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Prasannavadhana, A., Santosh Kumar, Prasad Thomas, Laxmi Narayan Sarangi, Santosh Kumar Gupta, Adyasha Priyadarshini, Viswas Konasagara Nagaleekar, and Vijendra Pal Singh. "Outer Membrane Proteome Analysis of Indian Strain ofPasteurella multocidaSerotype B:2 by MALDI-TOF/MS Analysis." Scientific World Journal 2014 (2014): 1–10. http://dx.doi.org/10.1155/2014/617034.
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Identification of outer membrane proteins (OMPs) is important to understand the bacteria structure and function, host-pathogen interaction, development of novel vaccine candidates, and diagnostic antigens. But till now the key antigens ofP. multocidaB:2 isolate causing haemorrhagic septicaemia (HS) in animals are not clearly defined. In this study, P52 strain ofP. multocidaserotype B:2 was grownin vitrounder iron-rich and iron-limited condition. The OMPs were extracted by sarkosyl method followed by SDS-PAGE and the proteins were identified by MALDI-TOF/MS analysis. In total, 22 proteins were identified, of which 7 were observed exclusively under iron-limited condition. Most of the high molecular weight proteins (TbpA, HgbA, HgbB, HasR, IroA, and HemR) identified in this study were involved in iron acquisition. Some hypothetical proteins (HP-KCU-10206, HP and AAUPMB 08244, HP AAUPMB 21592, HP AAUPMB 19766, AAUPMB 11295) were observed for the first time in this study which could be unique to serotype B:2. Further functionalin vivostudy of the proteins identified are required to explore the utility of these proteins in developing diagnostics and vaccine against HS.
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Azman, Adleen, Kalidasan Vasodavan, Narcisse Joseph, Suresh Kumar, Rukman A. Hamat, Syafinaz A. Nordin, Wan M. Aizat, Alex van Belkum, and Vasantha K. Neela. "Physiological and proteomic analysis of Stenotrophomonas maltophilia grown under the iron-limited condition." Future Microbiology 14, no. 16 (November 2019): 1417–28. http://dx.doi.org/10.2217/fmb-2019-0174.
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Aims: To study physiological and proteomic analysis of Stenotrophomonas maltophilia grown under iron-limited condition. Methods: One clinical and environmental S. maltophilia isolates grown under iron-depleted conditions were studied for siderophore production, ability to kill nematodes and alteration in protein expression using isobaric tags for relative and absolute quantification (ITRAQ). Results & conclusions: Siderophore production was observed in both clinical and environmental strains under iron-depleted conditions. Caenorhabditis elegans assay showed higher killing rate under iron-depleted (96%) compared with normal condition (76%). The proteins identified revealed, 96 proteins upregulated and 26 proteins downregulated for the two isolates under iron depletion. The upregulated proteins included several iron acquisition proteins, metabolic proteins and putative virulence proteins.
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Petrak, Jiri, Denisa Myslivcova, Petr Man, Radek Cmejla, Jana Cmejlova, and Daniel Vyoral. "Proteomic analysis of iron overload in human hepatoma cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 290, no. 5 (May 2006): G1059—G1066. http://dx.doi.org/10.1152/ajpgi.00469.2005.
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Iron-mediated organ damage is common in patients with iron overload diseases, namely, hereditary hemochromatosis. Massive iron deposition in parenchymal organs, particularly in the liver, causes organ dysfunction, fibrosis, cirrhosis, and also hepatocellular carcinoma. To obtain deeper insight into the poorly understood and complex cellular response to iron overload and consequent oxidative stress, we studied iron overload in liver-derived HepG2 cells. Human hepatoma HepG2 cells were exposed to a high concentration of iron for 3 days, and protein expression changes initiated by the iron overload were studied by two-dimensional electrophoresis and mass spectrometry. From a total of 1,060 spots observed, 21spots were differentially expressed by iron overload. We identified 19 of them; 11 identified proteins were upregulated, whereas 8 identified proteins showed a decline in response to iron overload. The differentially expressed proteins are involved in iron storage, stress response and protection against oxidative stress, protein folding, energy metabolism, gene expression, cell cycle regulation, and other processes. Many of these molecules have not been previously suggested to be involved in the response to iron overload and the consequent oxidative stress.
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Liu, MaFeng, Mi Huang, DeKang Zhu, MingShu Wang, RenYong Jia, Shun Chen, KunFeng Sun, et al. "Identifying the Genes Responsible for Iron-Limited Condition in Riemerella anatipestifer CH-1 through RNA-Seq-Based Analysis." BioMed Research International 2017 (2017): 1–10. http://dx.doi.org/10.1155/2017/8682057.
