions are only in the surface loops that do not affect folding (4,5,26), the IRE binding site has been suggested to be close to or the same as the binding cleft of the Fe-S cluster. Some residues predicted to reside in the putative cleft of iso-IRP1/c-aconitase are required for both aconitase function and iron regulation of mRNA function/RNA binding, based on site-directed mutagenesis and cross-linking studies (4,5,26). It has been hypothesized that the presence or absence of the Fe-S cluster modulates the extent to which the cleft is open and able to bind the IRE complex, and evidence supporting this model has been obtained (26,42,43). ...
... Iron regulates IRP1 by building an Fe-S cluster on the apoprotein, which blocks RNA binding activity. The Fe-S form of IRP1 is c-aconitase, which is the cytosolic isoform of the mitochondrial, [4Fe-4S] ironsulfur enzyme, aconitase (mt-aconitase) (4,5). The Fe-S cluster is a key determinant in selecting one of the two possible protein functions. ...
... This exquisite sensitivity of IRP to iron resources makes them prominent participants of metazoan iron homeostasis, but several other conditions (such as oxidative/nitrosative stress, hypoxia, hormone or cytokine production, or toxic metals) affect IRP activity. Most molecular and cellular properties of the IRP-IRE system have been recently and abundantly reviewed (1)(2)(3)(4). ...
... To improve the relatively crude model previously proposed to describe the structural changes affecting IRP1 when switching between its two activities (1,12,22,23), pure human recombinant IRP1 has been studied in the present work with methods affording valuable structural information in solution. Smallangle neutron scattering experiments have been combined with circular dichroism measurements to show that metal-depleted IRP1 adjusts to its RNA substrate or its [4Fe-4S] active center by locally folding around these elements. ...
... Tel.: 33-438785623; Fax: 33-438785872; E-mail: jean-marc.moulis@cea.fr. 1 The abbreviations used are: IRP, iron regulatory protein; IRE, ironresponsive element. ...
Metazoan iron regulatory protein 1 is a dual activity protein, being either an aconitase or a regulatory factor binding to
messenger RNA involved in iron homeostasis. Sequence comparisons and site-directed mutagenesis experiments have supported
a structural relationship between mitochondrial aconitase and iron regulatory protein 1. The structural properties of human
recombinant iron regulatory protein 1 have been probed in the present work. Although iron-free iron regulatory protein 1 displays
a significantly larger radius of gyration measured by small-angle neutron scattering than calculated for mitochondrial aconitase,
binding of either the [4Fe-4S] cluster needed for aconitase activity or of a RNA substrate turns iron regulatory protein 1
into a more compact molecule. These conformational changes are associated with the gain of secondary structural elements as
indicated by circular dichroism studies. They likely involve α-helices covering the substrate binding cleft of cytosolic aconitase,
and they suggest an induced fit mechanism of iron-responsive element recognition. These studies refine previously proposed
models of the “iron-sulfur switch” driving the biological function of human iron regulatory protein 1, and they provide a
structural framework to probe the relevance of the numerous cellular molecules proposed to affect its function.
... Iron metabolism is regulated at both the systemic and cellular levels [31]. In all vertebrates, the major protein involved in iron transport is TfR1 [32]. Differic-transferrin is taken up via the transferrin receptor. ...
... The homologous protein of TfR1, transferrin receptor 2 (TfR2), is ubiquitously expressed in hepatocytes [33,34]. At the cellular level, the regulation of the expression of proteins involved in iron metabolism and homeostasis, such as ferritin or the transferrin receptors, is coordinated through the interaction of iron sensing proteins, known as iron regulatory proteins (IRPs) or IRE-binding proteins, where IRE stands for iron-responsive elements [32]. The IRE/IRP regulatory system was first described in the late 1980s [31]. ...
Abstract:Iron is a critical metal for several vital biological processes. Most of the body’s iron isbound to hemoglobin in erythrocytes. Iron from senescent red blood cells is recycled by macrophagesin the spleen, liver and bone marrow. Dietary iron is taken up by the divalent metal transporter1 (DMT1) in enterocytes and transported to portal blood via ferroportin (FPN), where it is boundto transferrin and taken up by hepatocytes, macrophages and bone marrow cells via transferrinreceptor 1 (TfR1). While most of the physiologically active iron is bound hemoglobin, the majorstorage of most iron occurs in the liver in a ferritin-bound fashion. In response to an increased ironload, hepatocytes secrete the peptide hormone hepcidin, which binds to and induces internalizationand degradation of the iron transporter FPN, thus controlling the amount of iron released from thecells into the blood. This review summarizes the key mechanisms and players involved in cellularand systemic iron regulation.
(PDF) Molecular Sciences On Iron Metabolism and Its Regulation. Available from: https://www.researchgate.net/publication/351116651_Molecular_Sciences_On_Iron_Metabolism_and_Its_Regulation [accessed Apr 27 2021].
... 3Ј-noncoding regions of animal mRNAs encoding proteins of iron and oxidative metabolism, regulates synthesis of the encoded proteins posttranscriptionally. Iron regulatory proteins (IRPs) bind to the IREs to inhibit ribosome binding or protect mRNA from ribonuclease cleavage (1)(2)(3)(4)(5). The predicted secondary structures of the IRE family are hairpins with a six-nucleotide terminal loop (CAGUGN*, N* ϭ A, C, or U), interrupted by an internal loop/bulge (UGC/C) (ferritin-IRE) or a C-bulge (TfR, eALAS, and m-aconitase IREs), that is generally supported by enzymatic cleavage and chemical probing (6 -8); NMR spectroscopy shows a G-C base pair in the hairpin loop and in the internal loop/bulge (9 -12). ...
... Previous studies that compared IRP1 and IRP2 binding had shown that differential IRP binding occurred only with mutations in the hairpin loop (25-28), but not in natural IREs (20,(38)(39)(40). The hairpin loop is the most conserved part of the IREs; evolutionary divergence occurs in the stem and internal loop regions (1)(2)(3)(4)(5). Recent studies of IREs by NMR and other approaches, which showed significant structural differences in the internal loop/bulge and C-bulge IREs (11,12,41), stimulated reexamination of whether IRP1 and IRP2 differentially bind to the internal loop/bulge and C-bulge IREs. ...
A family of noncoding mRNA sequences, iron-responsive elements (IREs), coordinately regulate several mRNAs through binding
a family of mRNA-specific proteins, iron regulatory proteins (IRPs). IREs are hairpins with a constant terminal loop and base-paired
stems interrupted by an internal loop/bulge (in ferritin mRNA) or a C-bulge (in m-aconitase, erythroid aminolevulinate synthase,
and transferrin receptor mRNAs). IRP2 binding requires the conserved C-G base pair in the terminal loop, whereas IRP1 binding
occurs with the C-G or engineered U-A. Here we show the contribution of the IRE internal loop/bulge to IRP2 binding by comparing
natural and engineered IRE variants. Conversion of the internal loop/bulge in the ferritin-IRE to a C-bulge, by deletion of
U, decreased IRP2 binding by >95%, whereas IRP1 binding changed only 13%. Moreover, IRP2 binding to natural IREs with the
C-bulge was similar to the ΔU6 ferritin-IRE: >90% lower than the ferritin-IRE. The results predict mRNA-specific variation in IRE-dependent regulation in vivo and may relate to previously observed differences in iron-induced ferritin and m-aconitase synthesis in liver and cultured
cells. Variations in IRE structure and cellular IRP1/IRP2 ratios can provide a range of finely tuned, mRNA-specific responses
to the same (iron) signal.
... All cells use ferritin early in maturation with some specialized cells maintaining organismal iron reservoirs, such as hepatocytes, red cells of vertebrate embryos, and seed endosperm (1). The dual levels of gene expression (DNA and mRNA) emphasize the importance of ferritin in bacteria and animals; ferritin expression is coordinated with several other proteins of iron and oxygen homeostasis (5)(6)(7)(8)(9). Many studies have focused on Fe(II) entry and mineral formation in ferritin (10). ...
... Finally, cells with excess iron, to the point of toxicity, have degraded ferritin in lysosomes, suggesting that, at least in some cases, the ferritin protein itself had no direct role in iron release but was degraded to release the iron mineral (e.g., 15). Given the extremely complex regulatory system for ferritin production (5)(6)(7)(8)33), protein degradation seems likely to be only one of several mechanisms for releasing iron from the ferritin mineral. In the past, conserved amino acids in ferritin were attributed to requirements for protein folding/assembly or mineral formation. ...
Ferritin concentrates, stores, and detoxifies iron in most organisms. The iron is a solid, ferric oxide mineral (< or =4500 Fe) inside the protein shell. Eight pores are formed by subunit trimers of the 24 subunit protein. A role for the protein in controlling reduction and dissolution of the iron mineral was suggested in preliminary experiments [Takagi et al. (1998) J. Biol. Chem. 273, 18685-18688] with a proline/leucine substitution near the pore. Localized pore disorder in frog L134P crystals coincided with enhanced iron exit, triggered by reduction. In this report, nine additional substitutions of conserved amino acids near L134 were studied for effects on iron release. Alterations of a conserved hydrophobic pair, a conserved ion pair, and a loop at the ferritin pores all increased iron exit (3-30-fold). Protein assembly was unchanged, except for a slight decrease in volume (measured by gel filtration); ferroxidase activity was still in the millisecond range, but a small decrease indicates slight alteration of the channel from the pore to the oxidation site. The sensitivity of reductive iron exit rates to changes in conserved residues near the ferritin pores, associated with localized unfolding, suggests that the structure around the ferritin pores is a target for regulated protein unfolding and iron release.
... Because iron plays a central role in controlling cell survival and death, intracellular iron homeostasis is finely regulated. Iron regulatory proteins (IRP1 and IRP2) bind to the iron response element (IRE) in the untranslated region (UTR) of the target mRNAs associated with iron metabolism, in turn regulating the production of ferritin or the transferrin receptor involved in iron metabolism and homeostasis (58)(59)(60)(61). ...
