Expression, purification and preliminary crystallographic analysis of bacterial transmembrane protein EfeU for iron import.

The bacterial Efe system functions as an importer of free Fe2+ into cells independently of iron-chelating compounds such as siderophores and consisted of iron-binding protein EfeO, peroxidase EfeB, and transmembrane permease EfeU. While we and other researchers reported crystal structures of EfeO and EfeB, that of EfeU remains undetermined. In this study, we constructed expression system of EfeU derived from Escherichia coli, selected E. coli Rosetta-gami 2 (DE3) as an expression host, and succeeded in purification of the proteins which were indicated to form an oligomer by blue native PAGE. We obtained preliminary data of the X-ray crystallography, suggesting that expression and purification methods we established in this study enable structural analysis of the bacterial Efe system.

Sample preparation of blood plasma enables baseline separation of iron metalloproteins by SEC-GFAAS.

The analysis of human plasma for biomarkers holds promise to revolutionize disease diagnosis, but is hampered by the inherent complexity of the plasma proteome. One way to overcome this problem is to analyze plasma for a sub-proteome, such as the metalloproteome. Previous studies employing size-exclusion chromatography (SEC) coupled on-line to an inductively coupled plasma-atomic emission spectrometer (ICP-AES) have revealed that plasma contains ~12 copper, iron and zinc metalloproteins. This included the iron metalloproteins transferrin (Tf) and a recently identified haptoglobin-hemoglobin (Hp-Hb) complex, which is formed in plasma when red blood cells rupture. Since this SEC-ICP-AES method required a sample volume of 500 µL to generate diagnostically useful results, we sought to develop an alternative SEC-based hyphenated approach using a smaller SEC column (150 × 5 mm I.D.) and a graphite furnace atomic absorption spectrometer (GFAAS) as the iron-specific detector. A designed interface enabled the integration of the SEC system with the GFAAS. Baseline separation between the Hp-Hb complex and Tf was achieved by developing a sample preparation procedure which involved the chelating agent-based mobilization of Fe from Tf to a small molecular weight Fe complex. Spiking of human plasma (1.0 mL) with red blood cell lysate (1-2 µL) increased only the intensity of the Fe peak corresponding to the Hp-Hb complex, but not that of Tf. Since the developed SEC-GFAAS method requires only 50 µL of plasma for analysis, it can now be employed for the cost-effective quantification of the clinically relevant Hb-Hp complex in human plasma in <50 min.

Physiologically relevant reconstitution of iron-sulfur cluster biosynthesis uncovers persulfide-processing functions of ferredoxin-2 and frataxin.

Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich's ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich's ataxia therapies.

Structure of the human frataxin-bound iron-sulfur cluster assembly complex provides insight into its activation mechanism.

The core machinery for de novo biosynthesis of iron-sulfur clusters (ISC), located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich's ataxia (FRDA). Here the 3.2 Å resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates the interactions of FXN with other component proteins of the complex. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure reveals how FXN facilitates ISC production through stabilizing key loop conformations of NFS1 and ISCU at the protein-protein interfaces, and suggests how FRDA clinical mutations affect complex formation and FXN activation.

Purification of recombinant human thyroid peroxidase (hTPO) from AD293 mammalian cells.

Human thyroid peroxidase (hTPO) has been secretory expressed in AD293 mammalian cells. cDNA sequence of 'Gluc' (Gaussia luciferase) protein from Gaussia princeps was incorporated at the amino terminal of hTPO gene for secretion of targeted protein outside the mammalian cells. Augmentation of TPO clone in serum free mediums was investigated and a simplified purification procedure of hTPO has been reported here. Purified hTPO was further analyzed by SDS-PAGE and immunoblotting (western blotting). The relative molecular mass of hTPO was found to be 105kDa. This is the first report with respect to cost effective and simplified purification approach to get highest yield and purity of recombinant hTPO.

Characterization of the iron-binding properties of pyoverdine using electron-capture dissociation-tandem mass spectrometry.

Pyoverdines (PVD) are a group of siderophores produced by fluorescent Pseudomonads. Identification of PVD variants mostly relies on liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID). Here, both CID and the novel dissociation technique electron-capture dissociation (ECD) were applied to characterize PVD succinamide and its Fe(III)-chelated complex. The results clearly showed that ECD produced diagnostic side chain fragmentation of the PVD peptide chain and preserved the labile Fe(III) binding to the chromophore in contrast to CID. The ECD technique is therefore expected to support the understanding of strain-specific Fe(III) transport processes of PVDs.