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One of the important elements for most bacterial growth is iron, the bioavailability of which is limited in hosts. Riemerella anatipestifer (R. anatipestifer, RA), an important duck pathogen, requires iron to live. However, the genes involved in iron metabolism and the mechanisms of iron transport are largely unknown. Here, we investigated the transcriptomic effects of iron limitation condition on R. anatipestifer CH-1 using the RNA-Seq and RNA-Seq-based analysis. Data analysis revealed genes encoding functions related to iron homeostasis, including a number of putative TonB-dependent receptor systems, a HmuY-like protein-dependent hemin (an iron-containing porphyrin) uptake system, a Feo system, a gene cluster related to starch utilization, and genes encoding hypothetical proteins that were significantly upregulated in response to iron limitation. Compared to the number of upregulated genes, more genes were significantly downregulated in response to iron limitation. The downregulated genes mainly encoded a number of outer membrane receptors, DNA-binding proteins, phage-related proteins, and many hypothetical proteins. This information suggested that RNA-Seq-based analysis in iron-limited medium is an effective and fast method for identifying genes involved in iron uptake in R. anatipestifer CH-1.
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Schryvers, Anthony B., and B. Craig Lee. "Analysis of bacterial receptors for host iron-binding proteins." Journal of Microbiological Methods 18, no. 3 (October 1993): 255–66. http://dx.doi.org/10.1016/0167-7012(93)90040-o.
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Cullen, Paul A., Stuart J. Cordwell, Dieter M. Bulach, David A. Haake, and Ben Adler. "Global Analysis of Outer Membrane Proteins from Leptospira interrogans Serovar Lai." Infection and Immunity 70, no. 5 (May 2002): 2311–18. http://dx.doi.org/10.1128/iai.70.5.2311-2318.2002.
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ABSTRACT Recombinant leptospiral outer membrane proteins (OMPs) can elicit immunity to leptospirosis in a hamster infection model. Previously characterized OMPs appear highly conserved, and thus their potential to stimulate heterologous immunity is of critical importance. In this study we undertook a global analysis of leptospiral OMPs, which were obtained by Triton X-114 extraction and phase partitioning. Outer membrane fractions were isolated from Leptospira interrogans serovar Lai grown at 20, 30, and 37°C with or without 10% fetal calf serum and, finally, in iron-depleted medium. The OMPs were separated by two-dimensional gel electrophoresis. Gel patterns from each of the five conditions were compared via image analysis, and 37 gel-purified proteins were tryptically digested and characterized by mass spectrometry (MS). Matrix-assisted laser desorption ionization-time-of-flight MS was used to rapidly identify leptospiral OMPs present in sequence databases. Proteins identified by this approach included the outer membrane lipoproteins LipL32, LipL36, LipL41, and LipL48. No known proteins from any cellular location other than the outer membrane were identified. Tandem electrospray MS was used to obtain peptide sequence information from eight novel proteins designated pL18, pL21, pL22, pL24, pL45, pL47/49, pL50, and pL55. The expression of LipL36 and pL50 was not apparent at temperatures above 30°C or under iron-depleted conditions. The expression of pL24 was also downregulated after iron depletion. The leptospiral major OMP LipL32 was observed to undergo substantial cleavage under all conditions except iron depletion. Additionally, significant downregulation of these mass forms was observed under iron limitation at 30°C, but not at 30°C alone, suggesting that LipL32 processing is dependent on iron-regulated extracellular proteases. However, separate cleavage products responded differently to changes in growth temperature and medium constituents, indicating that more than one process may be involved in LipL32 processing. Furthermore, under iron-depleted conditions there was no concomitant increase in the levels of the intact form of LipL32. The temperature- and iron-regulated expression of LipL36 and the iron-dependent cleavage of LipL32 were confirmed by immunoblotting with specific antisera. Global analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of Leptospira.
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Carey-Fung, Oscar, Jesse T. Beasley, and Alexander A. T. Johnson. "Annotation and Molecular Characterisation of the TaIRO3 and TaHRZ Iron Homeostasis Genes in Bread Wheat (Triticum aestivum L.)." Genes 12, no. 5 (April 27, 2021): 653. http://dx.doi.org/10.3390/genes12050653.
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Effective maintenance of plant iron (Fe) homoeostasis relies on a network of transcription factors (TFs) that respond to environmental conditions and regulate Fe uptake, translocation, and storage. The iron-related transcription factor 3 (IRO3), as well as haemerythrin motif-containing really interesting new gene (RING) protein and zinc finger protein (HRZ), are major regulators of Fe homeostasis in diploid species like Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.), but remain uncharacterised in hexaploid bread wheat (Triticum aestivum L.). In this study, we have identified, annotated, and characterised three TaIRO3 homoeologs and six TaHRZ1 and TaHRZ2 homoeologs in the bread wheat genome. Protein analysis revealed that TaIRO3 and TaHRZ proteins contain functionally conserved domains for DNA-binding, dimerisation, Fe binding, or polyubiquitination, and phylogenetic analysis revealed clustering of TaIRO3 and TaHRZ proteins with other monocot IRO3 and HRZ proteins, respectively. Quantitative reverse-transcription PCR analysis revealed that all TaIRO3 and TaHRZ homoeologs have unique tissue expression profiles and are upregulated in shoot tissues in response to Fe deficiency. After 24 h of Fe deficiency, the expression of TaHRZ homoeologs was upregulated, while the expression of TaIRO3 homoeologs was unchanged, suggesting that TaHRZ functions upstream of TaIRO3 in the wheat Fe homeostasis TF network.
