Structure-function relationships in the ferritins.
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Amino Acid Sequence↗
Biomedical subjects
P M Harrison
Publications and source records attributed to P M Harrison.
N-acetylcysteine infusion in viral myocarditis: a case report.
N-acetylcysteine improves survival in established acute liver failure following paracetamol overdose by reducing the incidence of multiorgan failure. These benefits are thought to be related to decreased tissue hypoxia by the enhancement of both oxygen delivery and oxygen extraction. Similar findings have been recorded in critically ill patients from an alternative aetiology. The cardiovascular properties of N-acetylcysteine are to increase stroke volume index, and thus cardiac output, although there is no effect on cardiac output in normal subjects. N-acetylcysteine is known to improve myocardial contraction in a hamster model of chronic myocardial ischaemia, but such effects have not previously been described in humans. We report the beneficial circulatory effect of N-acetylcysteine in a patient with marked left ventricular dysfunction secondary to acute viral myocarditis.
Acetylcysteine↗
Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution.
Mammalian ferritins are 24-mers assembled from two types of polypeptide chain which provide the molecule with different functions. H(eavy) chains catalyse the first step in iron storage, the oxidation of iron(II). L(ight) chains promote the nucleation of the mineral ferrihydrite enabling storage of iron(III) inside the protein shell. We report here the comparison of the three-dimensional structures of recombinant human H chain (HuHF) and horse L chain (HoLF) ferritin homopolymers, which have been refined at 1.9 A resolution. There is 53% sequence identity between these molecules, and the two structures are very similar, the H and L subunit alpha-carbons superposing to within 0.5 A rms deviation with 41 water molecules in common. Nevertheless, there are significant important differences which can be related to differences in function. In particular, the centres of the four-helix bundles contain distinctive groups of hydrophilic residues which have been associated with ferroxidase activity in H chains and enhanced stability in L chains. L chains contain a group of glutamates associated with mineralisation within the iron storage cavity of the protein.
Amino Acid Sequence↗
Chemical synthesis, structural modeling, and biological activity of the epidermal growth factor-like domain of human cripto.
Cripto, also known as human teratocarcinoma-derived growth factor 1 (TDGF-1), contains a 40 amino acid region with some similarity to the epidermal growth factor (EGF) domain. However, sequence homology is largely restricted to the classical cysteine/glycine motif with only limited similarities in other regions. Significant differences to human EGF include the absence of all seven residues between the two N-terminal half-cystines and a five-residue shorter loop between the third and fourth half-cystines. We examine the hypothesis that, in spite of these differences, cripto can adopt the characteristic EGF-like 1-3, 2-4, 5-6 disulfide bond pattern. A comparative structural model of the growth factor cripto was constructed on the basis of its similarity to EGF, transforming growth factor alpha (TGF-alpha), and the EGF-like domain of human clotting factor IX. The predicted disulfide bridges and disulfide-bridged loops were analyzed and appear viable in the modeled structure. Moreover, to ascertain the importance of disulfide arrangement for cripto bioactivity, two 47-residue peptides were synthesized and then refolded using either a simple oxidative or a controlled sequential refolding protocol. The cripto peptides were tested for their ability to stimulate MAP-kinase activity, for inhibition of beta-casein induction, and for Shc phosphorylation in MDA-MB 453 human mammary carcinoma cells and HC-11 mouse mammary epithelial cells. Data suggest that cripto does adopt the 1-3, 2-4, 5-6 disulfide pattern and thus forms the classical EGF-like fold in spite of the significant deletions within the folding domain. The predicted structure of cripto shows some of the characteristics of both the ErbB1- and ErbB3/ErbB4-binding growth factors.
Amino Acid Sequence↗
Effects of modifications near the 2-, 3- and 4-fold symmetry axes on human ferritin renaturation.
Ferritin is a protein of 24 subunits which assemble into a shell with 432 point symmetry. It can be denatured reversibly in acidic guanidine hydrochloride, with the formation of poorly populated renaturation intermediates. In order to increase the accumulation of intermediates and to study the mechanism of ferritin renaturation, we analysed variants of the human ferritin H-chain altered at the N-terminus (delta(1-13)), near the 4-fold axis (Leu-169 --> Arg), the 3-fold axis (Asp-131 --> Ile + Glu-134 --> Phe) or the 2-fold axis (Ile-85 --> Cys). We also carried out specific chemical modifications of Cys-130 (near the 3-fold axis) and Cys-85 (near the 2-fold axis). Renaturation of the modified ferritins yielded assembly intermediates that differed in size and physical properties. Alterations of residues around the 2-, 4- and 3-fold axes produced subunit monomers, dimers and higher oligomers respectively. All these intermediates could be induced to assemble into ferritin 24-mers by concentrating them or by co-renaturing them with wild-type H-ferritin. The results support the hypothesis that the symmetric subunit dimers are the building blocks of ferritin assembly, and are consistent with a reassembly pathway involving the coalescence of dimers, probably around the 4-fold axis, followed by stepwise addition of dimers until the 24-mer cage is completed. In addition they show that assembly interactions are responsible for the large hysteresis of folding and unfolding plots. The implications of the studies for in vivo heteropolymer formation in vertebrates, which have two types of ferritin chain (H and L), are discussed.
