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E C Theil

Publications and source records attributed to E C Theil.

At least 37 records · Page 2Linked to original sources
PubMed1997

A sustainable solution for dietary iron deficiency through plant biotechnology and breeding to increase seed ferritin control.

OBJECTIVES: To stimulate novel sustainable solutions to the problem of the nutritional iron deficiency, we asked: How does Nature insure proper iron nutrition of embryos and neonatal animals? Estimates of iron deficiency world-wide are 30% of the population, with women and children at the greatest risk. Recent studies linking iron deficiency with impeded cognitive development emphasizes the enormity of the impact of iron deficiency. Sustainable solutions to the problem of dietary iron deficiency have been elusive. RESULTS: Data for storage iron was examined in seeds, developing plants, embryos and developing animals. In all cases, the common source of stored iron for development was ferritin. The protein component of ferritin concentrates iron billions of times above the solubility of the free metal ion. High conservation of ferritin sequences in bacteria, plants and animals and the specificity of ferritin bioavailability either added extrinsically or intrinsically enriched in a selected soybean cultivar, showed high efficacy in curing dietary iron deficiency in the rat model. Older data on ferritin were reevaluated in light of contemporary knowledge. CONCLUSIONS: Enhancement of natural seed ferritin content by biotechnology and breeding has the potential for a sustainable solution to the problem of global dietary iron deficiency.

Animals↗
PubMed1996

Ferritin gene organization: differences between plants and animals suggest possible kingdom-specific selective constraints.

Ferritin, a protein widespread in nature, concentrates iron approximately 10(11)-10(12)-fold above the solubility within a spherical shell of 24 subunits; it derives in plants and animals from a common ancestor (based on sequence) but displays a cytoplasmic location in animals compared to the plastid in contemporary plants. Ferritin gene regulation in plants and animals is altered by development, hormones, and excess iron; iron signals target DNA in plants but mRNA in animals. Evolution has thus conserved the two end points of ferritin gene expression, the physiological signals and the protein structure, while allowing some divergence of the genetic mechanisms. Comparison of ferritin gene organization in plants and animals, made possible by the cloning of a dicot (soybean) ferritin gene presented here and the recent cloning of two monocot (maize) ferritin genes, shows evolutionary divergence in ferritin gene organization between plants and animals but conservation among plants or among animals; divergence in the genetic mechanism for iron regulation is reflected by the absence in all three plant genes of the IRE, a highly conserved, noncoding sequence in vertebrate animal ferritin mRNA. In plant ferritin genes, the number of introns (n = 7) is higher than in animals (n = 3). Second, no intron positions are conserved when ferritin genes of plants and animals are compared, although all ferritin gene introns are in the coding region; within kingdoms, the intron positions in ferritin genes are conserved. Finally, secondary protein structure has no apparent relationship to intron/exon boundaries in plant ferritin genes, whereas in animal ferritin genes the correspondence is high. The structural differences in introns/exons among phylogenetically related ferritin coding sequences and the high conservation of the gene structure within plant or animal kingdoms of the gene structure within plant or animal kingdoms suggest that kingdom-specific functional constraints may exist to maintain a particular intron/exon pattern within ferritin genes. In the case of plants, where ferritin gene intron placement is unrelated to triplet codons or protein structure, and where ferritin is targeted to the plastid, the selection pressure on gene organization may relate to RNA function and plastid/nuclear signaling.

Amino Acid Sequence↗
PubMed1996

Purified ferritin and soybean meal can be sources of iron for treating iron deficiency in rats.

Ferritin and soybean meal were reevaluated as dietary treatments of iron deficiency in rats. Isotopes that had been used in the past were avoided because of contemporary knowledge of the physiological and structural complexity of ferritin protein and the solid iron mineral. Rats made anemic by iron-deficient diets were given equivalent amounts of iron as FeSO4, horse spleen ferritin, baked soybean meal, or soybean meal plus ferritin. Full recovery (89-109%) from anemia and increased tissue iron occurred after 28 d of treatment with any of the iron sources, which contrasts to past bioavailability studies using 59Fe-labeled ferritin and generally shorter periods of observation. Cultivar-specific variability was observed in soybean seed soluble iron and ferritin content (1.9-2.0 times the control cultivar, Arksoy), which was apparently heritable. The combined data suggest that manipulating ferritin expression and other soluble components of seed iron in soybeans and possibly other seeds, using Mendelian and biotechnological approaches, could contribute to a sustainable solution to global problems of iron deficiency.