Diabetic nephropathy (DN) is a chronic inflammatory disease that affects millions of diabetic patients worldwide. The key to treating of DN is early diagnosis and prevention. Once the patient enters the clinical proteinuria stage, renal damage is difficult to reverse. Therefore, developing early treatment methods is critical. DN pathogenesis results from various factors, among which the immune response and inflammation play major roles. Ferroptosis is a newly discovered type of programmed cell death characterized by iron-dependent lipid peroxidation and excessive ROS production. Recent studies have demonstrated that inflammation activation is closely related to the occurrence and development of ferroptosis. Moreover, hyperglycemia induces iron overload, lipid peroxidation, oxidative stress, inflammation, and renal fibrosis, all of which are related to DN pathogenesis, indicating that ferroptosis plays a key role in the development of DN. Therefore, this review focuses on the regulatory mechanisms of ferroptosis, and the mutual regulatory processes involved in the occurrence and development of DN and inflammation. By discussing and analyzing the relationship between ferroptosis and inflammation in the occurrence and development of DN, we can deepen our understanding of DN pathogenesis and develop new therapeutics targeting ferroptosis or inflammation-related regulatory mechanisms for patients with DN.
... Although conventional views agree that polyphenols only affect non-heme iron absorption, recent experiments have provided evidence that polyphenolic compounds can inhibit the absorption of heme and non-heme iron. It also showed that polyphenols have a dose-dependent inhibitory effect [9][10][11][12][13][14]. ...
DLLME, which is a method that minimizes organic solvent consumption and waste generation, is frequently used for trace analyte determination. In the present work, a simple, selective and sentsitive spectrophotometric method based on the dispersive liquid-liquid microextraction was reported. The procedure is based on the formation of a 1:1 complex between Fe3+ and a water-soluble Cu(II) phthalocyanine and then extraction of this complex into dichloromethane by dispersive effect of acetone. The experimental parameters that effecting the DLLME such as amount of extractive and disperser solvents, pH, salt concentration, Cu(II) phthalocyanine concentration and centrifuging time and rate were optimized. The linear range of the method is 0.4-70.0 ngmL-1 with a good correlation coefficient (R2) of 0.9912. The limits of detection (LOD) and quantification (LOQ) is 0.47 and 1.86 ngmL-1. The relative standart deviation (RSD, %) of the method for 40 ngmL-1 Fe3+ in sample solution (n=11) was 1.4% and the enrichment factor was calculated 240.
... However, the mechanism of the oxidation of these systems is very complex since there exist sharp differences in oxygen tolerance of the different FeS clusters. [26][27][28][29][30][31][32][33][34][35] In this context, an analysis, by means of the DFT, of differences in oxygen sensitivity of Fe 2 S 2 , Fe 3 S 4 and Fe 4 S 4 clusters coordinated by cysteine residues was carried out by Bruska et al. 31 [33][34][35] are accompanied by significant structural distortions. In view of the information presented above, we found it appealing to study how these three molecules interact with the free-standing Fe 2 S 2 , Fe 3 S 4 and Fe 4 S 4 clusters and how they change their structural, magnetic and electronic indicators among which the vertical detachment energies (VDEs) and electron affinities (EAs) have been measured. ...
We report results, based on density functional theory-generalized gradient approximation calculations, that shed light on how NO, CO, and O2 interact with Fe2S2, Fe3S4, and Fe4S4 clusters and how they modify their structural and electronic properties. The interest in these small iron sulfide clusters comes from the fact that they are at the protein cores and that elucidating fundamental aspects of their interaction with those light molecules which are known to modify their functionality may help in understanding complex behaviors in biological systems. CO and NO are found to bind molecularly, leading to moderate relaxations in the clusters, but nevertheless to changes in the spin-polarized electronic structure and related properties. In contrast, dissociative chemisorption of O2 is much more stable than molecular adsorption, giving rise to significant structural distortions, particularly in Fe4S4 that splits into two Fe2S2 subclusters. As a consequence, oxygen tends to strongly reduce the spin polarization in Fe and to weaken the Fe-Fe interaction inducing antiparallel couplings that, in the case of Fe4S4, clearly arise from indirect Fe-Fe exchange coupling mediated by O. The three molecules (particularly CO) enhance the stability of the iron-sulfur clusters. This increase is noticeably more pronounced for Fe2S2 than for the other iron-sulfur clusters of different compositions, a result that correlates with the fact that in recent experiments of CO reaction with Fe
m
S
m
(m = 1-4), the Fe2S2CO product results as a prominent one.
... salt ferric ammonium citrate (FAC) [17]. Iron-induced appearance of elevated Lf binding sites is blocked by cycloheximide but not by actinomycin D [17], suggesting the possibility that expression of these iron-dependent Lf-binding sites is governed by an iron-response element\iron-regulatory protein-dependent translational control mechanism [18]. It is unlikely that RHL-1 is responsible for the iron-dependent increase in Lf binding and endocytosis observed in FAC-treated cells for two reasons. ...
The major subunit [rat hepatic lectin-1 (RHL-1)] of the asialoglycoprotein (ASGP) receptor mediates endocytosis of the iron-binding protein lactoferrin (Lf) by isolated rat hepatocytes, yet iron loading of cultured adult rat hepatocytes increases the binding and endocytosis of Lf while greatly inhibiting the uptake of desialylated ligand. In the present study, we determined whether the iron-induced Lf-binding site is RHL-1 and examined the nature of the iron-induced block in ASGP receptor endocytic function. Isolated rat hepatocytes increased their non-haem iron content from 70 to 470 p.p. b. following incubation with ferric ammonium citrate (<=100 microgram/ml). These conditions blocked internalization of 125I-asialo-orosomucoid (ASOR) by approximately 90% but increased 125I-Lf endocytosis by 40%. ASOR and anti-RHL-1 sera blocked the binding and endocytosis of 125I-Lf on control cells but not on iron-loaded cells, indicating that the iron-induced Lf-binding site on hepatocytes is not RHL-1. Iron-loading of hepatocytes in the presence or absence of excess ASOR did not significantly alter the number of active ASGP receptors on the cell surface. In contrast, iron-loading decreased the number of active intracellular receptors by 40% and blocked the uptake of 125I-ASOR prebound to the cells by approximately 80%. Under these conditions, we found an iron-dependent evolution of 88 and 140 kDa RHL-1-containing, beta-mercaptoethanol-sensitive multimers that constituted up to 34 and 23%, respectively, of total immunodetectable RHL-1. We propose that iron-induced formation of cystinyl-linked RHL-1-containing multimers inhibits ASGP receptor movement between cell surface and interior and disrupts acylation of intracellular receptors.
... Iron regulation of ferritin mRNA translation dominates over transcriptional regulation in animals, which contrasts with plants. Animal ferritin mRNAs contain the well characterized iron-responsive elements (IREs), 1 which recognize iron regulatory proteins (IRPs) (5)(6)(7)(8)(9). A range of coordinated responses among a number of IRE-containing mRNAs is achieved through combinatorial interactions between iso-IREs and iso-IRPs to regulate the synthesis of ferritin, the transferrin receptor, erythroid aminolevulinate and mitochondrial aconitase (5, 16 -20). ...
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.
... Because iron can play an important role in oxidative stress and yet is essential for cellular growth, its uptake is tightly regulated in both yeast and mammalian systems (19,20). In S. cerevisiae, two plasma membrane proteins, Fre1p and Fre2p, reduce Fe 3ϩ to Fe 2ϩ , a function that is critical for iron uptake (20,21). ...
Saccharomyces cerevisiae lacking copper-zinc superoxide dismutase (sod1) shows a series of defects, including reduced rates of aerobic growth in synthetic glucose medium and reduced ability to
grow by respiration in glycerol-rich medium. In this work, we observed that addition of iron improved the respiratory growth
of the sod1 mutant and in glucose medium total intracellular iron content was higher in the sod1mutant than in wild type cells. Transcription of the high affinity iron transporter gene, FET3, was enhanced in thesod1 mutant, suggesting that iron transport systems were up-regulated. An sod1/fet3 double mutant showed increased sensitivity to oxygen and increased transcription of FET4, an alternative, low affinity, iron transporter. We propose that this increased iron demand in the sod1 mutant may be a reflection of the cells' efforts to reconstitute iron-sulfur cluster-containing enzymes that are continuously
inactivated in conditions of excess superoxide.
... A range of responses to cytoplasmic signals, such as iron, is possible among the mRNAs that contain IREs (11,12,39) despite the similarity of the iso-IRE secondary structure. All iso-IREs have the hairpin hexaloop (HL) with the conserved sequence CAGUGX and the helix distortion with a disordered C residue (4,5,7,(40)(41)(42). The range of physiological responses to iron among the mRNA with iso-IREs coincides with differences in binding of IRP2 (13). ...
Iron-responsive elements (IREs), a natural group of mRNA-specific sequences, bind iron regulatory proteins (IRPs) differentially and fold into hairpins [with a hexaloop (HL) CAGUGX] with helical distortions: an internal loop/bulge (IL/B) (UGC/C) or C-bulge. C-bulge iso-IREs bind IRP2 more poorly, as oligomers (n = 28-30), and have a weaker signal response in vivo. Two trans-loop GC base pairs occur in the ferritin IRE (IL/B and HL) but only one in C-bulge iso-IREs (HL); metal ions and protons perturb the IL/B [Gdaniec et al. (1998) Biochemistry 37, 1505-1512]. IRE function (translation) and physical properties (T(m) and accessibility to nucleases) are now compared for IL/B and C-bulge IREs and for HL mutants. Conversion of the IL/B into a C-bulge by a single deletion in the IL/B or by substituting the HL CG base pair with UA both derepressed ferritin synthesis 4-fold in rabbit reticulocyte lysates (IRP1 + IRP2), confirming differences in IRP2 binding observed for the oligomers. Since the engineered C-bulge IRE was more helical near the IL/B [Cu(phen)(2) resistant] and more stable (T(m) increased) and the HL mutant was less helical near the IL/B (ribonuclease T1 sensitive) and less stable (T(m) decreased), both CG trans-loop base pairs contribute to maximum IRP2 binding and translational regulation. The (1)H NMR spectrum of the Mg-IRE complex revealed, in contrast to the localized IL/B effects of Co(III) hexaammine observed previously, perturbation of the IL/B plus HL and interloop helix. The lower stability and greater helix distortion in the ferritin IL/B-IRE compared to the C-bulge iso-IREs create a combinatorial set of RNA/protein interactions that control protein synthesis rates with a range of signal sensitivities.