Circular Dichroism↗
Dinuclear center of ferritin: studies of iron binding and oxidation show differences in the two iron sites.
The ferroxidase activity of human ferritin has previously been associated with a diiron site situated centrally within the four-helix bundle of H-type chains (HuHF). However, direct information about the site of Fe(II) binding has been lacking, and events between Fe(II) binding and its oxidation have not previously been studied. A sequential stopped-flow assay has now been developed to enable the dissection of binding and oxidation. It depends on the ability of 1,10-phenanthroline to complex protein-bound Fe(II) and to distinguish it from the more immediately available free Fe(II). This approach, aided by the use of site-directed variants, indicates that in HuHF and the non-heme ferritin of Escherichia coli the first 48 Fe(II) atoms/molecule added are bound and oxidized at the dinuclear centers. At a constant iron concentration, the rate of Fe(II) oxidation was maximal for additions of 2 Fe(II) atoms/subunit, consistent with a two-electron oxidation of the Fe(II) pair. Although, at low Fe(II)/protein ratios, no cooperativity in Fe(II) binding was observed; a preferred order of binding was deduced [Fe(II) binding first at site A and then at site B]. Binding of Fe(II) at both sites was essential for fast oxidation. Modification of site A ligands resulted in slow iron binding and slow oxidation. Modification of site B did not prevent Fe(II) binding at site A but greatly reduced its oxidation rate. These differences may mean that dioxygen is initially bound to Fe(II) at site B.
Binding Sites↗
Plasma levels and hepatic mRNA expression of transforming growth factor-beta1 in patients with fulminant hepatic failure.
BACKGROUND/AIMS: Transforming growth factor-beta1 is an important cytokine involved in cell growth and inflammation which has been shown to be inhibitory to hepatic DNA synthesis. The aim of this study was to investigate the plasma levels and hepatic mRNA expression of transforming growth factor-beta1 in patients with fulminant hepatic failure in whom liver regeneration may be impaired. METHODS: Plasma levels of transforming growth factor-beta1 and human hepatocyte growth factor were measured in 57 fulminant hepatic failure patients and 20 healthy volunteers by ELISA. Northern blot analysis of transforming growth factor-beta1 and H3 histone, a marker for liver proliferation, was performed in liver tissue of 14 fulminant hepatic failure patients. RESULTS: The plasma levels of total transforming growth factor-beta1 in fulminant hepatic failure patients on admission (median 38.8 ng/ml, range 8.4-108 ng/ml) were significantly higher than those in control subjects (23.0 ng/ml, 8.5-34.9 ng/ml, p<0.001). Significantly higher levels were observed in non-A, non-B hepatitis patients (57.9 ng/ml, 38.8-108 ng/ml, n=10, p<0.001) compared to patients with paracetamol overdose (37.1 ng/ml, 8.4-72.5 ng/ml, n=47). In contrast, the plasma levels of free transforming growth factor beta1 were greater in paracetamol overdose (623 pg/ml, 46.7-1241 pg/ml, n=21) than in non-A, non-B hepatitis (131 pg/ml, 77.2-254 pg/ml, n=9), with both being higher than control (72.3 pg/ml, 28.7-108, n=7, p<0.001). The plasma levels of human hepatocyte growth factor in patients with paracetamol overdose (7.04 ng/ml, 1.00-62.4 ng/ml) were significantly higher than those in patients with non-A, non-B hepatitis (4.48 ng/ml, 0.74-9.10 ng/ml, p<0.05). Northern blots showed increased mRNA expression of transforming growth factor-beta1 in paracetamol-overdose patients (n=8, p<0.05), but not in patients with non-A non-B hepatitis (n=6), compared to controls (n=4). CONCLUSIONS: The increased circulating plasma TGF-beta1 in FHF may be part of the tissue repair process in fulminant hepatic failure. In patients with non-A, non-B hepatitis, the increased total transforming growth factor-beta1 together with a less elevated hepatocyte growth factor could be related to impaired liver regeneration in this group.