Anemia, Iron-Deficiency↗
PubMed1995

High resolution crystal structures of amphibian red-cell L ferritin: potential roles for structural plasticity and solvation in function.

Ferritin is a highly conserved multisubunit protein in animals, plants and microbes which assembles with cubic symmetry and transports hydrated iron ions and protons to and from a mineralized core in the protein interior. We report here the high resolution structures of recombinant amphibian red-cell L ferritin and two mutants solved under two sets of conditions. In one mutant, Glu56, 57, 58 and 60 were replaced with Ala, producing a lag phase in the kinetics of iron uptake. In the second mutant, His25 was replaced with Tyr with, at most, subtle effects on function. A molecule of betaine, used in the purification, is bound in all structures at the 2-fold axis near the recently identified heme binding site of bacterioferritin and horse spleen L ferritin. Comparisons of the five amphibian structures identify two regions of the molecule in which conformational flexibility may be related to function. The positions and interactions of a set of 10 to 18 side-chains, most of which are on the inner surface of the protein, are sensitive both to solution conditions and to the Glu-->Ala mutation. A subset of these side-chains and a chain of ordered solvent molecules extends from the vicinity of Glu56 to 58 and Glu60 to the 3-fold channel in the wild type protein and may be involved in the transport of either iron or protons. The "spine of hydration" is disrupted in the Glu-->Ala mutant. In contrast, H25Y mutation shifts the positions of backbone atoms between the site of the mutation and the 4-fold axis and side-chain positions throughout the structure; the largest changes in the position of backbone atoms are in the DE loop and E helix, approximately 10 A from the mutation site. In combination, these results indicate that solvation, structural plasticity and cooperative structural changes may play a role in ferritin function. Analogies with the structure and function of ion channel proteins such as annexins are noted.

Amphibians↗
PubMed1995

The importance of a single G in the hairpin loop of the iron responsive element (IRE) in ferritin mRNA for structure: an NMR spectroscopy study.

Noncoding sequences regulate the function of mRNA and DNA. In animal mRNAs, iron responsive elements (IREs) regulate the synthesis of proteins for iron storage, uptake and red cell heme formation. Folding of the IRE was indicated previously by reactivity with chemical and enzymatic probes. 1H- and 31P-NMR spectra now confirm the IRE folding; an atypical 31P-spectrum, differential accessibility of imino protons to solvents, multiple long-range NOEs and heat stable subdomains were observed. Biphasic hyperchromic transitions occurred (52 and 73 degrees C). A G-C base pair occurs in the hairpin loop (HL) (based on dimethylsulfate, RNAse T1 previously used, and changes in NMR imino proton resonances typical of G-C base pairs after G/A substitution). Mutation of the hairpin loop also decreased temperature stability and changed the 31P-NMR spectrum; regulation and protein (IRP) binding were previously shown to change. Alteration of IRE structure shown by NMR spectroscopy, occurred at temperatures used in studies of IRE function, explaining loss of IRP binding. The effect of the HL mutation on the IRE emphasizes the importance of HL structure in other mRNAs, viral RNAs (e.g. HIV-TAR), and ribozymes.

Animals↗
PubMed1995

Formation of the ferritin iron mineral occurs in plastids.

Ferritin in plants is a nuclear-encoded, multisubunit protein found in plastids; an N-terminal transit peptide targets the protein to the plastid, but the site for formation of the ferritin Fe mineral is unknown. In biology, ferritin is required to concentrate Fe to levels needed by cells (approximately 10(-7) M), far above the solubility of the free ion (10(-18) M); the protein directs the reversible phase transition of the hydrated metal ion in solution to hydrated Fe-oxo mineral. Low phosphate characterizes the solid-phase Fe mineral in the center of ferritin of the cytosolic animal ferritin, but high phosphate is the hallmark of Fe mineral in prokaryotic ferritin and plant (pea [Pisum sativum L.] seed) ferritin. Earlier studies using x-ray absorption spectroscopy showed that high concentrations of phosphate present during ferritin mineralization in vivo altered the local structure of Fe in the ferritin mineral so that it mimicked the prokaryotic type, whether the protein was from animals or bacteria. The use of x-ray absorption spectroscopy to analyze the Fe environment in pea-seed ferritin now shows that the natural ferritin mineral in plants has an Fe-P interaction at 3.26A, similar to that of bacterial ferritin; phosphate also prevented formation of the longer Fe-Fe interactions at 3.5A found in animal ferritins or in pea-seed ferritin reconstituted without phosphate. Such results indicate that ferritin mineralization occurs in the plastid, where the phosphate content is higher; a corollary is the existence of a plastid Fe uptake system to allow the concentration of Fe in the ferritin mineral.