... Iron±sulfur (Fe±S) clusters are abundant and pervasive cofactors that are essential for a wide variety of processes, including facilitation of electron transfer processes in oxidative phosphorylation, catalysis of enzymatic reactions in aconitase and dehydratases, and maintenance of structural integrity in the DNA repair enzyme endonuclease III (Beinert and Holm, 1997). In addition, Fe±S clusters are critical in enabling cells to sense intracellular iron and/or oxidant levels, as in the case of iron regulatory protein 1 (IRP1), a sensor of intracellular iron levels, and the bacterial proteins SoxR and FNR, sensors of intracellular oxygen species (Rouault and Klausner, 1996;Beinert and Kiley, 1999). Over the last few decades, much has been learned about the structure and function of Fe±S clusters, but, until recently, little was known about the in vivo cluster assembly process. ...
Iron-sulfur (Fe-S) clusters are cofactors found in many proteins that have important redox, catalytic or regulatory functions. In mammalian cells, almost all known Fe-S proteins are found in the mitochondria, but at least one is found in the cytosol. Here we report cloning of the human homologs to IscU and NifU, iron-binding proteins that play a critical role in Fe-S cluster assembly in bacteria. In human cells, alternative splicing of a common pre-mRNA results in synthesis of two proteins that differ at the N-terminus and localize either to the cytosol (IscU1) or to the mitochondria (IscU2). Biochemical analyses demonstrate that IscU proteins specifically associate with IscS, a cysteine desulfurase that is proposed to sequester inorganic sulfur for Fe-S cluster assembly. Protein complexes containing IscU and IscS can be found in the mitochondria as well as in the cytosol, implying that Fe-S cluster assembly takes place in multiple subcellular compartments in mammalian cells. The possible roles of the IscU proteins in mammalian cells and the potential implications of compartmentalization of Fe-S cluster assembly are discussed.
... However, there are many nonredox roles for Fe-S clusters that have been uncovered, suggesting that they are versatile cofactors (22,23). A relatively new role for iron-sulfur clusters that is of particular biological interest involves their participation as sensors of Fe levels or reactive oxygen species (ROS) in regulation of gene expression or enzymatic activity (22,(24)(25)(26)(27). For example, the Fe-S cluster in the transcription factor FNR (for fumarate nitrate reduction) appears to be involved in oxygen sensing (24). ...
The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine-2'-deoxyadenosine (OG*A) mismatches in DNA. MutY prevents DNA mutations caused by the misincorporation of A opposite OG by catalyzing the deglycosylation of the aberrant adenine. MutY is representative of a unique subfamily of DNA repair enzymes that also contain a [4Fe-4S]2+ cluster, which has been implicated in substrate recognition. Previously, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, or alanine. These experiments suggested that histidine coordination to the iron-sulfur cluster may be accommodated in MutY at position 199. Purification and enzymatic analysis of C199H and C199S forms indicated that these forms behave nearly identical to the WT enzyme. Furthermore, introduction of the C199H mutation in a truncated form of MutY (C199HT) allowed for crystallization and structural characterization of the modified [4Fe-4S] cluster coordination. The C199HT structure showed that histidine coordinated to the iron cluster although comparison to the structure of the WT truncated enzyme indicated that the occupancy of iron at the modified position had been reduced to 60%. Electron paramagnetic resonance (EPR) spectroscopy on samples of C199HT indicates that a significant percentage (15-30%) of iron clusters were of the [3Fe-4S]1+ form. Oxidation of the C199HT enzyme with ferricyanide increases the amount of the 3Fe cluster by approximately 2-fold. Detailed kinetic analysis on samples containing a mixture of [3Fe-4S]1+ and [4Fe-4S]2+ forms indicated that the reactivity of the [3Fe-4S]1+ C199HT enzyme does not differ significantly from that of the WT truncated enzyme. The relative resistance of the [4Fe-4S]2+ cluster toward oxidation, as well as the retention of activity of the [3Fe-4S]1+ form, may be an important aspect of the role of MutY in repair of DNA damage resulting from oxidative stress.
... The IRPs, coincidentally, are aconitase homologues and the amounts can vary depending on the cell type, iron status, oxygen status, etc. Similarly, IRE structures, but with specific mRNA features and IRP2 binding properties, occur in a number of other mRNAs involved in iron homeostasis to create a combinatorial array of interactions (93) that coordinately regulate synthesis of the proteins encoded in IRE-mRNAs (21,27,40,77,80,89,93). The mRNA-specific differences in IRE structure and IRP2 binding lead to a range or hierarchy of responses to iron or to anoxia (93). ...
Iron and oxygen are central to terrestrial life. Aqueous iron and oxygen chemistry will produce a ferric ion trillions of times less soluble than cell iron concentrations, along with radical forms of oxygen that are toxic. In the physiological environment, many proteins have evolved to transport iron or modulate the redox chemistry of iron that transforms oxygen in useful biochemical reactions. Only one protein, ferritin, evolved to concentrate iron to levels needed in aerobic metabolism. Reversible formation and dissolution of a solid nanomineral-hydrated, iron oxide is the main function of ferritin, which additionally detoxifies excess iron and possibly dioxygen and reactive oxygen. Ferritin is a large multifunctional, multisubunit protein with eight Fe transport pores, 12 mineral nucleation sites and up to 24 oxidase sites that produce mineral precursors from ferrous iron and oxygen. Regulation of ferritin synthesis in animals uses both DNA and mRNA controls and genes encoding two types of related subunits with: 1) catalytically active (H) or 2) inactive (L) oxidase sites. Ferritin with varying H/L ratios is related to cell-specific iron and oxygen homeostasis. H-ferritin oxidase activity accelerates rates of iron mineralization in ferritins and, in animals, ferritin produces H(2)O(2) as a byproduct. Properties of ferritin mRNA and ferritin protein pore structure are new targets for manipulating iron homeostasis. Recent observations of the high bioavailability of iron in soybean ferritin and efficient utilization of soybean and ferritin iron by iron-deficient animals, and of soybean iron by humans with borderline deficiency, indicate a role for ferritin in managing global iron deficiency in humans.
Magnetic iron oxide nanoparticles (MNPs) have been under intense investigation for at least the last five decades as they show enormous potential for many biomedical applications, such as biomolecule separation, MRI imaging, and hyperthermia. Moreover, a large area of research on these nanostructures is concerned with their use as carriers of drugs, nucleic acids, peptides, and other biologically active compounds, often leading to the development of targeted therapies. The uniqueness of MNPs is due to their nanometric size and unique magnetic properties.
Iron is central to respiration, photosynthesis, nitrogen fixation, and, in most organisms, the reduction of ribose to deoxyribose, rate limiting in DNA synthesis. Dioxygen, which allows high-efficiency bioenergetics, at the same time converts soluble ferrous ions to insoluble ferric ions; iron concentrations in cells are almost a trillion times the solubility of the free ferric ion under physiological conditions. Ferritin is the protein that concentrates ferric iron in all known organisms [reviewed in Theil (1987, 1990) Waldo and Theil (1996) Harrison and Lilley (1990)]. Induction of ferritin synthesis by iron also protects cells from oxidant stress (Balla et al. 1992). Gene regulation of ferritin synthesis is precise and complex and, in animals, is coordinately regulated with transferrin receptor (TfR) synthesis [reviewed in Theil (1990, 1993, 1994, 1997) Hentze and Kuhn (1996) Rouault and Klausner (1996) Klausner et al. (1993) Munro (1990)]. The problem of acquiring and concentrating iron is solved by the use of environmental iron to regulate expression of both iron-storage (ferritin) and iron-uptake (TfR) genes; changes in expression of ferritin and TfR are also regulated by growth factors and hormones and during cell differentiation.
Regulation of mRNA is likely an ancient form of genetic control, with the current rapid expansion of knowledge driven by advances in biotechnology. The regulation of ferritin mRNA by iron in animals, an older example of mRNA regulation, has been extended to other mRNAs of iron and oxidative metabolism. The regulatory element (IRE) is conserved in animal mRNAs encoding the transferrin receptor (TfR), m-aconitase, erythroid aminolevulinate synthase (eALAS) and Nramp2 (an ion transport protein), as well as ferritin. [1–4]. [The sequence identity of an IRE is >95% in the mRNAs of different animals, but is only 36–60% among different IRE containing mRNAs in the same animal]. An IRE can regulate either mRNA ribosome binding (translation) or degradation (stability/turnover). Control of ribosome binding is the mechanism of IRE-dependent regulation for ferritin, eALAS synthesis and m-aconitase, while control of nuclease binding/activity appears to be the mechanism of IRE-dependent regulation for TfR and Nramp2 synthesis. A common secondary structure is shared among the isoIREs which includes a hairpin loop (CAGUGX) and a base paired stem with an interruption or “hinge” in mid-stem. Stem base pair sequence is mRNA-specific as is the structure of the “hinge”. Among iso-IREs, the greatest variation occurs in the lower stem [1].
Transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1) are important proteins for cellular iron uptake, and both are regulated transcriptionally through the binding of hypoxia-inducible factor 1 (HIF-1) to hypoxia-responsive elements (HREs) under hypoxic conditions. These proteins are also regulated post-transcriptionally through the binding of iron regulatory protein 1 (IRP1) to iron-responsive elements (IREs) located in the mRNA untranslated region (UTR) to control cellular iron homeostasis. In iron-deficient cells, IRP1/IRE interactions stabilize TfR1 and DMT1 mRNAs, enhancing iron uptake. However, little is known about the impact of IRP1 on the regulation of cellular iron homeostasis under hypoxia. Thus, to investigate the role of IRP1 in hypoxic condition, overexpression and knockdown assays were performed using HepG2 cells. The overexpression of IRP1 suppressed the hypoxia-induced increase in TfR1 and DMT1 (+IRE) expression and reduced the stability of TfR1 and DMT1 (+IRE) mRNAs under hypoxia, whereas IRP1 knockdown further increased the hypoxia-induced expression of both proteins, preventing the decrease in IRE-dependent luciferase activity induced by hypoxia. Under hypoxic conditions, ferrous iron uptake, the labile iron pool (LIP), and total intracellular iron reduced when IRP1 was overexpressed and further increased when IRP1 was knocked down. IRP1 expression declined and TfR1/DMT1 (+IRE) expression increased with the time of hypoxia prolonged, whereas the binding of IRP1 to the IRE of TfR1/DMT1 mRNA maintained. In summary, IRP1 suppressed TfR1/DMT1 (+IRE) expression, limited the cellular iron content and decreased lactate dehydrogenase (LDH) release induced by hypoxia. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Objective: The objective was to predict the bioavailability of iron in children aged 4- to 6- years from Chinese traditional diets consumed commonly in Shandong rural region on the basis of the contents of nutritional factors known to promote or inhibit food iron absorption. Materials and methods: Iron absorption measurement from Chinese traditional diets was made in 10 subjects by using iron-stable isotope 57Fe, as tracer to label FeSO4. 57Fe and Dy were added to a biscuit, and administered orally 3 times a day for 2 days. The diet and feces samples were collected for 10 consecutive days after the intake of 57Fe. Atomic absorption spectrophotometer (AAS) and thermal ionization mass spectrometers (TIMS) were used for the determination of iron, calcium and stable iron isotope. The daily intake of ascorbic acid, fat, phytic acid and fiber was determined by biochemical analysis. Results: The absorption of iron was 6.06 ± 2.24%, much lower than the 10% reported in the West, which was adopted by Chinese authority to revise Chinese Recommended Dietary Allowance (RDAs) in 1988. The daily intake of iron was 10.29 mg, which was 85.8% of Chinese "Reference Nutrient Intakes (RNIs)" in 1998. The intakes of protein and ascorbic acid were only 56.70 and 19.00% of RNIs, respectively. Conclusion: Under the actual dietary pattern of Shandong rural region, the iron uptake of children is low, due to poor iron absorption and the insufficient daily iron intake.
The discovery of iron regulatory proteins (IRPs) has provided a molecular framework from which to more fully understand the coordinate regulation of vertebrate iron metab- olism. IRPs bind to iron responsive elements (IREs) in specific mRNAs and regulate their utilization. The targets of IRP action now appear to extend beyond proteins that function in the stor- age (ferritin) or cellular uptake (transferrin receptor) of iron to include those involved in other aspects of iron metabolism as well as in the tricarboxylic acid cycle. To date, it appears that IRPs modulate the utilization of six mammalian mRNAs. Current studies are aimed at defining the mechanisms responsible for the hierarchical regulation of these mRNAs by IRPs. In addition, much interest continues to focus on the signaling pathways through which IRP function is regulated. Multiple factors mod- ulate the RNA binding activity of IRP1 and/or IRP2 including iron, nitric oxide, phosphorylation by protein kinase C, oxidative stress and hypoxia/reoxygenation. Because IRPs are key mod- ulators of the uptake and metabolic fate of iron in cells, they are focal points for the modulation of cellular iron homeostasis in response to a variety of agents and circumstances. J. Nutr. 128: 2295-2298, 1998. The ability to obtain and safely use iron for various biochem- ical processes is a central requirement for nearly all forms of life. Iron is a component of proteins required for crucial cellular processes including respiration and cell division. However, the biological use of iron is limited by its low solubility as the uncomplexed metal and its propensity to participate in forma- tion of potentially lethal oxidizing agents. Given the important biological roles of iron it is not surprising that variations in body iron status influence human and animal health. Iron regulatory proteins (IRPs)4 and the proteins whose synthesis they regulate form a homeostatic network that allows mammals to make use of the essential properties of iron while reducing its potentially toxic effects.
IntroductionFerritin gene regulation I (DNA – plants)Ferritin gene regulation I1 (mRNA – animals): iso-IRES and iso-IRPsFemtin protein structure/functionConclusions
The ferritin superfamily of protein nanocages oxidizes ferrous ions to form hydrated ferric oxide nanominerals consisting of thousands of iron and oxygen atoms, in a central, internal cavity; varying amounts of phosphate are incorporated. Maxi-ferritins, found in all eukaryotes and many bacteria and Archea, have diameters of 10–12 nm, constructed from 24 self-assembling polypeptide subunits (four-α-helix bundles) with cavities of ∼8 nm, while mini-ferritins in bacteria assemble from 12 subunits, are ∼8 nm in diameter, and have cavities of ∼5 nm. Ferritins function in concentrating iron for protein co-factor synthesis (heme, FeS, etc.), recovering iron during senescence, and trapping excess iron and oxygen; mini-ferritins, also called Dps proteins, protect bacterial DNA from chemical/physical damage during stress, in some cases binding the DNA in bacterial “chromatin” structures. There are three types of Fe–protein interaction sites in the nanocages, although when mineralized, most of the iron atoms are in the inorganic phase: 1 – catalytic sites, related by simple DNA coding changes to di-iron oxygenase co-factors, that couple two ferrous with oxygen atoms into diferric peroxo and ferric oxy/hydroxo mineral precursors; 2 – nucleation sites on the cavity surface that initiate mineral formation; 3 – gated pores at triple subunit junctions, that control access of reductants and chelators to recover the mineral.
The spin-coupling model of zero-field splitting (ZFS) of tetrameric mixed-valent and monovalent clusters is developed. The matrix elements of the individual ZFS tensor operator of the second rank were calculated for tetramers of the general type in the representation of the total and intermediate spins. The spin-dependent correlations between ZFS parameters DS of the cluster states S and Di of individual ions si were obtained for [4Fe–4S]+ centers of native systems and synthetic model compounds with the high-spin Sgr=3/2 ground state. It was shown that the single-particle spin parameters of the [Fe3(II)Fe(III)] center essentially depend on the total, intermediate and local spins and on the exchange parameters. In the spin schemes with fixed intermediate spins, the correlation between the cluster total spin ZFS parameters, DS, and the individual ZFS parameters, Di, are determined by the total, intermediate and individual spins. Anisotropic cluster ZFS parameters DS strongly depend on isotropic Heisenberg exchange and double exchange inter-ion interactions due to exchange mixing of the states with different intermediate spins. It was shown that the cluster ZFS parameters DS, for the states with S=3/2, change their values and sign under the variation of the Heisenberg exchange and double exchange parameters during the cluster deformation. The single-particle ZFS parameters Di and the exchange effects determine the observed strong positive (DS=1.5–6 cm−1) and negative (DS=−1.7 to −5 cm−1) cluster ZFS splittings of the ground Sgr=3/2 states of the [4Fe–4S]+ clusters. Positive (negative) cluster ZFS parameters DS correspond to negative (positive) individual ZFS parameters D1−3 [Fe(II)]. The theory explains the observation of small negative effective hyperfine constants Ai for the Sgr=3/2 clusters with positive and negative cluster ZFS parameters DS. The correlations between the individual and cluster ZFS parameters were obtained for mixed-valent high-spin [4Fe–4S]+ clusters with Sgr=5/2, 7/2 and monovalent [4Fe–4S]0 cluster with Sgr=4.
The biological relevance of each of the three inorganic species – iron, oxygen, and nitric oxide (NO) – is crucial. Moreover,
their metabolic pathways cross each other and thus create a complex network of connections responsible for the regulation
of many essential biological processes. The iron storage protein ferritin, one of the main regulators of iron homeostasis,
influences oxygen and NO metabolism. Here, examples are given of the biological interactions of the ferritin molecule (ferritin
iron and ferritin shell) with reactive oxygen species (ROS) and NO. The focus is the regulation of ferritin expression by
ROS and NO. From these data, ferritin emerges as an important cytoprotective component of the cellular response to ROS and
NO. Also, by its ability to alter the amount of intracellular "free" iron, ferritin may affect the metabolism of ROS and NO.
It is proposed that this putative activity of ferritin may constitute a missing link in the regulatory loop between iron,
ROS, and NO.
Irreversible disassembly of the 4Fe-4S cluster in Chromatium
vinosum high-potential iron protein (HiPIP) has been investigated in the presence of a low concentration of guanidinium hydrochloride. From the dependence of degradation rate on [H+], it is deduced that at least three protons are required to trigger efficient cluster degradation. Under these conditions the protonated cluster shows broadened Mössbauer signals, but ΔE
Q (1.1 mm/s) and δ (0.44 mm/s) are similar to the native form. Collapse of the protonated transition state complex, revealed by rapid-quench Mössbauer experiments, occurs with a measured rate constant k
obs≈0.72±0.35 s–1 that is consistent with results from time-resolved electronic absorption and fluorescence (k
obs≈0.4±0.1 s–1) and EPR (k
obs≈0.62±0.18 s–1) measurements. Apparently, guanidinium hydrochloride serves to perturb the tertiary structure of the protein, facilitating protonation of the cluster, but not degradation per se. Release of iron ions occurs even more slowly with k
obs≈0.07±0.02 s–1, as determined by the appearance of the g=4.3 EPR signal. Proton-mediated cluster degradation is sensitive to the oxidation state of the cluster, with the oxidized state showing a two-fold slower rate in acidic solutions as a result of increased electrostatic repulsion with the cluster. Consistent results are obtained from absorption, fluorescence, Mössbauer and EPR measurements.
This review presents a historical perspective of Fe–S cluster biochemistry, ranging from the first identification of the Fe–S core, through the elucidation of their biological diversity of structure and function, to more recent considerations of the pathways of assembly and disassembly. The latter in particular is discussed in the context of biological utility in the regulation of genetic information.
Multidrug resistance (MDR) compromises the efficacy of chemotherapy. Many approaches have been used to reduce MDR; however, the results are poor. It has been reported that iron deprivation downregulates MDR genes. To investigate the relationship of iron with MDR and early growth response gene-1 (EGR1), we investigated the effect of iron deprivation on expression and/or function of multidrug resistance-1 (MDR1), early growth response gene-1 (EGR1), ferritin heavy chain gene (H-Fn) and MDR1-encoded P-glycoprotein (P-gp) in the K562 leukemic cell line.
The cells were stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and incubated with either FeCl(3) or the iron-chelating drug DFO. The mRNA levels of MDR1, EGR1 and H-Fn were detected by RT-PCR. The protein expression and function of P-gp were measured by immunohistochemical staining and flow cytometry, respectively.