Interaction of frataxin, an iron binding protein, with IscU of Fe-S clusters biogenesis pathway and its upregulation in AmpB resistant Leishmania donovani.

Leishmania donovani is a unicellular protozoon parasite that causes visceral leishmaniasis (VL), which is a fatal disease if left untreated. Certain Fe-S proteins of the TCA cycle and respiratory chain have been found in the Leishmania parasite but the precise mechanisms for their biogenesis and the maturation of Fe-S clusters remains unknown. Fe-S clusters are ubiquitous cofactors of proteins that perform critical cellular functions. The clusters are biosynthesized by the mitochondrial Iron-Sulphur Cluster (ISC) machinery with core protein components that include the catalytic cysteine desulphurase IscS, the scaffold proteins IscU and IscA, and frataxin as an iron carrier/donor. However, no information regarding frataxin, its regulation, or its role in drug resistance is available for the Leishmania parasite. In this study, we characterized Ld-frataxin to investigate its role in the ISC machinery of L. donovani. We expressed and purified the recombinant Ld-frataxin protein and observed its interaction with Ld-IscU by co-purification and pull-down assay. Furthermore, we observed that the cysteine desulphurase activity of the purified Ld-IscS protein was stimulated in the presence of Ld-frataxin and Ld-IscU, particularly in the presence of iron; neither Ld-frataxin nor Ld-IscU alone had significant effects on Ld-IscS activity. Interestingly, RT-PCR and western blotting showed that Ld-frataxin is upregulated in AmpB-resistant isolates compared to sensitive strains, which may support higher Fe-S protein activity in AmpB-resistant L. donovani. Additionally, Ld-frataxin was localized in the mitochondria, as revealed by digitonin fractionation and indirect immunofluorescence. Thus, our results suggest the role of Ld-frataxin as an iron binding/carrier protein for Fe-S cluster biogenesis that physically interacts with other core components of the ISC machinery within the mitochondria.

Iron-binding properties of sugar cane yeast peptides.

The extract of sugar-cane yeast (Saccharomyces cerevisiae) was enzymatically hydrolysed by Alcalase, Protex or Viscozyme. Hydrolysates were fractionated using a membrane ultrafiltration system and peptides smaller than 5kDa were evaluated for iron chelating ability through measurements of iron solubility, binding capacity and dialyzability. Iron-chelating peptides were isolated using immobilized metal affinity chromatography (IMAC). They showed higher content of His, Lys, and Arg than the original hydrolysates. In spite of poor iron solubility, hydrolysates of Viscozyme provided higher iron dialyzability than those of other enzymes. This means that more chelates of iron or complexes were formed and these kept the iron stable during simulated gastro-intestinal digestion in vitro, improving its dialyzability.

Effect of artemin on structural transition of ß-lactoglobulin.

Encysted embryos of Artemia are exceptionally resistant to severe environmental stress. This resistance is thought to depend in part on the existence of a protein termed artemin. There is only little information about the function of artemin. It has been reported artemin is a thermostable protein with RNA-binding ability. In addition, it reduces the extent of aggregation significantly and enhances the efficiency of refolding and activity recovery of carbonic anhydrase and horseradish peroxidase. In this study, the effect of artemin purified from Artemia urmiana on bovine ß-lactoglobulin (BLG) and its α-helical intermediate state has been evaluated by circular dichroism and intrinsic and extrinsic fluorescence spectroscopy. The results obtained in aqueous buffer show, artemin decreases the compactness of BLG structure and causes to the exposure of some hydrophobic groups. The results also indicate artemin has an inhibitory effect on ß-sheet→α-helix transition in the secondary structure of ß-lactoglobulin. Since this transition occurs during unfolding of ß-lactoglobulin, it seems artemin influences on the folding pathway of ß-lactoglobulin. This structural effect of artemin can result from its high surface hydrophobicity. Consequently, it is expected that artemin has chaperoning potency because of its effect on the folding of BLG.

Zirconia nanoparticles-coated column for the capillary electrochromatographic separation of iron-binding- and phosphorylated-proteins.