Biological Transport↗
PubMed1995

15N NMR and CD studies of the IRE (iron regulatory element in ferritin mRNA with single base substitution in the hairpin loop.

Noncoding sequences regulate mRNA and DNA function. IREs are a highly conserved family of noncoding mRNA sequences which coordinate ferritin mRNA translation and transferrin receptor mRNA stability by interactions with specific negative regulator protein, the IRP. RNA interactions with the IRP are modulated by cellular iron. The protein IRP binds to the entire IRE causing conformational changes in flanking region [Harrell et al. (1991) PNAS 88:4166-4170). The IRE+FL is the first RNA element encoding both positive and negative translational control and serves as a model mRNA regulatory elements. Folding of the IREs indicated previously by reactivity with chemical and enzymatic probes [E.C Theil (1994) Biochem. J., 304:1-11) is confirmed by using 1H, 15N and 31P (1) NMR) and CD to show that IRE secondary structure [hairpin-hexaloop (HL)/stem/internal loop or bulges] is folded, CD spectra display Mg2+ dependent structural changes in the temperature range used in the studies of IRE function. G/A substitution was shown by NMR and UV-vis to decrease stability of the folded IRE [H.Sierzputowska-Gracz et al. (1995) Nucl. Acids Res. 23:146-153]. A hairpin loop mutation affected only negative translational control.

Base Sequence↗
PubMed1994

Crystallization and structural analysis of bullfrog red cell L-subunit ferritins.

Ferritin is a 24 subunit protein that controls biomineralization of iron in animals, bacteria, and plants. Rates of mineralization vary among members of the ferritin family, particularly between L and H type subunits of animal ferritins which are differentially expressed in various cell types. To examine ferritin from a highly differentiated cell type and to clarify the relationship between ferritin structure and function, bullfrog red cell L ferritin has been cloned, overexpressed in E. coli, and crystallized under two conditions. Crystals were obtained at high ionic strength in the presence of MnCl2 at a concentration comparable to that of the protein and in the presence of MgCl2 at a concentration much higher than that of the protein. Under both crystallization conditions, the crystals are tetragonal bipyramids in the space group F432 with unit cell dimensions a = b = c = 182 +/- 0.5 A. Crystals obtained in the presence of manganese and ammonium sulfate diffract to 1.9 A, while those obtained in the presence of magnesium and sodium tartrate diffract to 1.6 A. Isomorphous crystals have been obtained under similar conditions for a site-directed mutant with a reduced mineralization rate in which Glu-57, -58, -59, and -61 are all replaced by Ala. The structure of wild type L-subunit with magnesium has been solved by molecular replacement using the calcium salt of human liver H subunit (Lawson et al., Nature (London) 349:541-544, 1991) as the model. The crystallographic R factor for the 6-2.2 A shell is 0.21. The overall fold of human H and bullfrog L ferritins is similar with an rms difference in backbone atomic positions of 0.97 A. The largest structural differences occur in the D helix and the loop connecting the D and E helices of the four helix bundle. Because red cell L ferritin and liver H ferritin show differences in both rates of mineralization and three-dimensional structure, more detailed comparisons of these structures are likely to shed new light on the relationship between conformation and function.

Animals↗
PubMed1994

Structure and function of IREs, the noncoding mRNA sequences regulating synthesis of ferritin, transferrin receptor and (erythroid) 5-aminolevulinate synthase.