DFO significantly reduced the intracellular iron level, and led to approximately 70% reduction of MDR1 mRNA, approximately 50% of reduction of H-Fn mRNA and approximately 30% reduction of P-gp protein in TPA-differentiated K562 cells. The P-gp pump function, measured by daunorubicin exclusion, was also reduced by DFO treatment.
These results suggest a close relationship between iron deprivation and reduced MDR1/P-gp expression and function. DFO may be used together with chemotherapeutic drugs to achieve better clinical efficacy.
Due to its character as an essential element for all forms of life, the biochemistry and physiology of iron has attracted very intensive interest for many decades. In more recent years, the ways that iron metabolism is regulated in mammalian and human organisms have been clarified, and many aspects of iron metabolism have been reviewed. In this article, some newer aspects concerning absorption and intracellular regulation of iron concentration are considered. These include a sorting of possible models for intestinal iron absorption, a description of ways for membrane passage of iron after release from transferrin during receptor-mediated endocytosis, a consideration of possible mechanisms for non-transferrin bound iron uptake and its regulation, and a review of recent knowledge on the properties of iron regulatory proteins and on regulation of iron metabolism by these proteins, changes of their own properties by non-iron-mediated influences, and regulatory events not mediated by these proteins. This somewhat heterogeneous collection of themes is a consequence of the intention to avoid repetition of the many aforementioned reviews already existing and to concentrate on newer findings generated within the last couple of years.
Iron regulatory protein 1 (IRP1) and IRP2 are cytoplasmic RNA binding proteins that are central regulators of mammalian iron homeostasis. We investigated the time-dependent effect of dietary iron deficiency on liver IRP activity in relation to the abundance of ferritin and the iron-sulfur protein mitochondrial aconitase (m-acon), which are targets of IRP action. Rats were fed a diet containing 2 or 34 mg iron/kg diet for 1-28 d. Liver IRP activity increased rapidly in rats fed the iron-deficient diet with IRP1 stimulated by d 1 and IRP2 by d 2. The maximal activation of IRP2 was five-fold (d 7) and three-fold (d 4) for IRP1. By d 4, liver ferritin subunits were undetectable and m-acon abundance eventually fell by 50% (P < 0.05) in iron-deficient rats. m-Acon abundance declined most rapidly from d 1 to 11 and in a manner that was suggestive of a cause and effect type of relationship between IRP activity and m-acon abundance. In liver, iron deficiency did not decrease the activity of cytosolic aconitase, catalase or complex I of the electron transport chain nor was there an effect on the maximal rate of mitochondrial oxygen consumption with the use of malate and pyruvate as substrates. Thus, the decline in m-acon abundance in iron deficiency is not reflective of a global decrease in liver iron-sulfur proteins nor does it appear to limit ATP production. Our results suggest a novel role for m-acon in cellular iron metabolism. We conclude that, in liver, iron deficiency preferentially affects the activities of IRPs and the targets of IRP action.
The ferritin IRE, a highly conserved (96-99% in vertebrates) mRNA translation regulatory element in animal mRNA, was studied by molecular modeling (using MC-SYM and DOCKING) and by NMR spectroscopy. Cobalt(III) hexammine was used to model hydrated Mg2+. IRE isoforms in other mRNAs regulate mRNA translation or stability; all IREs bind IRPs (iron regulatory proteins). A G.C base pair, conserved in ferritin IREs, spans an internal loop/bulge in the middle of an A-helix and, combined with a dynamic G.U base pair, formed a pocket suitable for Co(III) hexammine binding. On the basis of the effects of Co(III) hexammine on the 1H NMR spectrum and results of automatic docking into the IRE model, the IRE bound Co(III) hexammine at the pocket in the major groove; Mg2+ may bind to the IRE at the same site on the basis of an analogy to Co(III) hexammine and on the Mg2+ inhibition of Cu-(phen)2 cleavage at the site. Distortion of the IRE helix by the internal loop/bulge near a conserved unpaired C required for IRP binding and adjacent to an IRP cross-linking site suggests a role for the pocket in ferritin IRE/IRP interactions.
How and where iron exits from ferritin for cellular use is unknown. Twenty-four protein subunits create a cavity in ferritin where iron is concentrated >10(11)-fold as a mineral. Proline substitution for conserved leucine 134 (L134P) allowed normal assembly but increased iron exit rates. X-ray crystallography of H-L134P ferritin revealed localized unfolding at the 3-fold axis, also iron entry sites, consistent with shared use sites for iron exit and entry. The junction of three ferritin subunits appears to be a dynamic aperture with a "shutter" that cytoplasmic factors might open or close to regulate iron release in vivo.
Several major advances in our understanding of the structure, function and properties of biological iron-sulfur clusters have occurred in the past year. These include a new structural type of cluster in the inappropriately named prismane protein, the establishment of redox-mediated [Fe2S2]2+ <--> [Fe4S4]2+ cluster conversions, and the characterization of valence-delocalized [Fe2S2]+ and all ferrous clusters with [Fe2S2]0, [Fe3S4]2- and [Fe4S4]0 cores. The emergence of novel types of redox, regulatory and enzymatic roles have also raised the possibility of iron-sulfur clusters mediating two electron redox processes, coupling proton and electron transfer, and catalyzing disulfide reduction and reductive cleavage of S-adenosylmethionine via sulfur-based cluster chemistry.
In a previous study, site-directed mutagenesis experiments identified three of the four ligands to the [2Fe-2S] cluster in animal ferrochelatase as conserved cysteines in the COOH-terminal extension, Cys-403, Cys-406, and Cys-411 in human ferrochelatase (Crouse, B. R., Sellers, V. M., Finnegan, M. G., Dailey, H. A. & Johnson, M. K. (1996) Biochemistry 35, 16222-16229). The nature of the fourth ligand was left unresolved, and spectroscopic studies raised the possibility of one noncysteinyl, oxygenic ligand. In this work, we report two lines of evidence that strongly suggest the fourth ligand is a cysteine residue. Cysteine at position 196 in human recombinant ferrochelatase when changed to a serine results in an inactive enzyme that is lacking the [2Fe-2S] cluster. Furthermore, whole cell EPR studies demonstrate that in the C196S mutant the cluster fails to assemble. Additionally, the cloning and expression of Drosophila melanogaster ferrochelatase has allowed the identification, by EPR and UV-visible spectroscopy, of a [2Fe-2S]2+ cluster with properties analogous to those of animal ferrochelatases. The observation that Drosophila ferrochelatase contains only four conserved cysteines at positions 196, 403, 406, and 411, is in accord with the proposal that these residues function as cluster ligands. In the case of the ferrochelatase iron-sulfur cluster ligands, NH2-Cys-X206-Cys-X2-Cys-X4-Cys-COOH, the position distant from other ligands may lead to a spatial positioning of the cluster near the enzyme active site or at the interface of two domains, thereby explaining the loss of enzyme activity that accompanies cluster degradation and reinforcing the idea that the cluster functions as a regulatory switch.
The translation or stability of the mRNAs from ferritin, maconitase, erythroid aminoevulinate synthase and the transferrin receptor is controlled by the binding of two iron regulatory proteins to a family of hairpin-forming RNA sequences called iron-responsive elements (IREs). The determination of high-resolution nuclear magnetic resonance (NMR) structures of IRE variants suggests an unusual hexaloop structure, leading to an intra-loop G-C base pair and a highly exposed loop guanine, and a special internal loop/bulge in the ferritin IRE involving a shift in base pairing not predicted with standard algorithms.
Cleavage of synthetic 55- and 30-mer RNA oligonucleotides corresponding to the ferritin IRE with complexes based on oxoruthenium(IV) shows enhanced reactivity at a hexaloop guanine and at a guanine adjacent to the internal loop/bulge with strong protection at a guanine in the internal loop/bulge. These results are consistent with the recent NMR structures. The synthetic 55-mer RNA binds the iron-regulatory protein from rabbit reticulocyte lysates. The DNA analogs of the 55- and 30-mers do not show the same reactivity pattern.
The chemical reactivity of the guanines in the ferritin IRE towards oxoruthenium(IV) supports the published NMR structures and the known oxidation chemistry of the metal complexes. The results constitute progress towards developing stand-alone chemical nucleases that reveal significant structural properties and provide results that can ultimately be used to constrain molecular modeling.
The discovery of iron regulatory proteins (IRPs) has provided a molecular framework from which to more fully understand the coordinate regulation of vertebrate iron metabolism. IRPs bind to iron responsive elements (IREs) in specific mRNAs and regulate their utilization. The targets of IRP action now appear to extend beyond proteins that function in the storage (ferritin) or cellular uptake (transferrin receptor) of iron to include those involved in other aspects of iron metabolism as well as in the tricarboxylic acid cycle. To date, it appears that IRPs modulate the utilization of six mammalian mRNAs. Current studies are aimed at defining the mechanisms responsible for the hierarchical regulation of these mRNAs by IRPs. In addition, much interest continues to focus on the signaling pathways through which IRP function is regulated. Multiple factors modulate the RNA binding activity of IRP1 and/or IRP2 including iron, nitric oxide, phosphorylation by protein kinase C, oxidative stress and hypoxia/reoxygenation. Because IRPs are key modulators of the uptake and metabolic fate of iron in cells, they are focal points for the modulation of cellular iron homeostasis in response to a variety of agents and circumstances.
Iron-sulfur clusters are prosthetic groups that are required for the function of numerous enzymes in the cell, including enzymes important in respiration, photosynthesis, and nitrogen fixation. Here we report cloning of the human homolog of NifS, a cysteine desulfurase that is proposed to supply the inorganic sulfur in iron-sulfur clusters. In human cells, different forms of NifS that localize either to mitochondria or to the cytosol and nucleus are synthesized from a single transcript through initiation at alternative inframe AUGs, and initiation site selection varies according to the pH of the medium or cytosol. Thus, a novel form of translational regulation permits rapid redistribution of NifS proteins into different compartments of the cell in response to changes in metabolic status.