Acetaminophen↗
The prion folding problem.
Prion diseases are neurodegenerative disorders in which dramatic conformational change in the structure of the prion protein is the fundamental event. This structural transition involves the loss of substantial alpha-helical content and the acquisition of beta-sheet structure. A convergence of recent biological and structural studies argues that the mechanism underlying the prion diseases is truly unprecedented.
Biopolymers↗
The disulphide beta-cross: from cystine geometry and clustering to classification of small disulphide-rich protein folds.
Small disulphide-rich protein folds (SDFs) tend to have less, regular secondary structure than larger protein folds and are thus problematic in protein structure taxonomy and prediction. We report regularities for disulphide-bridged beta-sheet and for cystine clustering that are particularly relevant to such proteins. The repertoire of cystine conformations results in preferences in disulphide distribution between/within beta-sheets. For example, disulphides seldom bridge between beta-sheets with antiparallel orientation for the flanking polypeptide segments, as the separations between packed sheets are such that the only rotamers that straddle them easily are those that generally require parallel orientation. A left-handed chirality preference is described for the intervening connection for disulphides bridging between berta-strands in different sheets in such a parallel orientation. Geometrical analysis of clusters of two cystine residues has shown that closely clustered cystine residues tend to have approximately orthogonal relative orientation. A positive orientation of this type is most often accommodated by a recurrent motif of disulphide-bridged beta-sheet that we call the disulphide beta-cross. The consensus features of this motif are described. It occurs in non-homologous proteins with a variety of folds, subsuming partial similarities previously noted by several other workers. Further examples are discussed, such as a two-cystine/two-beta-hairpin assembly common to hirudin and the three-fingered toxin folds. We suggest that the consensus features enable it to act as a good folding nucleus. We classify similar three-cystine arrangements that may be described as a ladder or stack, that tend to contain a disulphide beta-cross and that recur in folds that can otherwise be quite different. The preferences for disulphide-beta-sheet distribution and for cystine clusters contribute to an array of partial similarities for SDFs, many of which incorporate the disulphide beta-cross. It is suggested that SDF taxonomy cannot properly be considered without using both the relationship between clustered cystine residues and that between cystine residues and the regular secondary structures that they connect (here, we study beta in particular). The implications for SDF classification are demonstrated.
Cystine↗
The ferritins: molecular properties, iron storage function and cellular regulation.
The iron storage protein, ferritin, plays a key role in iron metabolism. Its ability to sequester the element gives ferritin the dual functions of iron detoxification and iron reserve. The importance of these functions is emphasised by ferritin's ubiquitous distribution among living species. Ferritin's three-dimensional structure is highly conserved. All ferritins have 24 protein subunits arranged in 432 symmetry to give a hollow shell with an 80 A diameter cavity capable of storing up to 4500 Fe(III) atoms as an inorganic complex. Subunits are folded as 4-helix bundles each having a fifth short helix at roughly 60 degrees to the bundle axis. Structural features of ferritins from humans, horse, bullfrog and bacteria are described: all have essentially the same architecture in spite of large variations in primary structure (amino acid sequence identities can be as low as 14%) and the presence in some bacterial ferritins of haem groups. Ferritin molecules isolated from vertebrates are composed of two types of subunit (H and L), whereas those from plants and bacteria contain only H-type chains, where 'H-type' is associated with the presence of centres catalysing the oxidation of two Fe(II) atoms. The similarity between the dinuclear iron centres of ferritin H-chains and those of ribonucleotide reductase and other proteins suggests a possible wider evolutionary linkage. A great deal of research effort is now concentrated on two aspects of ferritin: its functional mechanisms and its regulation. These form the major part of the review. Steps in iron storage within ferritin molecules consist of Fe(II) oxidation, Fe(III) migration and the nucleation and growth of the iron core mineral. H-chains are important for Fe(II) oxidation and L-chains assist in core formation. Iron mobilisation, relevant to ferritin's role as iron reserve, is also discussed. Translational regulation of mammalian ferritin synthesis in response to iron and the apparent links between iron and citrate metabolism through a single molecule with dual function are described. The molecule, when binding a [4Fe-4S] cluster, is a functioning (cytoplasmic) aconitase. When cellular iron is low, loss of the [4Fe-4S] cluster allows the molecule to bind to the 5'-untranslated region (5'-UTR) of the ferritin m-RNA and thus to repress translation. In this form it is known as the iron regulatory protein (IRP) and the stem-loop RNA structure to which it binds is the iron regulatory element (IRE). IREs are found in the 3'-UTR of the transferrin receptor and in the 5'-UTR of erythroid aminolaevulinic acid synthase, enabling tight co-ordination between cellular iron uptake and the synthesis of ferritin and haem. Degradation of ferritin could potentially lead to an increase in toxicity due to uncontrolled release of iron. Degradation within membrane-encapsulated "secondary lysosomes' may avoid this problem and this seems to be the origin of another form of storage iron known as haemosiderin. However, in certain pathological states, massive deposits of "haemosiderin' are found which do not arise directly from ferritin breakdown. Understanding the numerous inter-relationships between the various intracellular iron complexes presents a major challenge.