The synthesis of least three proteins involved in iron metabolism is coordinately regulated in animals through noncoding sequences in mRNA, the IREs; the transcription of the genes encoding the proteins are also regulated. Cellular iron is the best known effector of changes in regulation of mRNA with IREs. A hairpin loop is the secondary structure of IRES which conserve the hairpin loop sequence, CAGUGU/C. However, variable stem sequences, apparently related to mRNA-specific function, create a family of IRE regulatory sequences. At least three types of proteins recognize IRE regions: (1) Nucleases which degrade mRNAs with 3' noncoding IRES; the IRE/IRE-BP stabilizes mRNAs with 3' noncoding IRES (transferrin receptor mRNA). (2) Initiation factors/ribosomes; the IRE/IRE-BP blocks ribosome binding of mRNAs with 5' noncoding IREs (ferritin, eALAS mRNAs). (3) Initiation factors to enhance translation (ferritin mRNA) when the IRE-BP does not bind; the ferritin IRE is thus both a negative and positive control element depending on which type of protein is bound. The IRE in ferritin mRNA is the most studied IRE to date. Site-directed mutagenesis shows that sites throughout the IRE alter negative control and IRE-BP binding reflecting the fact that the footprint of the IRE-BP is over the entire IRE. Base paired flanking regions (FL) which are ferritin IRE specific, enhance the effects of IRE-BP binding on negative control. Positive control is altered by modifying the single sites in stem/internal loop but not in the hairpin loop.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Aminolevulinate Synthetase↗
PubMed1994

Posttranscriptional regulation of ferritin during nodule development in soybean.

During soybean (Glycine max) nodule development, induced ferritin mRNA concentration remains elevated while the protein concentration decreases 4- to 5-fold (M. Ragland and E.C. Theil [1993] Plant Mol Biol 21: 555-560). Investigation of posttranscriptional regulation of nodule ferritin during development showed that ferritin mRNA was efficiently translated based on polyribosome size in vivo, protein synthesis (0.8% of total protein) in vitro, and protein synthesis in intact nodules. Ferritin, a plastid protein, was processed in both immature and mature nodules. In chimeric mRNA, soybean ferritin mRNA sequences blocked the function of the iron regulatory element (IRE), the cis regulatory element of animal ferritin mRNA; the IRE regulates chimeric animal mRNAs. The absence of translational regulation of ferritin in plants contrasts with ferritin regulation in animals. Thus, ferritin regulation has diverged during evolution, whereas structure of the mature protein has been conserved. Ferritin in mature soybean nodules is apparently regulated after translation, possibly in analogy with such plastid proteins as chlorophyll-binding proteins D1, CP43, LHCI, and LHCII, the small subunit of ribulose-bisphosphate carboxylase, and apoplastocyanin. An autocatalytic mechanism observed in vivo for degradation of plastid protein D1 and in vitro for pea ferritin during iron release could explain the ferritin decreases in mature nodules.

Animals↗
PubMed1993

Formation of iron(III)-tyrosinate is the fastest reaction observed in ferritin.

Rapid mineralization of ferritin, characteristic of protein with H-type subunits, coincides with formation of a specific Fe(III)-tyrosinate complex. The pseudo-first-order rate constant for Fe(II) oxidation by H-subunit-type ferritin has now been shown to be 700-900 times greater than any previously reported for ferritin; kox = 1000 s-1 for formation of the specific Fe(III)-tyrosinate complex (A550nm) or formation of less defined Fe(III)-oxo multinuclear complexes (A420nm). Formation of multinuclear Fe(III)-oxo complexes and O2 consumption were biphasic. In the first phase, up to 50 Fe atoms/ferritin molecule were rapidly oxidized, accompanied by formation of the Fe(III)-tyrosinate complex; saturation of the sites which formed the Fe(III)-tyrosinate complex also required 50 Fe/ferritin molecule. The sigmoidal shape of the curve obtained by plotting the initial rate of oxidation during the rapid phase of mineralization versus added [Fe(II)] suggested a more complex reaction pathway of ferroxidation than previously described. During the second phase of mineralization, Fe(III)-tyrosinate decreased, but multinuclear Fe(III)-oxo complexes and O2 consumption continued to increase at a slower rate. Recovery of the rapid oxidation pathway paralleled recovery of the site for Fe(III)-tyrosinate formation; full regeneration of the Fe(III)-tyrosinate sites was gradual over a period of 12 h, as if the movement of Fe(III) along the biomineralization pathway in the protein was slow and was accompanied by conformational changes which affected the Fe(III)-tyrosinate site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The influence of the base-paired flanking region on structure and function of the ferritin mRNA iron regulatory element.