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Kucera, Jiri, Eva Pakostova, Oldrich Janiczek, and Martin Mandl. "Changes in Acidithiobacillus ferrooxidans Ability to Reduce Ferric Iron by Elemental Sulfur." Advanced Materials Research 1130 (November 2015): 97–100. http://dx.doi.org/10.4028/www.scientific.net/amr.1130.97.
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Ferric iron may act as a thermodynamically favourable electron acceptor during elemental sulfur oxidation byAcidithiobacillus ferrooxidansin extremely acidic anoxic environments. A loss of anaerobic ferric iron reduction ability has been observed in ferrous iron-grownA. ferrooxidansCCM 4253 after aerobic passaging on elemental sulfur. In this study, iron-oxidising cells aerobically adapted from ferrous iron to elemental sulfur were still able to anaerobically reduce ferric iron, however, following aerobic passage on elemental sulfur it could not. Preliminary quantitative proteomic analysis of whole cell lysates of the passage that lost anaerobic ferric iron-reducing activity resulted in 150 repressed protein spots in comparison with the antecedent culture, which retained the activity. Identification of selected protein spots by tandem mass spectrometry revealed physiologically important proteins including rusticyanin and outer-membrane cytochrome Cyc2, which are involved in iron oxidation. Other proteins were associated with sulfur metabolism such as sulfide-quinone reductase and proteins encoded by the thiosulfate dehydrogenase and heterodisulfide reductase complex operons. Furthermore, proteomic analysis identified proteins directly related to anaerobiosis. The results indicate the importance of iron-oxidising system components for anaerobic sulfur oxidation in the studied microbial strain.
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Marchewka, Damian, Irena Roterman, Magdalena Strus, Klaudyna Śpiewak, and Grzegorz Majka. "Structural Analysis of the Lactoferrin Iron Binding Pockets." bams 8, no. 4 (December 2012): 351–59. http://dx.doi.org/10.1515/bams-2012-0024.
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ABSTRACT Lactoferrin (Lf) - member of the transferrin family of proteins responsible for many different functions in the body of mammals participates in regulation of free iron level in the body fluids making the protein bacteriostatic. The main goal of studies was to test the suitability of molecular dynamic simulation to study structural changes in the tertiary structure of lactoferrin. According to ConSurf Server analysis one of the most conservative amino acids was found not only in iron- but also in carbohydrates- binding pockets which may suggest a significant impact of carbohydrates on the functions performed by lactoferrin. Pocket-Finder program applied to find iron-binding pockets revealed the potential Fe binding area. The stability of the ligand deprived protein was verified performing the 50 ns dynamic simulation using the Gromacs program. The tertiary structure changes during the simulation were observed in N-lob solely. No structural changes were observed in C-lob iron-binding pocket.
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Khan, Mateen A. "Analysis of Ion and pH Effects on Iron Response Element (IRE) and mRNA-Iron Regulatory Protein (IRP1) Interactions." Current Chemical Biology 14, no. 2 (November 19, 2020): 88–99. http://dx.doi.org/10.2174/2212796814999200604121937.
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Background: Cellular iron uptake, utilization, and storage are tightly controlled through the action of iron regulatory proteins (IRPs). IRPs achieve this control by binding to IREs-mRNA in the 5'- or 3'-end of mRNAs that encode proteins involved in iron metabolism. The interaction of iron regulatory proteins with mRNAs containing an iron responsive element plays a central role in this regulation. The IRE RNA family of mRNA regulatory structures combines absolutely conserved protein binding sites with phylogenetically conserved base pairs that are specific to each IREs and influence RNA/protein stability. Our previous result revealed the binding and kinetics of IRE RNA with IRP1. The aim of the present study is to gain further insight into the differences in protein/RNA stability as a function of pH and ionic strength. Objective: To determine the extent to which the binding affinity and stability of protein/RNA complex was affected by ionic strength and pH. Methods: Fluorescence spectroscopy was used to characterize IRE RNA-IRP protein interaction. Results: Scatchard analysis revealed that the IRP1 protein binds to a single IRE RNA molecule. The binding affinity of two IRE RNA/IRP was significantly changed with the change in pH. The data suggests that the optimum binding of RNA/IRP complex occurred at pH 7.6. Dissociation constant for two IRE RNA/IRP increased with an increase in ionic strength, with a larger effect for FRT IRE RNA. This suggests that numerous electrostatic interactions occur in the ferritin IRE RNA/IRP than ACO2 IRE RNA/IRP complex. Iodide quenching shows that the majority of the tryptophan residues in IRP1 are solvent-accessible, assuming that most of the tryptophan residues contribute to protein fluorescence. Conclusion: The results obtained from this study clearly indicate that IRE RNA/IRP complex is destabilized by the change in pH and ionic strength. These observations suggest that both pH and ion are important for the assembly and stability of the IRE RNA/IRP complex formation.