Animals↗
Identification of the ferroxidase centre of Escherichia coli bacterioferritin.
The bacterioferritin (BFR) of Escherichia coli takes up iron in the ferrous form and stores it within its central cavity as a hydrated ferric oxide mineral. The mechanism by which oxidation of iron (II) occurs in BFR is largely unknown, but previous studies indicated that there is ferroxidase activity associated with a site capable of forming a dinuclear-iron centre within each subunit [Le Brun, Wilson, Andrews, Harrison, Guest, Thomson and Moore (1993) FEBS Lett. 333, 197-202]. We now report site-directed mutagenesis experiments based on a putative dinuclear-metal-ion-binding site located within the BFR subunit. The data reveal that this dinuclear-iron centre is located at a site within the four-alpha-helical bundle of each subunit of BFR, thus identified as the ferroxidase centre of BFR. The metal-bound form of the centre bears a remarkable similarity to the dinuclear-iron sites of the hydroxylase subunit of methane mono-oxygenase and the R2 subunit of ribonucleotide reductase. Details of how the dinuclear centre of BFR is involved in the oxidation mechanism were investigated by studying the inhibition of iron (II) oxidation by zinc (II) ions. Data indicate that zinc (II) ions bind at the ferroxidase centre of apo-BFR in preference to iron (II), resulting in a dramatic reduction in the rate of oxidation. The mechanism of iron (II) oxidation is discussed in the light of this and previous work.
Amino Acid Sequence↗
Iron(II) oxidation by H chain ferritin: evidence from site-directed mutagenesis that a transient blue species is formed at the dinuclear iron center.
The iron storage molecule, ferritin, consists of an iron core surrounded by a shell of 24 protein subunits, which, in mammals, are of two types, H and L. Prior to storage of iron as a hydrous ferric oxide within the protein shell, Fe(II) is catalytically oxidized at dinuclear centers within H chains. When 48 Fe(II) atoms/molecule were added to 1 microM recombinant human H chain apoferritin (apo-HuHF), in 0.1 M Mes (pH 6.5), oxidation was 80% complete within about 0.2 s while 99% of the Fe(II) was oxidized within 10 s. A broad visible absorption band (400-800 nm, with a maximum at 650 nm) appeared during the fast phase of Fe(II) oxidation. It reached a plateau at 0.2-0.3 s and then declined while Fe(II) oxidation proceeded to completion and absorbance in the near-UV (300-400 nm) increased. The transient visible species was not observed when Tyr-34 was replaced by phenylalanine or when other conserved amino acids at the ferroxidase centers were substituted by residues which are unable to bind iron or which alter the charge balance. When a second increment of 48 iron atoms was added, 10 min after the first, the visible absorbance was absent and the rate of oxidation slower. Restoration of full oxidative activity took over 24 h. The data indicate that the fast oxidation of Fe(II) by apo-HuHF and the transient visible absorbance associated with it are due to Fe(II) oxidation at the ferroxidase centers.
Binding Sites↗
Site-directed replacement of the coaxial heme ligands of bacterioferritin generates heme-free variants.
The bacterioferritin (BFR) of Escherichia coli is a heme-containing iron storage molecule. It is composed of 24 identical subunits, which form a roughly spherical protein shell surrounding a central iron storage cavity. Each of the 12 heme moieties of BFR possesses bis-methionine axial ligation, a heme coordination scheme so far only found in bacterioferritins. Members of the BFR family contain three partially conserved methionine residues (excluding the initiating methionine) and in this study each was substituted by leucine and/or histidine. The Met52 variants were devoid of heme, whereas the Met31 and Met86 variants possessed full heme complements and were spectroscopically indistinguishable from wild-type BFR. The heme-free Met52 variants appeared to be correctly assembled and were capable of accumulating iron both in vivo and in vitro. No major differences were observed in the overall rate of iron accumulation for BFR-M52H, BFR-M52L, and the wild-type protein. The iron contents of the Met52 variants, as isolated, were at least 4 times greater than for wild-type BFR. This study is consistent with the reported location of the BFR heme site at the 2-fold axis and shows that heme is unnecessary for BFR assembly and iron uptake.