Ferritin and transferrin receptors are co-ordinately regulated by the same RNA-protein interaction: the conserved iron regulatory element (IRE) in mRNA and the IRE-binding protein (IRE-BP/IRP/FRP/P-90). The 28 nucleotide IRE in ferritin mRNA is a single copy, with base-paired flanking regions (FL), located near the 5' cap. In the transferrin receptor mRNA, the IRE is located in the 3' untranslated region, as five variable copies and lacking predicted base-paired flanking regions; an alternate predicted structure without IREs has similar stability. When iron is scarce, ferritin mRNA does not form polyribosomes whereas the transferrin receptor mRNA is translated; when iron is abundant, ferritin mRNA forms polyribosomes and the transferrin receptor mRNA is degraded. To investigate structures which contribute to differences in the regulation of the two mRNAs, the effect of mutation of the ferritin FL was studied. Changes in structure (changes in reactivity with RNase V1 and RNase S1. Fe-bleomycin) and changes in function (translation in rabbit reticulocyte extracts) were compared for mutant and wild-type FL sequences in ferritin mRNA. The disruption of a triplet of base-pairs in the FL had diminished regulation; a second mutation to restore the triplet base-pairs conferred wild-type translational regulation. Conformation of the mutant RNA-IRE-BP complex was also different. We show that the triplet of base-pairs is conserved; the triplet is also the location of IRE-BP-dependent conformational changes in the FL structure previously observed. Increasing FL base-pairs had no effect on function. Structural changes associated with altered function included bleomycin sites in the IRE, suggesting an alternate conformation of the hairpin, and different base-stacking (V1 sensitivity) in the FL. The function of the FL, which is altered by mutation of phylogenetically conserved triplet base-pairs, may be enhancement of formation of a particular IRE stem-loop-protein interaction.

Animals↗
PubMed1993

Formation of an Fe(III)-tyrosinate complex during biomineralization of H-subunit ferritin.

An iron(III)-tyrosinate complex was identified in ferritin by ultraviolet-visible and resonance Raman spectroscopies. Previously, a specific amino acid side chain coordinated to iron in ferritin was not known. Ferritin protein was overexpressed in Escherichia coli from complementary DNA sequences of bullfrog red cell ferritin. The purple iron(III)-tyrosinate intermediate that formed during the first stages of iron uptake was replaced by the amber multinuclear iron(III)-oxo complexes of fully mineralized ferritin. Only the H subunit formed detectable amounts of the iron(III)-tyrosinate complex, which may explain the faster rates of iron biomineralization in H- compared to L-type ferritin.

Amino Acid Sequence↗
PubMed1993

Ferritin (mRNA, protein) and iron concentrations during soybean nodule development.

To study how iron-rich nodules concentrate and store iron, ferritin (mRNA, protein) was analyzed in developing soybean nodules and compared to nitrogenase (mRNA/activity) and leghemoglobin (mRNA, protein, heme). Both ferritin mRNA and protein concentrations increased early in nodulation. Later in nodulation ferritin protein declined, in contrast to the mRNA, as nitrogenase (mRNA and activity) increased and leghemoglobin (mRNA and protein) accumulated. A precursor/product relationship between iron stored in ferritin and iron in nitrogenase or leghemoglobin is suggested. The uncoordinated changes in ferritin mRNA and protein during nodulation contrast with nitrogenase mRNA and nitrogenase activity suggesting possible translational and posttranscriptional effects on ferritin expression.

Ferritins↗
PubMed1993

The IRE (iron regulatory element) family: structures which regulate mRNA translation or stability.