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van Vliet, Arnoud H. M., Karl G. Wooldridge, and Julian M. Ketley. "Iron-Responsive Gene Regulation in a Campylobacter jejuni fur Mutant." Journal of Bacteriology 180, no. 20 (October 15, 1998): 5291–98. http://dx.doi.org/10.1128/jb.180.20.5291-5298.1998.
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ABSTRACT The expression of iron-regulated systems in gram-negative bacteria is generally controlled by the Fur protein, which represses the transcription of iron-regulated promoters by using Fe2+ as a cofactor. Mutational analysis of the Campylobacter jejuni fur gene was carried out by generation of a set of mutant copies of fur which had a kanamycin or chloramphenicol resistance gene introduced into the regions encoding the N and C termini of the Fur protein. The mutated genes were recombined into the C. jejuni NCTC 11168 chromosome, and putative mutants were confirmed by Southern hybridization. C. jejuni mutants were obtained only when the resistance genes were transcribed in the same orientation as the fur gene. The C. jejuni fur mutant grew slower than the parental strain. Comparison of protein profiles of fractionated C. jejuni cells grown in low- or high-iron medium indicated derepressed expression of three iron-regulated outer membrane proteins with molecular masses of 70, 75, and 80 kDa. Characterization by N-terminal amino acid sequencing showed the 75-kDa protein to be identical to CfrA, a Campylobacter colisiderophore receptor homologue, whereas the 70-kDa protein was identified as a new siderophore receptor homologue. Periplasmic fractions contained four derepressed proteins with molecular masses of 19, 29, 32, and 36 kDa. The 19-kDa protein has been previously identified, but its function is unknown. The cytoplasmic fraction contained two iron-repressed and two iron-induced proteins with molecular masses of 26, 55, 31, and 40 kDa, respectively. The two iron-repressed proteins have been previously identified as the oxidative stress defense proteins catalase (KatA) and alkyl hydroperoxide reductase (AhpC). AhpC and KatA were still iron regulated in the fur mutant, suggesting the presence of Fur-independent iron regulation. Further analysis of the C. jejuni iron and Fur regulons by using two-dimensional gel electrophoresis demonstrated the total number of iron- and Fur-regulated proteins to be lower than for other bacterial pathogens.
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Juniar, Linda, Vida Adlfar, Michael Hippler, Hideaki Tanaka, and Genji Kurisu. "Crystallographic analysis and phasing of iron-assimilating protein 1 (FEA1) from Chlamydomonas reinhardtii." Acta Crystallographica Section F Structural Biology Communications 77, no. 5 (April 28, 2021): 134–39. http://dx.doi.org/10.1107/s2053230x21003952.
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As an essential component of protein cofactors, iron is important for all photosynthetic organisms. In Chlamydomonas reinhardtii, iron levels are strictly controlled by proteins such as iron-assimilating protein 1 (FEA1). This periplasmic protein is expressed under conditions of iron deficiency, but its mechanisms of function remain unknown. Because FEA1 has no amino-acid similarity to protein structures in the Protein Data Bank, its crystal structure cannot be solved by molecular replacement. Here, recombinant FEA1 protein lacking the N-terminal signal sequence was successfully purified and crystals of apo FEA1 were obtained by hanging-drop vapor diffusion. Neither selenomethionine substitution nor heavy-atom derivatization was successful; therefore, the phase problem of FEA1 crystals was solved by the native sulfur SAD method using long-wavelength X-rays (2.7 Å). Laser-cutting technology was used to increase the signal-to-noise ratio and derive an initial structure. This study will lead to further structural studies of FEA1 to understand its function and its links to the iron-assimilation pathway.
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Tran, Phu V., Srikanth Dakoji, Kathryn H. Reise, Kathleen K. Storey, and Michael K. Georgieff. "Fetal iron deficiency alters the proteome of adult rat hippocampal synaptosomes." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 305, no. 11 (December 1, 2013): R1297—R1306. http://dx.doi.org/10.1152/ajpregu.00292.2013.