A ZrO(2) nanoparticles (ZrO(2)NPs)-coated column was prepared through a sol-gel process using zirconium(iv) oxychloride, which reacted with silanol groups of the fused-silica capillary. The condensation reaction was carried out at 350 °C for 8 h. Electroosmotic flow (EOF) measurements and scanning electron microscopy (SEM) images were used to characterize the ZrO(2)NPs fabricated on the inner wall of the capillary. Below the pI value (pH 5-6), cathodic EOF elucidated that the phosphate buffer adsorbs tightly on the zirconia surface, resulting in a negatively charged surface. In this work, iron-binding proteins, phosphorylated proteins and glycoproteins were selected as the model compounds. The effects of pH, concentration, buffer type and the organic modifier were studied to optimize the separation efficiency. Iron-binding proteins exhibited a retention time for myoglobin (Mb) < hemoglobin (Hb), which corresponded to the binding constants for ZrO(2)NPs. The α- and ß-subunit of Hb could be separated in borate buffer (20 mM, pH 9.0) with MeOH (20%, v/v). Greater affinity of α-casein and bovine serum albumin (BSA) for the stationary phase as the pH decreased was found by comparison with that of conalbumin (ConA) and transferrin (Tf). Interestingly, 14 peaks for glycoisoforms of ovalbumin (OVA) were observed using borate buffer (40 mM, pH 9.0). The established method was also applied to the determination of analytes in the egg whites of chicken and duck eggs.

Cloning, purification, crystallization and X-ray crystallographic analysis of Ignicoccus hospitalis neelaredoxin.

Superoxide reductases (SORs) are metalloproteins which constitute the most recently identified oxygen-detoxification system in anaerobic and microaerobic bacteria and archaea. SORs are involved in scavenging superoxide radicals from the cell by catalyzing the reduction of superoxide ({\rm O}_{2};{\bullet -}) to hydrogen peroxide and are characterized by a catalytic nonhaem iron centre coordinated by four histidine ligands and one cysteine ligand. Ignicoccus hospitalis, a hyperthermophilic crenarchaeon, is known to have a neelaredoxin-type SOR that keeps toxic oxygen species levels under control. Blue crystals of recombinant I. hospitalis oxidized neelaredoxin (14.1 kDa, 124 residues) were obtained. These crystals diffracted to 2.4 A resolution in-house at room temperature and belonged to the hexagonal space group P6(2)22 or P6(4)22, with unit-cell parameters a = b = 108, c = 64 A. Cell-content analysis indicated the presence of one monomer in the asymmetric unit.

Purification, crystallization and X-ray crystallographic analysis of Archaeoglobus fulgidus neelaredoxin.

Neelaredoxins are a type of superoxide reductase (SOR), which are blue 14 kDa metalloproteins with a catalytic nonhaem iron centre coordinated by four histidines and one cysteine in the ferrous form. Anaerobic organisms such as Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing archaeon, have developed defence mechanisms against toxic oxygen species in which superoxide reductases play a key role. SOR is responsible for scavenging toxic superoxide anion radicals (O(2)(*-)), catalysing the one-electron reduction of superoxide to hydrogen peroxide. Crystals of recombinant A. fulgidus neelaredoxin in the oxidized form (13.7 kDa, 125 residues) were obtained using polyethylene glycol and ammonium sulfate. These crystals diffracted to 1.9 A resolution and belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 75.72, c = 185.44 A. Cell-content analysis indicated the presence of a tetramer in the asymmetric unit, with a Matthews coefficient (V(M)) of 2.36 A(3) Da(-1) and an estimated solvent content of 48%. The three-dimensional structure was determined by the MAD method and is currently under refinement.

Lunasin peptide purified from Solanum nigrum L. protects DNA from oxidative damage by suppressing the generation of hydroxyl radical via blocking fenton reaction.

Oxidative DNA damage is the most critical factor implicated in carcinogenesis and other disorders. However, the protective effects of lunasin against oxidative DNA damage have not yet reported. In this study, we report here the protective effect of lunasin purified from Solanum nigrum L. against oxidative DNA. Lunasin protected DNA from the oxidative damage induced by Fe(2+) ion and hydroxyl radical. To better understand the mechanism for the protective effect of lunasin against DNA damage, the abilities to chelate Fe(2+), scavenge the generated hydroxyl radical and block the generation of hydroxyl radical were evaluated. Although it did not scavenge generated hydroxyl radical, lunasin blocked the generation of hydroxyl radical by chelating Fe(2+) ion. We conclude that lunasin protects DNA from oxidation by blocking fenton reaction between Fe(2+) and H(2)O(2) by chelating Fe(2+) and that consumption of lunasin may play an important role in the chemoprevention for the oxidative carcinogenesis.

