Elizabeth C. Theil

Structure of dual function iron regulatory protein 1 complexed with ferritin IRE-RNA.

Iron regulatory protein 1 (IRP1) binds iron-responsive elements (IREs) in messenger RNAs (mRNAs), to repress translation or degradation, or binds an iron-sulfur cluster, to become a cytosolic aconitase enzyme. The 2.8 angstrom resolution crystal structure of the IRP1:ferritin H IRE complex shows an open protein conformation compared with that of cytosolic aconitase. The extended, L-shaped IRP1 molecule embraces the IRE stem-loop through interactions at two sites separated by ∼30 angstroms, each involving about a dozen protein:RNA bonds. Extensive conformational changes related to binding the IRE or an iron-sulfur cluster explain the alternate functions of IRP1 as an mRNA regulator or enzyme.

Combinatorial mRNA Regulation: Iron Regulatory Proteins and Iso-iron-responsive Elements (Iso-IREs)

Combinations of RNA elements (mRNA specific) with binding proteins give a wide range of responses to biological signals from iron, oxygen, NO, or growth factors. Combinatorial regulation of transcription to coordinate synthesis of groups of proteins is well known and is exemplified by steroid hormone-responsive genes (1). Combinatorial regulation of mRNA utilization to coordinate synthesis of groups of proteins is unique currently to iron and oxygen metabolism in animals (2–15) (see Fig. 1). The RNA elements are called iso-iron-responsive elements (iso-IREs), and the binding proteins, called iso-iron regulatory proteins (iso-IRPs), are aconitase homologues. Examples of iso-IRE mRNAs are ferritin to concentrate iron, TfR and DMT-1 for iron uptake, and ferroportin (Fpn1/IREG1/MTP1) for iron efflux. Several proteins for oxygen metabolism are also encoded in iso-IRE mRNAs, exemplified by aminolevulinate synthase (eALAS) in heme synthesis and mt-aconitase in the trichloroacetic acid cycle. Signals that control isoIRE/iso-IRP binding include iron, oxygen, hydrogen peroxide, NO, and activators of protein kinase C. When IRE regulation of mRNA function was last described in a Minireview (1990) only two IRE-mRNAs (ferritin and TfR) were known (2), in contrast to the many IRE mRNAs currently known. Iron was the only known signal, and knowledge of structure was limited to RNA sequence and secondary structure determined by prediction and enzymatic/chemical probes (2). Annotation of the literature in the intervening period is in Refs. 4–7. Now much is known about IRE tertiary structure (7). Multiple signaling pathways are known to converge on the IRE/IRP interaction (4, 6, 8–11). The combinatorial RNA/protein family and the effects of the RNA protein complex on protein synthesis are illustrated in Fig. 1. The result of the RNA-binding protein specificity, for the different iso-IRE-containing mRNAs, is quantitative differences in the expression of proteins that are finely tuned over a wide range. Such precise control over the synthesis of each of the proteins relates to the central role of the proteins in normal cell biology: iron trafficking, heme synthesis, and cellular ATP production. The flexibility of regulation using the IRE/IRP mRNA/protein interactions is illustrated by the liver where the same amount of iron induces ferritin synthesis up to 100-fold (2), but mitochondrial aconitase is only induced 2–3-fold (3); the difference likely relates to a narrow tolerance of cells to concentration changes in trichloroacetic acid cycle enzymes. Differences in the iso-IRE binding in each mRNA suggest a higher percentage of ferritin mRNA will be bound to IRPs than mt-aconitase mRNA (see Fig. 3), allowing quantitative variations in the response of protein synthesis to signals. An alternate mechanism for IRE/IRP control of protein synthesis is regulated mRNA turnover, illustrated by the TfR IRE.

Living with iron (and oxygen): questions and answers about iron homeostasis.