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Fetal and neonatal iron deficiency results in cognitive impairments in adulthood despite prompt postnatal iron replenishment. To systematically determine whether abnormal expression and localization of proteins that regulate adult synaptic efficacy are involved, we used a quantitative proteomic approach (isobaric tags for relative and absolute quantitation, iTRAQ) and pathway analysis to identify dysregulated proteins in hippocampal synapses of fetal iron deficiency model. Rat pups were made iron deficient (ID) from gestational day 2 through postnatal day (P) 7 by providing pregnant and nursing dams an ID diet (4 ppm Fe) after which they were rescued with an iron-sufficient diet (200 ppm Fe). This paradigm resulted in a 40% loss of brain iron at P15 with complete recovery by P56. Synaptosomes were prepared from hippocampi of the formerly iron-deficient (FID) and always iron-sufficient controls rats at P65 using a sucrose gradient method. Six replicates per group that underwent iTRAQ labeling and LC-MS/MS analysis for protein identification and comparison elucidated 331 differentially expressed proteins. Western analysis was used to confirm findings for selected proteins in the glutamate receptor signaling pathway, which regulates hippocampal synaptic plasticity, a cellular process critical for learning and memory. Bioinformatics were performed using knowledge-based Interactive Pathway Analysis. FID synaptosomes show altered expression of synaptic proteins-mediated cellular signalings, supporting persistent impacts of fetal iron deficiency on synaptic efficacy, which likely cause the cognitive dysfunction and neurobehavioral abnormalities. Importantly, the findings uncover previously unsuspected pathways, including neuronal nitric oxide synthase signaling, identifying additional mechanisms that may contribute to the long-term biobehavioral deficits.
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Moreno-Fernandez, Jorge, María J. M. Alférez, Inmaculada López-Aliaga, and Javier Díaz-Castro. "Role of Fermented Goat Milk on Liver Gene and Protein Profiles Related to Iron Metabolism during Anemia Recovery." Nutrients 12, no. 5 (May 8, 2020): 1336. http://dx.doi.org/10.3390/nu12051336.
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Despite the crucial role of the liver as the central regulator of iron homeostasis, no studies have directly tested the modulation of liver gene and protein expression patterns during iron deficiency instauration and recovery with fermented milks. Fermented goat milk consumption improves the key proteins of intestinal iron metabolism during iron deficiency recovery, enhancing the digestive and metabolic utilization of iron. The aim of this study was to assess the influence of fermented goat or cow milk consumption on liver iron homeostasis during iron-deficiency anemia recovery with normal or iron-overload diets. Analysis included iron status biomarkers, gene and protein expression in hepatocytes. In general, fermented goat milk consumption either with normal or high iron content up-regulated liver DMT1, FPN1 and FTL1 gene expression and DMT1 and FPN1 protein expression. However, HAMP mRNA expression was lower in all groups of animals fed fermented goat milk. Additionally, hepcidin protein expression decreased in control and anemic animals fed fermented goat milk with normal iron content. In conclusion, fermented goat milk potentiates the up-regulation of key genes coding for proteins involved in iron metabolism, such as DMT1, and FPN1, FTL1 and down-regulation of HAMP, playing a key role in enhanced iron repletion during anemia recovery, inducing a physiological adaptation of the liver key genes and proteins coordinated with the fluctuation of the cellular iron levels, favoring whole-body iron homeostasis.
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Burdo, Joseph R., Ian A. Simpson, Sharon Menzies, John Beard, and James R. Connor. "Regulation of the Profile of Iron-Management Proteins in Brain Microvasculature." Journal of Cerebral Blood Flow & Metabolism 24, no. 1 (January 2004): 67–74. http://dx.doi.org/10.1097/01.wcb.0000095800.98378.03.
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The distribution of brain iron is heterogeneous, but the mechanism by which these regional differences are achieved and maintained is unknown. In this study, the authors test two hypotheses related to brain iron transport. The first is that there is regional variability in the profile of proteins associated with iron transport and storage in the brain microvasculature. The second hypothesis is that the iron status of the brain will dictate the response of the protein profile in the microvasculature to changes in systemic iron status. The profile analysis consists of transferrin (iron transport), ferritin (iron storage), transferrin receptor (iron uptake), and divalent metal transporter 1 (release of iron from endosomes). An additional protein involved in cellular iron efflux, ferroportin, was not detected in brain microvasculature. The results show that there are significantly higher levels of these proteins in the microvasculature from each area of the brain compared to a whole brain homogenate, but no regional differences within the microvasculature. The levels of ferritin observed in the microvasculature indicate that the microvascular endothelial cells have significant iron storage capacity. There are no significant changes in the regional protein profiles in response to systemic iron manipulation when brain iron status was normal. In contrast, in Belgrade rats, whose brain is iron deficient, the expression of both divalent metal transporter 1 and transferrin receptor was increased compared with control in almost all brain regions examined, but not transferrin or ferritin. These findings indicate that regional brain iron heterogeneity is not maintained by differences in microvascular iron-management protein levels. The results also indicate that brain iron status dictates the response of the microvascular protein profile to systemic iron manipulation.
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Morrissey, Julie A., Alan Cockayne, Jane Hammacott, Keith Bishop, Amy Denman-Johnson, Philip J. Hill, and Paul Williams. "Conservation, Surface Exposure, and In Vivo Expression of the Frp Family of Iron-Regulated Cell Wall Proteins in Staphylococcus aureus." Infection and Immunity 70, no. 5 (May 2002): 2399–407. http://dx.doi.org/10.1128/iai.70.5.2399-2407.2002.