Amino Acid Sequence↗
Tyrosyl radical formation during the oxidative deposition of iron in human apoferritin.
The radical chemistry of ferritin is incompletely understood. The present study was undertaken to investigate the production of radicals in H-chain recombinant human ferritin (HuHF) and mixed H/L-chain horse spleen ferritin (HoSF) and the potential role of radicals in the oxidative deposition of iron in these proteins. Radical production follows distinct pathways for the two proteins; an intact H-chain ferroxidase site is required for radical generation in both of them, however. With the H-chain HuHF, an EPR spectrum characteristic of a tyrosyl radical is seen following Fe2+ oxidation by O2 and, based on measurements with site-directed variants, is suggested to arise from residue Tyr-34 located in the vicinity of the ferroxidase site. The observation of this radical correlates with the observation of a 400-600 nm absorbance seen in stopped-flow kinetics studies which seems to require the presence of Tyr-34 (Bauminger et al. (1993) Biochem. J. 296, 709-714). The data are inconsistent, however, with the Tyr-34 radical being critically important in the protein-catalyzed mechanism of iron oxidation. Unlike HuHF, the radicals observed in L-chain-rich HoSF appear to arise from hydroxyl radical damage to the protein through Fenton chemistry. These latter radicals also appear to be centered on aromatic amino acids and may be derived from histidine.
Animals↗
Ferritin does not accumulate iron oxidized by caeruloplasmin.
Ferritin is an iron-storage protein ubiquitous in mammals, plants and bacteria. It can be reconstituted, in vitro, from the apoprotein and Fe(II) salts in the presence of dissolved oxygen. Recently it has been reported that caeruloplasmin can facilitate apoferritin reconstitution and that iron oxidized by caeruloplasmin is sequestered within the ferritin shell. Here we show that the primary effect of adding caeruloplasmin to horse spleen ferritin during reconstitution is the competition between the two molecules for the iron. This competition results in overall increased rates of iron oxidation and a mixture of products, namely iron-containing ferritin and iron hydroxy polymers attached to caeruloplasmin. Iron oxidized by caeruloplasmin is not incorporated, to any significant extent, into horse spleen ferritin.
Animals↗
Analysis and classification of disulphide connectivity in proteins. The entropic effect of cross-linkage.
An analysis and a classification of protein disulphide connectivity in a set of distinct sequences are presented. We analyse the number of disulphides per sequence, the number of disulphides per residue and the length of disulphide cross-linked loops. Observed connectivities are classified according to the different possible types of arrangement. In addition, we classify disulphide connectivity by physical models describing the arrangement of multiple disulphides. Firstly, we consider whether the features of native connectivity arrangement are describable by the likelihood of diffusive contact in the unfolded state. This is referred to as the diffusion model, and was originated by Kauzmann. A second model, effectively the inverse of the diffusion model, describes native connectivity arrangement as dominated by the entropic stabilisation effect of cross-linkage. This is referred to as the entropic model. Additionally, we compare the distribution of disulphide cross-linked loops and of loops formed by disulphide-like contacts. For short sequences (less than about 75 residues) native connectivities tend to have entropically more-stabilising arrangement features, whilst for longer sequences (greater than about 200 residues) the diffusion model is appropriate. We introduce the concept of arrangement entropy as a measure of the complexity of a connectivity.
Disulfides↗
Direct observation of the iron binding sites in a ferritin.
X-Ray analysis of the ferritin of Escherichia coli (Ec-FTN) and of Ec-FTN crystals soaked in (NH4)2Fe(SO4)2 has revealed the presence of three iron-binding sites per subunit. Two of these form a di-iron site in the centre of the subunit as has been proposed for the 'ferroxidase centres' of human ferritin H chains. This di-iron site, lying within the 4-alpha-helix bundle, resemble those of ribonucleotide reductase, methane monoxygenase and haemerythrin. The third iron is bound by ligands unique to Ec-FTN on the inner surface of the protein shell. It is speculated that this state may represent the nucleation centre of a novel type of Fe(III) cluster, recently observed in Ec-FTN.
Bacterial Proteins↗
Serum nitrates as markers of postoperative morbidity.
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Biomarkers↗