Mechanisms to regulate iron homeostasis are very likely billions of years older than those for oxygen homeostasis since contemporary microbes regulate iron in the absence of oxygen and may model ancient organisms that lived before atmospheric oxygen appeared.1 Moreover, proteins that manage iron and oxidants such as the mini-ferritins in contemporary bacteria, also called Dps (DNA protection during stress) proteins, are expressed in anaerobic archea.2 Mini-ferritins are 12 subunit protein cages from archea and bacteria that contrast with maxi-ferritins, the 24 subunit cages from bacteria, animals and plants, which use iron and dioxygen as substrates, by consuming iron and hydrogen peroxide as substrates to make the mineral inside protein nanocages. Such peroxide-consuming ferritins may be progenitors of modern ferritins and may have contributed to the transition from anaerobic to aerobic life with iron and oxygen. Iron and oxygen homeostasis, in animals, integrate DNA/mRNA controls; regulation of these two processes intersect along many pathways. Iron homeostasis occurs within cells and between cells to confer balance throughout tissues and the organism. Each cell or tissue type will have a different iron set point for homeostasis that reflects the specific role of each cell type. For example, animal red blood cells need much more iron than epithelial cells because of the synthesis of hemoglobin.3 Similarly, in plants, leaves contain much more iron than flowers because of the synthesis of ferredoxins important in photosynthesis.4 Growing animals or plants will also have different requirements for iron than aging animals or senescent plants. Thus, the conditions that cause changes in iron homeostasis may vary depending on age or specialized function, explaining, in part, quantitatively different results that can be obtained with cultured cell lines derived from different tissues. Metal ion homeostasis has common features shared by all the metal ions that include cell uptake, cell efflux, and intracellular transport. However, the chemical properties of iron require additional iron–specific homeostatic features compared to other metals, because of the hydrolytic properties of ferric ion under physiological condition. Hydrated ferric ions are relatively strong acids; protons in water coordinated to ferric ions have a pKa ≈ 3. The conjugate bases of hydrated ferric ions form multinuclear species rapidly, accounting for the low solubility of aqueous ferric ions (10−18 M) and for rust formation. In addition, living cells and organisms use much more iron than other metals. For example, the human body contains ~3.5 g of iron compared to 100 mg of copper. Iron concentrations in cells are much higher than the solubility of free ferric ions in aqueous solution in air. Average iron concentrations in cells are ~10−4 M requiring, since the solubility of free ferric ions in water/plasma is 10−18 M, a concentration gradient of 100 trillion between aqueous, external environments and intracellular environments. The concentration difference is achieved using transporters (membrane), intracellular carriers and concentrators, cellular exporters, and, in multicellular organisms, extracellular carriers. Except for iron concentrators such as ferritins, the uses of receptors, transporters, and intracellular carriers is shared with other metal ions, such as copper, manganese, etc. The ferritins, which concentrate and store intracellular iron at concentrations far above the solubility of the free ion, are unique for iron homeostasis, compared to other metal ions. Many different chemical species of iron exist in the environment that are available for biological absorption. Examples include iron–protoporphyrin IX, iron chelates, inorganic iron salts, polynuclear iron, and, at least in animals, iron minerals stored in ferritin.5,6 Iron acquisition related to homeostasis is discussed elsewhere in this issue. Studies on iron homeostasis are rarely related to the chemical species of iron used. The working model of iron in transit is Fe(II) bound to chaperone or carrier sites of varying kinetic and equilibrium stabilities. Thus, this review will focus on iron homeostasis independently of iron speciation.

Opening protein pores with chaotropes enhances Fe reduction and chelation of Fe from the ferritin biomineral

Iron is concentrated in ferritin, a spherical protein with a capacious cavity for ferric nanominerals of <4,500 Fe atoms. Global ferritin structure is very stable, resisting 6 M urea and heat (85°C) at neutral pH. Eight pores, each formed by six helices from 3 of the 24 polypeptide subunits, restrict mineral access to reductant, protons, or chelators. Protein-directed transport of Fe and aqueous Fe3+ chemistry (solubility ≈10−18 M) drive mineralization. Ferritin pores are “gated” based on protein crystals and Fe chelation rates of wild-type (WT) and engineered proteins. Pore structure and gate residues, which are highly conserved, thus should be sensitive to environmental changes such as low concentrations of chaotropes. We now demonstrate that urea or guanidine (1–10 mM), far below concentrations for global unfolding, induced multiphasic rate increases in Fe2+-bipyridyl formation similar to conservative substitutions of pore residues. Urea (1 M) or the nonconservative Leu/Pro substitution that fully unfolded pores without urea both induced monophasic rate increases in Fe2+ chelation rates, indicating unrestricted access between mineral and reductant/chelator. The observation of low-melting ferritin subdomains by CD spectroscopy (melting midpoint 53°C), accounting for 10% of ferritin α-helices, is unprecedented. The low-melting ferritin subdomains are pores, based on percentage helix and destabilization by either very dilute urea solutions (1 mM) or Leu/Pro substitution, which both increased Fe2+ chelation. Biological molecules may have evolved to control gating of ferritin pores in response to cell iron need and, if mimicked by designer drugs, could impact chelation therapies in iron-overload diseases.

Ferritins: iron/oxygen biominerals in protein nanocages.

Ferritin protein nanocages that form iron oxy biominerals in the central nanometer cavity are nature’s answer to managing iron and oxygen; gene deletions are lethal in mammals and render bacteria more vulnerable to host release of antipathogen oxidants. The multifunctional, multisubunit proteins couple iron with oxygen (maxi-ferritins) or hydrogen peroxide (mini-ferritins) at catalytic sites that are related to di-iron sites oxidases, ribonucleotide reductase, methane monooxygenase and fatty acid desaturases, and synthesize mineral precursors. Gated pores, distributed symmetrically around the ferritin cages, control removal of iron by reductants and chelators. Gene regulation of ferritin, long known to depend on iron and, in animals, on a noncoding messenger RNA (mRNA) structure linked in a combinatorial array to functionally related mRNA of iron transport, has recently been shown to be linked to an array of proteins for antioxidant responses such as thioredoxin and quinone reductases. Ferritin DNA responds more to oxygen signals, and ferritin mRNA responds more to iron signals. Ferritin genes (DNA and RNA) and protein function at the intersection of iron and oxygen chemistry in biology.