Hereditary hyperferritinemia-cataract syndrome (HHCS) is a novel genetic disorder characterized by elevated serum ferritin and early onset cataract formation. The excessive ferritin production in HHCS patients arises from aberrant regulation of L-ferritin translation caused by mutations within the iron-responsive element (IRE) of the L-ferritin transcript. IREs serve as binding sites for iron regulatory proteins (IRPs), iron-sensing proteins that regulate ferritin translation. Previous observations suggested that each unique HHCS mutation conferred a characteristic degree of hyperferritinemia and cataract severity in affected individuals. Here we have measured the in vitro affinity of the IRPs for the mutant IREs and correlated decreases in binding affinity with clinical severity. Thermodynamic analysis of these IREs has also revealed that although some HHCS mutations lead to changes in the stability and secondary structure of the IRE, others appear to disrupt IRP-IRE recognition with minimal effect on IRE stability. HHCS is a noteworthy example of a human genetic disorder that arises from mutations within a protein-binding site of an mRNA cis-acting element. Analysis of the effects of these mutations on the energetics of the RNA-protein interaction explains the phenotypic variabilities of the disease state.
A family of non-coding sequences in the mRNA (iso-IREs [iron-responsive elements]) regulate synthesis of key proteins in animal iron and oxidative metabolism such as ferritin and mitochondrial aconitase. Differential recognition between iso-IREs and iso-IRPs (iron regulatory proteins) regulates the translation or degradation of the IRE-containing mRNAs. IREs are hairpin loop structures with an internal loop/bulge or bulge that influence the binding of the iso-IRPs. The iso-IRPs have sequence homology to the aconitases and at least one IRP can be converted to an aconitase. Signals that target the iso-IRE/iso-IRP interactions in mRNA include environmental iron, O2, nitric oxide, H2O2, ascorbate, growth factors, and protein kinase C-dependent IRP phosphorylation. Iso-IRE structural specificity suggests a means of pharmacologically targeting mRNA function with chemicals such as Fe-bleomycin and other transition metal complexes that could be extended to other mRNAs with specific structures. With the iso-IRE/iso-IRP system, nature has evolved coordinated combinatorial control of iron and oxygen metabolism that may exemplify control of mRNAs in other metabolic pathways, viral reproduction, and oncogenesis.
A report from the World Health Organization estimates that 46% of the world's 5- to 14-year-old children are anemic. In addition, 48% of the world's pregnant women are anemic. A majority of these cases of anemia are due to iron deficiency. Our aim here is to review the latest data on iron regulatory mechanisms, iron sources and requirements. Human and animal studies have shown that amino acids and peptides influence iron absorption from the intestinal lumen. Inter-organ transport and uptake of nonheme iron is largely performed by the complex transferring-transferring receptor system. Moreover, the discovery of cytoplasmic iron regulatory proteins (IRPs) has provided a molecular framework from which we understand the coordination of cellular iron homeostasis in mammals. IRPs and the iron responsive elements (IREs) to which they bind allow mammals to make use of the essential properties of iron while reducing its potentially toxic effect. Physiologic iron requirements are three times higher in pregnancy than they are in menstruating women (approximately 1200 mg must be acquired from the body's iron store or from the diet by the end of pregnancy). The administration of iron supplements weekly instead of daily in humans has been proposed and is being actively investigated as a viable means of controlling iron deficiency in populations, including pregnant women.
Synthesis of proteins for iron homeostasis is regulated by specific, combinatorial mRNA/protein interactions between RNA stem-loop structures (iron-responsive elements, IREs) and iron-regulatory proteins (IRP1 and IRP2), controlling either mRNA translation or stability. The transferrin receptor 3'-untranslated region (TfR-3'-UTR) mRNA is unique in having five IREs, linked by AU-rich elements. A C-bulge in the stem of each TfR-IRE folds into an IRE that has low IRP2 binding, whereas a loop/bulge in the stem of the ferritin-IRE allows equivalent IRP1 and IRP2 binding. Effects of multiple IRE interactions with IRP1 and IRP2 were compared between the native TfR-3'-UTR sequence (5xIRE) and RNA with only 3 or 2 IREs. We show 1) equivalent IRP1 and IRP2 binding to multiple TfR-IRE RNAs; 2) increased IRP-dependent nuclease resistance of 5xIRE compared with lower IRE copy-number RNAs; 3) distorted TfR-IRE helix structure within the context of 5xIRE, detected by Cu-(phen)(2) binding/cleavage, that coincides with ferritin-IRE conformation and enhanced IRP2 binding; and 4) variable IRP1 and IRP2 expression in human cells and during development (IRP2-mRNA predominated). Changes in TfR-IRE structure conferred by the full length TfR-3'-UTR mRNA explain in part evolutionary conservation of multiple IRE-RNA, which allows TfR mRNA stabilization and receptor synthesis when IRP activity varies, and ensures iron uptake for cell growth.
Regulation of gene expression is essential for the homeostasis of an organism, playing a pivotal role in cellular proliferation, differentiation, and response to specific stimuli. Multiple studies over the last two decades have demonstrated that the modulation of mRNA stability plays an important role in regulating gene expression. The stability of a given mRNA transcript is determined by the presence of sequences within an mRNA known as cis-elements, which can be bound by trans-acting RNA-binding proteins to inhibit or enhance mRNA decay. These cis-trans interactions are subject to a control by a wide variety of factors including hypoxia, hormones, and cytokines. In this review, we describe mRNA biosynthesis and degradation, and detail the cis-elements and RNA-binding proteins known to affect mRNA turnover. We present recent examples in which dysregulation of mRNA stability has been associated with human diseases including cancer, inflammatory disease, and Alzheimer's disease.
Ferritin, a major form of endogenous iron in food legumes such as soybeans, is a novel and natural alternative for iron supplementation strategies where effectiveness is limited by acceptability, cost, or undesirable side effects. A member of the nonheme iron group of dietary iron sources, ferritin is a complex with Fe3+ iron in a mineral (thousands of iron atoms inside a protein cage) protected from complexation. Ferritin illustrates the wide range of chemical and biological properties among nonheme iron sources. The wide range of nonheme iron receptors matched to the structure of the iron complexes that occurs in microorganisms may, by analogy, exist in humans. An understanding of the chemistry and biology of each type of dietary iron source (ferritin, heme, Fe2+ ion, etc.), and of the interactions dependent on food sources, genes, and gender, is required to design diets that will eradicate global iron deficiency in the twenty-first century.
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.
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.
Iron regulation of the human transferrin receptor gene was examined in murine cells transformed with chimeric constructs containing the human transferrin receptor gene's promoter and either the structural gene for bacterial chloramphenicol acetyltransferase or the human transferrin receptor cDNA. The activity of the transferrin receptor gene's promoter with the heterologous indicator gene was found to be approximately equal to 3-fold higher in cells treated with the iron chelator desferrioxamine than in cells treated with the iron source, hemin. A higher degree of iron regulation was seen in the expression of the human transferrin receptor cDNA driven by its own promoter. The receptor cDNA under the control of the simian virus 40 early promoter was also iron-regulated. Several human transferrin receptor transcripts differing in their 3' end were produced in the murine cells regardless of the promoter used, with the shorter transcripts being relatively unregulated by iron. Deletion of cDNA corresponding to most of the 3' untranslated portion of the mRNA for the receptor ablated the iron regulation. We conclude that at least two genetic elements exist for the regulation of the transferrin receptor gene by iron. One has its locus in the DNA upstream of the transferrin receptor gene's transcription start site, and the other is dependent upon the integrity of the sequences in the 3' end of the gene.
Expression of the human transferrin receptor (hTR) and its mRNA is strongly induced by iron deprivation. By measuring transcription elongation rates, levels of hTR-specific nuclear RNA, and mRNA half-lives, we found this regulation to occur posttranscriptionally in the cytoplasm. Analysis of hTR cDNA mutants with deletions in the 3' untranslated region revealed the existence of two distinct domains, both of which are essential for regulation in mouse L cells. The regulated phenotype correlates with the presence of a stem-loop structure predicted by a computer algorithm. Expression of point and deletion mutants affecting the stem-loop confirmed the requirement for this secondary structure in regulation. The 3' untranslated region of hTR cDNA was sufficient to confer iron-dependent regulation on another gene.
The iron responsive element binding protein (IRE-BP) regulates iron storage and uptake in response to iron. This control results
from the interaction of the IRE-BP with the iron responsive element (IRE), a conserved sequence/structure element located
near the 5′ end of all ferritin mRNAs and in the 3′ UTR of transferrin receptor mRNAs. Proteolysis was used to probe for functional
elements of the IRE-BP. Partial chymotrypsin digestion generates a simple digestion pattern yielding fragments of 68, 56,
41, and 30 kDa. The 68 and 30 kDa fragments are derived from a single cleavage at Trp623. Further cleavages of the 68 kDa polypeptide yield the 56 and 41 kDa peptides. A combination of UVcrosslinking and chymotrypsin
digestion was used to localize an RNA binding element within the C-terminus of the 68 kDa fragment, between amino acid residues
480 and 623. This region includes cysteine residues 503 and 506 which have been shown to be required for iron-sulfur cluster
assembly and for iron regulation of the IRE-BP. Proteolytic fragments of the IRE-BP that contain this RNA binding region can
be crosslinked to the IRE but do not bind with high affinity, suggesting that elements within the IRE-BP, in addition to those
located between residues 480 and 623, are required for high affinity binding to the IRE.
Iron-regulatory protein (IRP) is a master regulator of cellular iron homeostasis. Expression of several genes involved in iron uptake, storage, and utilization is regulated by binding of IRP to iron-responsive elements (IREs), structural motifs within the untranslated regions of their mRNAs. IRP-binding to IREs is controlled by cellular iron availability. Recent work revealed that nitric oxide (NO) can mimic the effect of iron chelation on IRP and on ferritin mRNA translation, whereas the stabilization of transferrin receptor mRNA following NO-mediated IRP activation could not be observed in gamma-interferon/lipopolysaccharide-stimulated murine macrophages. In this study, we establish the function of NO as a signaling molecule to IRP and as a regulator of mRNA translation and stabilization. Fibroblasts with undetectable levels of endogenous NO synthase activity were stably transfected with a cDNA encoding murine macrophage inducible NO synthase. Synthesis of NO activates IRE binding, which in turn represses ferritin mRNA translation and stabilizes transferrin receptor mRNA against targeted degradation. Furthermore, iron starvation and NO release are shown to be independent signals to IRP. The posttranscriptional control of iron metabolism is thus intimately connected with the NO pathways.