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ABSTRACT Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis identified two conserved, immunogenic Staphylococcus aureus cell wall proteins, of 40 and 87 kDa, expressed under iron-restricted growth conditions in vitro and in vivo. N-terminal sequencing and subsequent genome analysis showed that these proteins are encoded by adjacent monocistronic open reading frames designated frpA and frpB, respectively. Studies with an S. aureus fur mutant confirmed that expression of FrpA and FrpB is regulated by Fur but that there also appears to be differential expression of these proteins in different iron-restricted media in vitro. FrpA and FrpB share some amino acid sequence homology with each other and with a putative S. aureus membrane protein, FrpC. frpC is the first gene of a Fur-regulated operon encoding four proteins of unknown function (FrpC, -D, -G, and -H) and the binding protein (FrpE) and permease (FrpF) of a putative iron transporter. Antisense mutagenesis and bioassays showed that FrpA and FrpB are not required for growth of S. aureus under iron-restricted conditions in vitro and do not appear to be involved in the transport of iron from siderophores or in binding of hemin. Further phenotypic analysis suggested that FrpA may be involved in adhesion of S. aureus to plastic in vitro. Binding of S. aureus to microtiter wells was found to be iron regulated, and iron-restricted S. aureus containing antisense frpA or frpAB but not frpB constructs showed reduced binding compared to vector construct controls.
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Parker, Dane, Ruth M. Kennan, Garry S. Myers, Ian T. Paulsen, and Julian I. Rood. "Identification of a Dichelobacter nodosus Ferric Uptake Regulator and Determination of Its Regulatory Targets." Journal of Bacteriology 187, no. 1 (January 1, 2005): 366–75. http://dx.doi.org/10.1128/jb.187.1.366-375.2005.
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ABSTRACT The expression of iron regulated genes in bacteria is typically controlled by the ferric uptake regulator (Fur) protein, a global transcriptional repressor that regulates functions as diverse as iron acquisition, oxidative stress, and virulence. We have identified a fur homologue in Dichelobacter nodosus, the causative agent of ovine footrot, and shown that it complements an Escherichia coli fur mutant. Homology modeling of the D. nodosus Fur protein with the recently solved crystal structure of Fur from Pseudomonas aeruginosa indicated extensive structural conservation. As Southern hybridization analysis of different clinical isolates of D. nodosus indicated that the fur gene was present in all of these strains, the fur gene was insertionally inactivated to determine its functional role. Analysis of these mutants by various techniques did not indicate any significant differences in the expression of known virulence genes or in iron-dependent growth. However, we determined several Fur regulatory targets by two-dimensional gel electrophoresis coupled with mass spectrometry. Analysis of proteins from cytoplasmic, membrane, and extracellular fractions revealed numerous differentially expressed proteins. The transcriptional basis of these differences was analyzed by using quantitative reverse transcriptase PCR. Proteins with increased expression in the fur mutant were homologues of the periplasmic iron binding protein YfeA and a cobalt chelatase, CbiK. Down-regulated proteins included a putative manganese superoxide dismutase and ornithine decarboxylase. Based on these data, it is suggested that in D. nodosus the Fur protein functions as a regulator of iron and oxidative metabolism.
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Ramírez, Pablo, Nicolas Guiliani, Lissette Valenzuela, Simon Beard, and Carlos A. Jerez. "Differential Protein Expression during Growth of Acidithiobacillus ferrooxidans on Ferrous Iron, Sulfur Compounds, or Metal Sulfides." Applied and Environmental Microbiology 70, no. 8 (August 2004): 4491–98. http://dx.doi.org/10.1128/aem.70.8.4491-4498.2004.
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ABSTRACT A set of proteins that changed their levels of synthesis during growth of Acidithiobacillus ferrooxidans ATCC 19859 on metal sulfides, thiosulfate, elemental sulfur, and ferrous iron was characterized by using two-dimensional polyacrylamide gel electrophoresis. N-terminal amino acid sequencing and mass spectrometry analysis of these proteins allowed their identification and the localization of the corresponding genes in the available genomic sequence of A. ferrooxidans ATCC 23270. The genomic context around several of these genes suggests their involvement in the energetic metabolism of A. ferrooxidans. Two groups of proteins could be distinguished. The first consisted of proteins highly upregulated by growth on sulfur compounds (and downregulated by growth on ferrous iron): a 44-kDa outer membrane protein, an exported 21-kDa putative thiosulfate sulfur transferase protein, a 33-kDa putative thiosulfate/sulfate binding protein, a 45-kDa putative capsule polysaccharide export protein, and a putative 16-kDa protein of unknown function. The second group of proteins comprised those downregulated by growth on sulfur (and upregulated by growth on ferrous iron): rusticyanin, a cytochrome c 552, a putative phosphate binding protein (PstS), the small and large subunits of ribulose biphosphate carboxylase, and a 30-kDa putative CbbQ protein, among others. The results suggest in general a separation of the iron and sulfur utilization pathways. Rusticyanin, in addition to being highly expressed on ferrous iron, was also newly synthesized, as determined by metabolic labeling, although at lower levels, during growth on sulfur compounds and iron-free metal sulfides. During growth on metal sulfides containing iron, such as pyrite and chalcopyrite, both proteins upregulated on ferrous iron and those upregulated on sulfur compounds were synthesized, indicating that the two energy-generating pathways are induced simultaneously depending on the kind and concentration of oxidizable substrates available.