Loops and Bulge/Loops in Iron-responsive Element Isoforms Influence Iron Regulatory Protein Binding FINE-TUNING OF mRNA REGULATION?

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.

Ferritin protein nanocages use ion channels, catalytic sites, and nucleation channels to manage iron/oxygen chemistry

The ferritin superfamily is composed of ancient, nanocage proteins with an internal cavity, 60% of total volume, that reversibly synthesize solid minerals of hydrated ferric oxide; the minerals are iron concentrates for cell nutrition as well as antioxidants due to ferrous and oxygen consumption during mineralization. The cages have multiple iron entry/exit channels, oxidoreductase enzyme sites, and, in eukaryotes, Fe(III)O nucleation channels with clustered exits that extend protein activity to include facilitated mineral growth. Ferritin protein cage differences include size, amino acid sequence, and location of the active sites, oxidant substrate and crystallinity of the iron mineral. Genetic regulation depends on iron and oxygen signals, which in animals includes direct ferrous signaling to RNA to release and to ubiquitin-ligases to degrade the protein repressors. Ferritin biosynthesis forms, with DNA, mRNA and the protein product, a feedback loop where the genetic signals are also protein substrates. The ferritin protein nanocages, which are required for normal iron homeostasis and are finding current use in the delivery of nanodrugs, novel nanomaterials, and nanocatalysts, are likely contributors to survival and success during the transition from anaerobic to aerobic life.

NMR reveals pathway for ferric mineral precursors to the central cavity of ferritin

Ferritin is a multimeric nanocage protein that directs the reversible biomineralization of iron. At the catalytic ferroxidase site two iron(II) ions react with dioxygen to form diferric species. In order to study the pathway of iron(III) from the ferroxidase site to the central cavity a new NMR strategy was developed to manage the investigation of a system composed of 24 monomers of 20 kDa each. The strategy is based on 13C-13C solution NOESY experiments combined with solid-state proton-driven 13C-13C spin diffusion and 3D coherence transfer experiments. In this way, 75% of amino acids were recognized and 35% sequence-specific assigned. Paramagnetic broadening, induced by iron(III) species in solution 13C-13C NOESY spectra, localized the iron within each subunit and traced the progression to the central cavity. Eight iron ions fill the 20-Å-long iron channel from the ferrous/dioxygen oxidoreductase site to the exit into the cavity, inside the four-helix bundle of each subunit, contrasting with short paths in models. Magnetic susceptibility data support the formation of ferric multimers in the iron channels. Multiple iron channel exits are near enough to facilitate high concentration of iron that can mineralize in the ferritin cavity, illustrating advantages of the multisubunit cage structure.

LOCALIZED UNFOLDING AT THE JUNCTION OF THREE FERRITIN SUBUNITS : A MECHANISM FOR IRON RELEASE?

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 >1011-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.

Bach1 repression of ferritin and thioredoxin reductase1 is heme-sensitive in cells and in vitro and coordinates expression with heme oxygenase1, beta-globin, and NADP(H) quinone (oxido) reductase1.

Ferritin gene transcription is regulated by heme as is ferritin mRNA translation, which is mediated by the well studied mRNA·IRE/IRP protein complex. The heme-sensitive DNA sequence in ferritin genes is the maf recognition/antioxidant response element present in several other genes that are induced by heme and repressed by Bach1. We now report that chromatin immunoprecipitated with Bach1 antiserum contains ferritin DNA sequences. In addition, overexpression of Bach1 protein in the transfected cells decreased ferritin expression, indicating insufficient endogenous Bach1 for full repression; decreasing Bach1 with antisense RNA increased ferritin expression. Thioredoxin reductase1, a gene that also contains a maf recognition/antioxidant response element but is less studied, responded similarly to ferritin, as did the positive controls heme oxygenase1 and NADP(H) quinone (oxido) reductase1. Bach1-DNA promoter interactions in cells were confirmed in vitro with soluble, recombinant Bach1 protein and revealed a quantitative range of Bach1/DNA stabilities: ferritin L ∼ ferritin H ∼ β-globin, β-globin ∼ 2-fold >heme oxygenase1 = quinone reductase β-globin ∼ 4-fold >thioredoxin reductase1. Such results indicate the possibility that modulation of cellular Bach1 concentrations will have variable effects among the genes coordinately regulated by maf recognition/antioxidant response elements in iron/oxygen/antioxidant metabolism.