We describe a new procedure to Identify RNA or DNA binding sites in proteins, based on a combination of UV cross-linking and
single-hit chemical peptlde cleavage. Site-directed mutagenesis is used to create a series of mutants with single Asn-Gly
sequences in the protein to be analysed. Recombinant mutant proteins are incubated with their radiolabelled target sequence
and UV Irradiated. Covalently linked RNA- or DNA-proteln complexes are digested with hydroxylamine and labelled peptides identified
by SDS-PAGE and autoradiography. The analysis requires only small amounts of protein and is achieved within a relatively short
time. Using this method we mapped the site at which human iron regulatory protein (IRP) is UV cross-linked to iron responsive
element RNA to amino acid residues 116–151.
The iron-responsive element-binding protein (IRE-BP) binds to specific stem-loop RNA structures known as iron-responsive elements (IREs) present in a variety of cellular mRNAs (e.g., those encoding ferritin, erythroid 5-aminolevulinate synthase, and transferrin receptor). Expression of these genes is regulated by interaction with the IRE-BP. The IRE-BP is identical in sequence to cytosolic aconitase, and the function of the protein is determined by the presence or absence of an Fe-S cluster. The protein either functions as an active aconitase when the Fe-S cluster is present or as an RNA-binding protein when the protein lacks this cluster. Aconitase activity and IRE-binding activity are mutually exclusive, and interconversion between the two activities is determined by intracellular Fe concentrations. Mapping of the RNA-binding site of the IRE-BP by UV cross-linking studies defines a major contact site between IRE and protein in the active-site region. Modeling based on probable structural similarities between the previously crystallized mitochondrial aconitase and the IRE-BP predicts that these residues would be accessible to the IRE only were there a major change in the predicted conformation of the protein when cells are iron-depleted.
Iron regulatory factor (IRF) Is a cytoplasmlc mRNA-blndlng protein that coordinates post-transcrlptlonally the expression
of several important proteins in iron metabolism. Binding of IRF to Iron-responsive elements (IRE) in the 5′ untranslated
region (UTR) of ferrltln and erythrold 5-aminolevullnic acid-synthase mRNAs inhibits their translation, whereas binding to
IREs in the 3′ UTR of transfer-Tin receptor (TfR) mRNA prevents the degradation of this mRNA. IRF binds RNA strongly after
Iron deprivation, but is Inactive, yet present, under conditions of high cellular iron supply. Recently, IRF was also shown
to have aconltase activity indicating the existence of an Fe-S cluster In the protein. In the current study we expressed human
IRF In Insect cells from recombinant baculovirus and analysed IRE-binding and aconltase activities under various culture conditions.
Newly made apoprotein, synthesized in the absence of Iron, was fully active in IRE-bindlng, but showed no aconitase activity.
In contrast, IRF made by cells grown in high iron medium bound RNA poorly, but exhibited high aconitase activity with a Km of 9.2 μM for cls-aconltate. Apo-IRF was converted In vitro to active aconitase by Fe-S cluster-generating conditions, and under the same conditions lost Its RNA-blndlng capacity. These
results indicate that the two activities are mutually exclusive and controlled through formation of the Fe-S cluster.
The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. Our results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis.
In recent reports attention has been drawn to the extensive amino acid homology between pig heart, yeast, and Escherichia coli aconitases (EC 4.2.1.3) and the iron-responsive element binding protein (IRE-BP) of mammalian cells [Rouault, T. A., Stout, C. D., Kaptain, S., Harford, J. B. & Klausner, R. D. (1991) Cell 64, 881-883.; Hentze, M. W. & Argos, P. (1991) Nucleic Acids Res. 19, 1739-1740.; Prodromou, C., Artymiuk, P. J. & Guest, J. R. (1992) Eur. J. Biochem. 204, 599-609]. Iron-responsive elements (IREs) are stem-loop structures located in the untranslated regions of mRNAs. IRE-BP is required in the posttranscriptional regulation of ferritin mRNA translation and stabilization of transferrin receptor mRNA. In spite of substantial homology between the amino acid sequences of mammalian mitochondrial aconitase and IRE-BP, the mitochondrial protein does not bind IREs. However, there is a second aconitase, found only in the cytosol of mammalian tissues, that might serve as an IRE-BP. To test this possibility, we have prepared sufficient quantities of the heretofore poorly characterized beef liver cytosolic aconitase. This enzyme is isolated largely in its active [4Fe-4S] form and has a turnover number similar to that of mitochondrial aconitase. The EPR spectra of the two enzymes are markedly different. The amino acid composition, molecular weight, isoelectric point, and the sequences of six random peptides clearly show that these physicochemical and structural characteristics are identical to those of IRE-BP, and that c-aconitase is distinctly different from m-aconitase. In addition, both cytosolic aconitase and IRE-BP can have aconitase activity or function as IRE-BPs, as shown in the following paper and elsewhere [Zheng, L. Kennedy, M. C., Blondin, G. A., Beinert, H. & Zalkin, H. (1992) Arch. Biochem. Biophys., in press]. This leads us to the conclusion that cytosolic aconitase is IRE-BP.
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.
The crystal structures of mitochondrial aconitase with isocitrate and nitroisocitrate bound have been solved and refined to R factors of 0.179 and 0.161, respectively, for all observed data in the range 8.0-2.1 A. Porcine heart enzyme was used for determining the structure with isocitrate bound. The presence of isocitrate in the crystals was corroborated by Mössbauer spectroscopy. Bovine heart enzyme was used for determining the structure with the reaction intermediate analogue nitroisocitrate bound. The inhibitor binds to the enzyme in a manner virtually identical to that of isocitrate. Both compounds bind to the unique Fe atom of the [4Fe-4S] cluster via a hydroxyl oxygen and one carboxyl oxygen. A H2O molecule is also bound, making Fe six-coordinate. The unique Fe is pulled away approximately 0.2 A from the corner of the cubane compared to the position it would occupy in a symmetrically ligated [4Fe-4S] cluster. At least 23 residues from all four domains of aconitase contribute to the active site. These residues participate in substrate recognition (Arg447, Arg452, Arg580, Arg644, Gln72, Ser166, Ser643), cluster ligation and interaction (Cys358, Cys421, Cys424, Asn258, Asn446), and hydrogen bonds supporting active site side chains (Ala74, Asp568, Ser571, Thr567). Residues implicated in catalysis are Ser642 and three histidine-carboxylate pairs (Asp100-His101, Asp165-His147, Glu262-His167). The base necessary for proton abstraction from C beta of isocitrate appears to be Ser642; the O gamma atom is proximal to the calculated hydrogen position, while the environment of O gamma suggests stabilization of an alkoxide (an oxyanion hole formed by the amide and side chain of Arg644). The histidine-carboxylate pairs appear to be required for proton transfer reactions involving two oxygens bound to Fe, one derived from solvent (bound H2O) and one derived from substrate hydroxyl. Each oxygen is in contact with a histidine, and both are in contact with the side chain of Asp165, which bridges the two sites on the six-coordinate Fe.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
Ferritin is a ubiquitous iron-storage protein found in the cells of animals, plants, molds, and bacteria which it protects from toxic intracellular levels of iron. Ferritin stores iron within a hollow protein shell formed by subunits of two types, H and L. The 5' untranslated regions of the two subunit mRNAs contain an almost identical 28-nucleotide sequence which regulates translation by binding to a specific cell sap protein. When cell iron level is low, this repressor protein obstructs translation of stored ferritin mRNAs, whereas increased iron levels release this protein, thus permitting extensive ferritin subunit synthesis to respond rapidly. Similar motifs in the 3' untranslated region of transferrin receptor mRNA interact with this protein to regulate breakdown of the mRNA and thus change the receptor population. Finally, transcription of the H and L genes can be independently increased by iron and other factors. In the case of iron, synthesis of the L-mRNA is increased preferentially since ferritin shells with a preponderance of L-subunits store iron more efficiently. Thus regulation of ferritin synthesis at the translational and transcriptional levels and by transferrin receptor mRNA abundance at the level of breakdown provide a coordinated mechanism for protecting cells against the effects of excess iron.
The synthesis of both transferrin receptor (TfR) and ferritin is regulated post-transcriptionally by iron. This is mediated
by iron responsive elements (IRES) in the 5′- and 3′-untranslated regions, respectively, of TfR and ferritin mRNAs. Although
these IRES have different sequences, they both form a characteristic stem-loop. We used competttion assays and partial peptide
mapping of UV-crosslinked ferritin and TfR IRE-protein complexes to show that the cytosolic protein binding to the ferritin
5′-IRE, the iron-responsive element binding protein (IRE-BP), also binds to TfR 3′-IRES. To identify the structural requirements
necessary for RNA-protein binding, ferritin IRE RNAs were synthesized which contained altered secondary structures and base
substitutions. Affinities of these RNAs for IRE-BP were assayed in RNA-protein binding gels. Substitutions disrupting base-pairing
of the stem prevented IRE-BP binding. Substitutions which restored base-pairing also restored IRE-BP binding. We conclude
that the IRE-BP
binds to both ferritin and TfR IREs and recognizes a particular IRE conformation.
An increasing number of iron-sulfur (Fe-S) proteins are found in which the Fe-S cluster is not involved in net electron transfer, as it is in the majority of Fe-S proteins. Most of the former are (de)hydratases, of which the most extensively studied is aconitase. Approaches are described and discussed by which the Fe-S cluster of this enzyme could be brought into states of different structure, ligation, oxidation and isotope composition. The species, so obtained, provided the basis for spectroscopic and chemical investigations. Results from studies by protein chemistry, EPR, Mössbauer, 1H, 2H and 57Fe electron-nuclear double resonance spectroscopy are described. Conclusions, which bear on the electronic structure of the Fe-S cluster, enzyme-substrate interaction and the enzymatic mechanism, were derived from a synopsis of the recent work described here and of previous contributions from several laboratories. These conclusions are discussed and summarized in a final section.
The crystal structure of the 80,000 Da Fe-S enzyme aconitase has been solved and refined at 2.1 A resolution. The protein contains four domains; the first three from the N-terminus are closely associated around the [3Fe-4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Association of the larger C-terminal domain with the first three domains creates an extensive cleft leading to the Fe-S cluster. Residues from all four domains contribute to the active site region, which is defined by the Fe-S cluster and a bound SO4(2-) ion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well as several bound water molecules. Three of these side chains reside on a three-turn 3(10) helix in the first domain. The SO4(2-) ion is bound 9.3 A from the center of the [3Fe-4S] cluster by the side chains of 2 Arg and 1 Gln residues. Each of 3 His side chains in the putative active site is paired with Asp or Glu side chains.