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Hartmann, Carolin, Martin Elsner, Reinhard Niessner, and Natalia P. Ivleva. "Nondestructive Chemical Analysis of the Iron-Containing Protein Ferritin Using Raman Microspectroscopy." Applied Spectroscopy 74, no. 2 (February 7, 2019): 193–203. http://dx.doi.org/10.1177/0003702818823203.
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Ferritin is a ubiquitous intracellular iron storage protein of animals, plants, and bacteria. The cavity of this protein acts like a reaction chamber for natural formation and storage of nano-sized particles via biomineralization. Knowledge of the chemical composition and structure of the iron core is highly warranted in the fields of nano technologies as well as biomolecules and medicine. Here, we show that Raman microspectroscopy (RM) is a suitable nondestructive approach for an analysis of proteins containing such nano-sized iron oxides. Our approach addresses: (1) synthesis of suitable reference materials, i.e., ferrihydrite, maghemite and magnetite nanoparticles; (2) optimization of parameters for Raman spectroscopic analysis; (3) comparison of Raman spectra from ferritin with apoferritin and our reference minerals; and (4) validation of Raman analysis by X-ray diffraction and Mössbauer spectroscopy as two independent complementary approaches. Our results reveal that the iron core of natural ferritin is composed of the iron(III) hydroxide ferrihydrite (Fe2O3 ∙ 0.5 H2O).
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Han, Yu, Kun Zang, Changshui Liu, Yingjie Li, and Qingjun Ma. "The putative siderophore-interacting protein from Vibrio anguillarum: protein production, analysis, crystallization and X-ray crystallographic studies." Acta Crystallographica Section F Structural Biology Communications 74, no. 5 (April 16, 2018): 283–87. http://dx.doi.org/10.1107/s2053230x18005125.
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Siderophore-interacting proteins (SIPs) play an important role in iron acquisition in many bacteria. SIPs release iron from the internalized ferric siderophore complex by reducing ferric iron to ferrous iron, but how the iron is reduced is not well understood. Here, a sip gene was identified in the genome of Vibrio anguillarum 775. To further understand the catalytic mechanism of the protein, the SIP was overexpressed in Escherichia coli Rosetta (DE3) cells, purified and crystallized for X-ray diffraction analysis. The crystal diffracted to 1.113 Å resolution and belonged to space group P21, with unit-cell parameters a = 64.63, b = 58.47, c = 70.65 Å, β = 114.19°.
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Bäumler, Andreas J., Tracy L. Norris, Todd Lasco, Wolfgang Voigt, Rolf Reissbrodt, Wolfgang Rabsch, and Fred Heffron. "IroN, a Novel Outer Membrane Siderophore Receptor Characteristic of Salmonella enterica." Journal of Bacteriology 180, no. 6 (March 15, 1998): 1446–53. http://dx.doi.org/10.1128/jb.180.6.1446-1453.1998.
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ABSTRACT Speciation in enterobacteria involved horizontal gene transfer. Therefore, analysis of genes acquired by horizontal transfer that are present in one species but not its close relatives is expected to give insights into how new bacterial species were formed. In this study we characterize iroN, a gene located downstream of theiroBC operon in the iroA locus ofSalmonella enterica serotype Typhi. Like iroBC, the iroN gene is present in all phylogenetic lineages ofS. enterica but is absent from closely related species such as Salmonella bongori or Escherichia coli. Comparison of the deduced amino acid sequence of iroN with other proteins suggested that this gene encodes an outer membrane siderophore receptor protein. Mutational analysis in S. enterica and expression in E. coli identified a 78-kDa outer membrane protein as the iroN gene product. When introduced into an E. coli fepA cir fiu aroB mutant on a cosmid, iroN mediated utilization of structurally related catecholate siderophores, includingN-(2,3-dihydroxybenzoyl)-l-serine, myxochelin A, benzaldehyde-2,3-dihydroxybenzhydrazone, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-l-lysine, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-l-lysine amide, and enterochelin. These results suggest that theiroA locus functions in iron acquisition in S. enterica.