Ga3+ as a mechanistic probe in Fe3+ transport: characterization of Ga3+ interaction with FbpA.

The obligate human pathogens Haemophilus influenzae, Neisseria gonorrhoeae, and N. meningitidis utilize a highly conserved, three-protein ATP-binding cassette transporter (FbpABC) to shuttle free Fe(3+) from the periplasm and across the cytoplasmic membrane. The periplasmic binding protein, ferric binding protein (FbpA), is capable of transporting other trivalent cations, including Ga(3+), which, unlike Fe(3+), is not redox-active. Because of a similar size and charge as Fe(3+), Ga(3+) is widely used as a non-redox-active Fe(3+) substitute for studying metal complexation in proteins and bacterial populations. The investigations reported here elucidate the similarities and differences in FbpA sequestration of Ga(3+) and Fe(3+), focusing on metal selectivity and the resulting transport function. The thermodynamic binding constant for Ga(3+) complexed with FbpA at pH 6.5, in 50 mM 4-morpholineethanesulfonic acid, 200 mM KCl, 5 mM KH(2)PO(4) was determined by UV-difference spectroscopy as log K'eff=13.7+/-0.6. This represents a 10(5)-fold weaker binding relative to Fe(3+) at identical conditions. The unfolding/refolding behavior of Ga(3+) and Fe(3+) holo-FbpA were also studied using a matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy technique, stability of unpurified proteins from rates of H/D exchange (SUPREX). This analysis indicates significant differences between Fe(3+) and Ga(3+) sequestration with regard to protein folding behavior. A series of kinetic experiments established the lability of the Ga(3+)FbpA-PO(4) assembly, and the similarities/differences of stepwise loading of Fe(3+) into apo- or Ga(3+)-loaded FbpA. These biophysical characterization data are used to interpret FbpA-mediated Ga(3+) transport and toxicity in cell culture studies.

A novel role for thioredoxin reductase in the iron metabolism of S. cerevisiae.

Intracellular levels of iron are tightly regulated. Saccharomyces cerevisiae uses well-defined pathways to extract iron molecules from the environment. Once inside the cell, the iron molecules must be transferred to target sites via an intracellular iron transporter. Although analogous carriers have been described for other metals, such as copper, an iron transporter has yet to be identified. We used two-dimensional gel electrophoresis and mass spectrometry techniques to attempt to identify the iron transporter from cytosolic fraction of S. cerevisiae. In this study, we identified the iron-binding activity of thioredoxin reductase, and our data suggest a potential role for this enzyme in intracellular iron transport.

Binding of yeast frataxin to the scaffold for Fe-S cluster biogenesis, Isu.

Friedreich ataxia is caused by reduced activity of frataxin, a conserved iron-binding protein of the mitochondrial matrix, thought to supply iron for formation of Fe-S clusters on the scaffold protein Isu. Frataxin binds Isu in an iron-dependent manner in vitro. However, the biological relevance of this interaction and whether in vivo the interaction between frataxin and Isu is mediated by adaptor proteins is a matter of debate. Here, we report that alterations of conserved, surface-exposed residues of yeast frataxin, which have deleterious effects on cell growth, impair Fe-S cluster biogenesis and interaction with Isu while altering neither iron binding nor oligomerization. Our results support the idea that the surface of the beta-sheet, adjacent to the acidic, iron binding ridge, is important for interaction of Yfh1 with the Fe-S cluster scaffold and point to a critical role for frataxin in Fe-S cluster biogenesis.

Identification of an iron-responsive protein that is antigenic in patients with Chlamydia trachomatis genital infections.

Chlamydia trachomatis is an important cause of immune-mediated damage to the reproductive tract of infected patients. Certain chlamydial antigens and host genetic factors have been identified as contributing to immunopathological events, but a comprehensive understanding of specific components involved in destructive vs. protective immune responses to chlamydial infections is far from clear. In this study, it is shown that C. trachomatis-infected patients generate antibodies against an iron-responsive chlamydial protein, YtgA. The identity of YtgA was confirmed by mass spectrometry following two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. This finding underscores a necessity to examine patient sera samples to identify chlamydial antigens that are likely encountered and important to the immune response during human infections.

Expression and one-step purification of recombinant Arabidopsis thaliana frataxin homolog (AtFH).