Iron regulatory elements (IREs) are a family of 28 nucleotide, non-coding elements which regulate the translation of ferritin mRNA (iron storage), erythroid delta-aminolevulinic acid synthase mRNA (heme synthesis) and the stability of the transferrin receptor (TfR) mRNA (iron uptake). IREs in the 5' end control translation (ribosome binding) and IREs in the 3' end control turnover (degradation). The specific regulator protein, the IRE-BP, is a member of the aconitase family but binds RNA only in the apo form without the Fe-S cluster. Cellular iron alters the IRE/IRE-BP interaction leading to translation of ferritin and eALAS mRNAs but degradation of the TfR mRNA. IRE function requires proximity to the 5' cap, achieved either by a short leader (eALAS) or a long, base-pairing flanking region (FL) (ferritin); a conserved triplet of FL base pairs enhances repression of ferritin mRNA. TfR mRNA has five AU-rich IREs which can also form an alternate structure with inter-IRE base pairs, in the absence of the IRE-BP. Ferritin IREs regulate both translation repression (negative control-IRE-BP dependent) and enhancement (positive control-initiation factor dependent); IRE-BP binding induces conformational changes in the FL. IREs use CAGUGU/C to form a hairpin loop with specific variations in the stem such as internal or bulge loops. A current structural model obtained using metallonucleases (1,10-phenanthroline-Cu, Fe-EDTA, Fe-bleomycin) and a preliminary analysis of the NMR spectrum, is a distorted helix with folds. The effect of cellular iron, Fe-S clusters and heme on the IRE-BP/RNA is not completely understood.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

A possible role for the conserved trimer interface of ferritin in iron incorporation.

Ferritin is a large protein, highly conserved among higher eukaryotes, which reversibly stores iron as a mineral of hydrated ferric oxide. Twenty-four polypeptides assemble to form a hollow coat with the mineral inside. Multiple steps occur in iron core formation. First, Fe2+ enters the protein. Then, several alternate paths may be followed which include oxidation at site(s) on the protein, oxidation on the core surface, and mineralization. Sequence variations occur among ferritin subunits which are classified as H or L; Fe2+ oxidation at sites on the protein appears to be H-subunit-specific or protein-specific. Other steps of ferritin core formation are likely to involve conserved sites in ferritins. Since incorporation of Fe2+ into the protein must precede any of the other steps in core formation, it may involve sites conserved among the various ferritin proteins. In this study, accessibility of Fe2+ to 1,10-phenanthroline, previously shown to be inaccessible to Fe2+ inside ferritin, was used to measure Fe2+ incorporation in two different ferritins under various conditions. Horse spleen ferritin (L/H = 10-20:1) and sheep spleen ferritin (L/H = 1:1.6) were compared. The results showed that iron incorporation measured as inaccessibility of Fe2+ to 1,10-phenanthroline increased with pH. The effect was the same for both proteins, indicating that a step in iron core formation common among ferritins was being measured. Conserved sites previously proposed for different steps in ferritin core formation are at the interfaces of pairs and trios of subunits. Dinitrophenol cross-links, which modify pairs of subunits and affect iron oxidation, had no effect on Fe2+ incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The iron regulatory region of ferritin mRNA is also a positive control element for iron-independent translation.

The iron regulatory element (IRE) in the 5'-untranslated region of ferritin mRNA interacts with a specific regulator protein (P-90, IRE-BP, or FRP) to block translation. High cellular iron changes the IRE/P-90 interaction to relax the translational block and allow polyribosome formation. We now show that the IRE and base-paired flanking regions also enhance translation in the absence of P-90, explaining the high translational efficiency of deregulated ferritin mRNA observed previously. The effect of the IRE on translational efficiency was examined by comparing four sets of mRNAs: (1) +/- IRE in animal (frog) ferritin, regulated translationally by iron in vivo; (2) +/- animal IRE fused with plant (soybean) ferritin, regulated transcriptionally by iron in vivo; (3) repositioned IRE in animal ferritin; (4) mutated IRE in animal ferritin with G16A substitution, which decreases P-90 binding (negative control). The IRE region increased translational efficiency of both the animal ferritin and the heterologous IRE/soybean ferritin fusion mRNAs; the effect was observed in cell-free translation systems from either plants (wheat germ) or animals (rabbit reticulocyte). Repositioning the IRE further from the 5' cap eliminated positive control of translation. The single base mutation had no effect, indicating that positive and negative translational control involves different sections of the IRE region. Thus, the IRE region in ferritin mRNA encodes both positive translational control and, when combined with the regulator protein P-90, negative translational control.

Animals↗