A cytosolic protein, named iron-responsive element-binding protein (IRE-BP), is sensitive to cellular iron concentration. At low cytosolic iron level, IRE-BP is activated and binds to stem-loop untranslated regions (IRE regions) of transferrin and ferritin mRNAs, activating and inhibiting their translations, respectively. This concerted mechanism permits a fine control of iron homeostasis in the cell. The activity of IRE-BP can be measured by its binding to IRE regions, using a protein band-shift electrophoretic assay. Damage to cells by oxidative stress is known to be mediated by iron. We observed that IRE-BP is rapidly activated by exposure of V79 Chinese hamster ovary cells to H2O2. However, if cell extracts are exposed to H2O2 IRE-BP activation is not observed. Therefore, the activation is not a direct consequence of the H2O2 attack to IRE-BP. The in vivo IRE-BP-activation by H2O2 is not prevented by hydroxyl radical scavengers or by the iron chelator 1,10-phenanthroline, indicating that Fenton reaction is not involved in the process. In fact, simultaneous exposure of cells to H2O2 and 1,10-phenanthroline produces an even stronger activation than exposure to H2O2 alone. The interpretation of the mechanism of IRE-BP activation by oxidative stress is hampered by the fact that the mechanism of IRE-BP modulation by cytosolic iron has not been established. It has been recently shown that the iron-sulfur cluster in IRE-BP must be completely disassembled in order for activation to occur and that this is triggered by low iron in the cell. It is likely that IRE-BP senses Fe(II) and that its oxidation to Fe(III) by H2O2 or chelation by 1,10-phenanthroline set up a program for increasing iron uptake. The physiological consequences of this activation still has to be assessed.
Cellular iron metabolism comprises pathways of iron-protein synthesis and degradation, iron uptake via transferrin receptor (TfR) or release to the extracellular space, as well as iron deposition into ferritin and remobilization from such stores. Different cell types, depending on their rate of proliferation and/or specific functions, show strong variations in these pathways and have to control their iron metabolism to cope with individual functions. Studies with cultured cells have revealed a specific cytoplasmic protein, called 'iron regulatory protein' (IRP) (previously known as IRE-BP or IRF), that plays a key role in iron homoeostasis by regulating coordinately the synthesis of TfR, ferritin, and erythroid 5-aminolevulinate synthase (eALAS). Present in all tissues analysed, IRP is identical with the [4Fe-4S] cluster containing cytoplasmic aconitase. Under conditions of iron chelation, IRP is an apo-protein which binds with high affinity to specific RNA stem-loop elements (IREs) located 5' of the initiation codon in ferritin and eALAS mRNA, and 3' in the untranslated region of TfR mRNA. At 5' sites IRF blocks mRNA translation, whereas 3' it inhibits TfR mRNA degradation. Both effects compensate for low intracellular iron concentrations. Under high iron conditions, IRP is converted to the holo-protein and dissociates from mRNA. This reverses the control towards less iron uptake and more iron storage. Iron can therefore be considered as a feedback regulator of its own metabolism. It has recently become evident that nitric oxide, produced by macrophages and other cell types in response to interferon-gamma, induces the IRE-binding activity of IRF. Moreover measurements of the RNA-binding activity of IRP in tissue extracts may provide valuable information on iron availability.
The iron-responsive element binding protein/cytosolic aconitase functions as either an RNA binding protein that regulates the uptake, sequestration, and utilization of iron or an enzyme that interconverts citrate and isocitrate. These mutually exclusive functions are regulated by changes in cellular iron levels. By site-directed mutagenesis we show that (i) ligation of a [4Fe-4S] cluster is necessary to inactivate RNA binding and activate enzyme function in vivo, (ii) three of four arginine residues of the aconitase active site participate in RNA binding, and (iii) aconitase activity is not required for iron-mediated regulation of RNA binding.
All cells have to adjust uptake, utilization and storage of iron according to the availability and their requirement for this essential metal. Progress in recent years has led to the elucidation of the molecular control mechanisms that co-ordinate the uptake, utilization and storage of iron in mammalian cells and has highlighted the role of a newly-identified regulatory protein, the iron regulatory factor (IRF). IRF is a cytoplasmic protein that senses the intracellular iron level and responds by adjusting its function. When the iron level is low, it binds to so-called 'iron responsive elements' (IREs) contained in the mRNAs encoding proteins involved in iron metabolism and erythroid haem synthesis. When levels of cellular iron rise, IRF converts into the enzyme aconitase and looses its ability to bind to IREs. We discuss both functions of this Janus face protein and describe how its function is controlled by the status of an iron sulphur cluster in the IRF protein. We also speculate about how an IRF-mediated regulation may relate to certain medical disorders.
The nifS gene product (NIFS) is a pyridoxal phosphate binding enzyme that catalyzes the desulfurization of L-cysteine to yield L-alanine and sulfur. In Azotobacter vinelandii this activity is required for the full activation of the nitrogenase component proteins. Because the nitrogenase component proteins, Fe protein and MoFe protein, both contain metalloclusters which are required for their respective activities, it is suggested that NIFS participates in the biosynthesis of the nitrogenase metalloclusters by providing the inorganic sulfur required for Fe-S core formation [Zheng, L., White, R. H., Cash, V. L. Jack, R. F., & Dean, D. R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2754-2758]. In the present study the mechanism for the desulfurization of L-cysteine catalyzed by NIFS was determined in the following ways. First, the substrate analogs, L-allylglycine and vinylglycine, were shown to irreversibly inactivate NIFS by formation of a gamma-methylcystathionyl or cystathionyl residue, respectively, through nucleophilic attack by an active site cysteinyl residue on the corresponding analog-pyridoxal phosphate adduct. Second, this reactive cysteinyl residue, which is required for L-cysteine desulfurization activity, was identified as Cys325 by the specific alkylation of that residue and by site-directed mutagenesis experiments. Third, the formation of an enzyme-bound cysteinyl persulfide was identified as an intermediate in the NIFS-catalyzed reaction. Fourth, evidence was obtained for an enamine intermediate in the formation of L-alanine.(ABSTRACT TRUNCATED AT 250 WORDS)
The translation of ferritin and erythroid 5-aminolevulinate synthase mRNAs is regulated via a specific high-affinity interaction between an iron-responsive element in the 5' untranslated region of ferritin and erythroid 5-aminolevulinate synthase mRNAs and a 98-kDa cytoplasmic protein, the iron-regulatory factor. Iron-regulatory factor was expressed in vaccinia-virus-infected HeLa cells (hIRFvac) and in Escherichia coli (hIRFeco). An N-terminal histidine tag allowed a rapid one-step purification of large quantities of soluble recombinant protein. Both hIRFvac and hIRFeco bound specifically to iron-responsive elements and were immunoprecipitated by iron-regulatory-factor antibodies. Using in-vitro-transcribed chloramphenicol-acetyltransferase mRNAs bearing an iron-responsive element in the 5' untranslated region, specific repression of chloramphenicol-acetyltransferase translation by hIRFvac and hIRFeco was demonstrated in wheat-germ extract. In addition, hIRFvac and hIRFeco were shown to display aconitase activity. Treatment of hIRFvac and hIRFeco with FeSO4 resulted in a drastic reduction in iron-responsive-element-binding of iron-regulatory factor, but caused a strong stimulation of its aconitase activity. The results establish that recombinant iron-regulatory factor is a bifunctional protein; after purification, it binds to iron-responsive elements and represses translation in vitro. Following iron treatment, iron-responsive-element binding is lost and aconitase activity is gained. No eukaryotic co-factor seems to be required for the conversion of the iron-responsive-element binding to the aconitase form of the protein.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
Biological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme composed of two separately purifiable component proteins encoded by the structural genes nifH, nifD, and nifK. Deletion of the Azotobacter vinelandii nifS gene lowers the activities of both nitrogenase component proteins. Because both nitrogenase component proteins have metallocluster prosthetic groups that are composed of iron- and sulfur-containing cores, this result indicated that the nifS gene product could be involved in the mobilization of the iron or sulfur required for metallocluster formation. In the present work, it is shown that NIFS is a pyridoxal phosphate-containing homodimer that catalyzes the formation of L-alanine and elemental sulfur by using L-cysteine as substrate. NIFS activity is extremely sensitive to thiol-specific alkylating reagents, which indicates the participation of a cysteinyl thiolate at the active site. Based on these results we propose that an enzyme-bound cysteinyl persulfide that requires the release of the sulfur from the substrate L-cysteine for its formation ultimately provides the inorganic sulfide required for nitrogenase metallocluster formation. The recent discovery of nifS-like genes in non-nitrogen-fixing organisms also raises the possibility that the reaction catalyzed by NIFS represents a universal mechanism that involves pyridoxal phosphate chemistry, in the mobilization of the sulfur required for metallocluster formation.
Iron must cross biological membranes to reach essential intracellular enzymes. Two proteins in the plasma membrane of yeast—a
multicopper oxidase, encoded by the FET3 gene, and a permease, encoded by the FTR1 gene—were shown to mediate high-affinity iron uptake. FET3 expression was required for FTR1 protein to be transported to the plasma membrane. FTR1 expression was required for apo-FET3 protein to be loaded with copper and thus acquire oxidase activity. FTR1 protein also
played a direct role in iron transport. Mutations in a conserved sequence motif of FTR1 specifically blocked iron transport.
Iron-sulfur clusters are prosthetic groups commonly found in proteins that participate in oxidation-reduction reactions and catalysis. Here, we focus on two proteins that contain iron-sulfur clusters, the fumarate nitrate reduction (FNR) protein of Escherichia coli and mammalian iron-responsive-element-binding protein 1 (IRP1), both of which function as direct sensors of oxygen and iron levels. Assembly and disassembly of iron-sulfur clusters is the key to sensing in these proteins and we speculate that iron-sulfur clusters might be found in other regulatory proteins that sense levels of iron and/or oxygen.