Frataxin, a nuclear-encoded mitochondrial protein, has been proposed to participate in Fe-S cluster assembly, mitochondrial energy metabolism, respiration, and iron homeostasis. However, its precise function remains elusive. Frataxin is highly conserved in living organisms with no major structural changes, in particular at the C-terminal protein domain, suggesting that it plays a key function in all organisms. Recently, a plant gene, AtFH, with significant homology to other members of the frataxin family has been described. To gain insight on the frataxin role in plants, the frataxin domain was expressed in Escherichia coli BL21-codonPlus (DE3)-RIL cells and purified using a Ni-chelating column. The purified protein, added to a mixture containing Fe(II) and H2O2, attenuates the Fenton reaction indicating that the recombinant plant frataxin is functional. The procedure described here produced high yield of 99% pure protein through only one chromatographic step, suitable for further structure-function studies.

Transport of divalent transition-metal ions is lost in small-intestinal tissue of b/b Belgrade rats.

Belgrade rats exhibit microcytic, hypochromic anemia and systemic iron deficiency due to a glycine-to-arginine mutation at residue 185 in a metal ion transporter of a divalent metal transporter/divalent cation transporter/solute carrier 11 group A member 2 or 3 (DMT1/DCT1/SLC11A2), a member of the natural-resistance-associated macrophage protein (Nramp) family. By use of rabbit duodenal tissue, a calcein fluorescence assay has previously been developed to assess transport of divalent metal ions across the small-intestinal brush border membrane (BBM). The assay was readily applied here to rat BBM to learn if it detects DMT1 activity. The results demonstrate protein-mediated transport across the BBM of all tested ions: Mn(2+), Fe(2+), and Ni(2+). Transport into BBM vesicles (BBMV) from (b/b) Belgrade rats was below the detection limit. BBMV of +/b origin had substantial activity. The kinetic rate constant for Ni(2+) membrane transport for +/b BBMV was within the range for normal rabbit tissue. Vesicles from +/b basolateral membranes (BLM) showed similar activity to BBMV while b/b BLM vesicles (BLMV) lacked transport activity. Immunoblots using isoform-specific antibodies demonstrated that intestinal levels of b/b DMT1 were increased compared to +/b DMT1, reflecting iron deficiency. Immunoblots on BBMV indicated that lack of activity in b/b vesicles was not due to a failure of DMT1 to localize to the BBMV; an excess of specific isoforms was present compared to +/b BBMV or duodenal extracts. Immunoblots from BLMV also exhibited enrichment in DMT1 isoforms, despite their distinct origin. Immunofluorescent staining of thin sections of b/b and +/b proximal intestines confirmed that DMT1 localized similarly in mutant and control enterocytes and showed that DMT1 isoforms have distinct distributions within intestinal tissue.

Increased DMT1 but not IREG1 or HFE mRNA following iron depletion therapy in hereditary haemochromatosis.

BACKGROUND AND AIMS: While upregulation of divalent metal transporter 1 (DMT1) and iron regulated gene 1 (IREG1) within duodenal enterocytes is reported in patients with hereditary haemochromatosis (HH), these findings are controversial. Furthermore, the effect of HFE, the gene mutated in HH, on expression of these molecules is unclear. This study examines duodenal expression of these three molecules in HH patients (prior to and following phlebotomy), in patients with iron deficiency (ID), and in controls. METHODS: DMT1, IREG1, and HFE mRNA were measured in duodenal tissue of C282Y homozygous HH patients, in ID patients negative for the C282Y mutation with a serum ferritin concentration less than 20 microg/l, and in controls negative for C282Y and H63D mutations with normal iron indices, using real time polymerase chain reaction. RESULTS: DMT1 and IREG1 mRNA levels were not significantly different in non-phlebotomised (untreated) HH patients compared with controls. DMT1 expression was significantly increased in HH patients who had undergone phlebotomy therapy (treated) and in patients with ID compared with controls. IREG1 was significantly increased in ID patients relative to controls, and while IREG1 expression was 1.8-fold greater in treated HH patients, this was not statistically significant. HFE mRNA expression was not significantly different in any of the groups investigated relative to controls. CONCLUSIONS: These findings demonstrate that untreated HH patients do not have increased duodenal DMT1 and IREG mRNA, but rather phlebotomy increases expression of these molecules, reflecting the effect of phlebotomy induced erythropoiesis. Finally, HFE appears to play a minor role in the regulation of iron absorption by the duodenal enterocyte.