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Dill, Brian D., Sophie Dessus-Babus, and Jane E. Raulston. "Identification of iron-responsive proteins expressed by Chlamydia trachomatis reticulate bodies during intracellular growth." Microbiology 155, no. 1 (January 1, 2009): 210–19. http://dx.doi.org/10.1099/mic.0.022731-0.
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The obligate intracellular bacterium Chlamydia trachomatis serovar E is the most prevalent cause of bacterial sexually transmitted disease. With an established requirement for iron, the developmental cycle arrests at the intracellular reticulate body stage during iron restriction, resulting in a phenomenon termed persistence. Persistence has implications in natural infections for altered expression of virulence factors and antigens, in addition to a potential role in producing chronic infection. In this study, chlamydial proteins in iron-restricted, infected HEC-1B cells were radiolabelled during mid-developmental cycle growth, harvested, and separated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Of ∼250 radiolabelled protein species visualized, densitometric analysis revealed 25 proteins that increased in expression under iron restriction compared to iron-sufficient control samples; ten protein species identified by mass spectrometry are involved in the oxidative damage response (alkyl hydroperoxide reductase, 6-phosphogluconolactonase and acyl carrier protein synthase), transcription (RNA polymerase subunit alpha and transcription anti-termination factors NusA and NusG), protein modification (peptide deformylase and trigger factor), and virulence (Chlamydia protein associating with death domains, CADD). Transcript-level expression patterns of ahpC, devB, cadd, fabF and ct538 were measured by quantitative RT-PCR throughout the developmental cycle, and each gene examined demonstrated a significant but small mid-cycle increase in transcript level in iron-restricted cultures compared to iron-replete controls. Taken together, these data suggest that the primary response of chlamydiae to reduced iron availability is to increase expression of proteins involved in protection against oxidative damage via iron-catalysed generation of reactive oxygen species and adaptation to stress by increasing expression of transcriptional machinery and other stress-responsive proteins.
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Santhakumar, Sreenithi, Rekha Athiyarath, Anne George Cherian, Vinod Abraham, Biju George, and Eunice Sindhuvi Edison. "Communication of Placental Iron Trafficking Proteins with Maternal and Fetal Iron Regulators." Blood 132, Supplement 1 (November 29, 2018): 3629. http://dx.doi.org/10.1182/blood-2018-99-116344.
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Abstract During pregnancy, iron is a primary requisite micronutrient for the developing fetal-placental unit and increased maternal erythrocyte mass expansion. Iron deficiency anemia in pregnancy (IDAP) remains a constant public health problem in our country where all of them receive routine iron supplementation.The mechanism involved in iron regulatory pathway across the placenta is more complex and less understood. Here we examined the hematological and biochemical parameters of mother and fetus, placental mRNA and protein expression of iron trafficking proteins in iron replete and deplete pregnant women. Subjects who presented to the department of community health for child birth were screened and consenting patients (age<35 years and a gestational age≥36weeks) were included in the study. Maternal and cord blood samples were collected and a cut placental tissue (~0.5cm) was obtained at the time of delivery. Hematological and biochemical parameters were analysed according to standard methods. Serum ferritin was measured using chemiluminescent immunoassay. Maternal and cord blood serum hepcidin and GDF15 levels were measured using ELISA (DRG Diagnostics and Ray Biotech, respectively). Placental tissue RNA extracted using PARIS kit (Ambion) and cDNA was synthesised using RT2 first strand Kit (QIAGEN). The relative quantification of twenty two genes involved in iron uptake, export, transport and regulation was done using Custom RT2 PCR Array (SA Biosciences, QIAGEN). Western blot analysis was performed for significantly expressed genes. Statistical analysis was performed using SPSS software. A total of one hundred and thirty eight pregnant women (n=138) were included in the study; 14 subjects were excluded due to unavailability of either maternal or cord blood serum samples. A cut off of ferritin<30ng/mL was taken to define iron deficiency since all of them were on regular iron supplementation during pregnancy. Of the 124 subjects, 28 subjects had iron deficiency anaemia (Hb<10.5g/dL and Ferritin<30ng/mL) and 52 were iron replete (Hb>10.5g/dL and Ferritin>30ng/mL). Forty four women who did not fulfil either of the criteria were not included for analysis. The demographic, hematological and biochemical parameters of the groups are tabulated (Table.1). In the IDAP, even though the maternal serum ferritin levels were reduced, their fetal ferritin levels did not differ significantly from the control fetuses [IDAP fetuses- 139.8ng/mL, 15.4-300.5 vs Control fetuses - 143.4ng/mL, 13.1-461; p= 0.72]. No significant association was observed between ferritin and hepcidin levels either in the maternal or fetal serum. Interestingly, we found a negative correlation between maternal serum GD15 levels and fetal hepcidin: ferritin ratio (r=-0.439; p=0.025). Placental gene expression and protein expression was further analyzed (Table.2). Significantly higher gene expression of placental IREB2 [p=0.011], and ferroportin [p=0.000] were observed in iron deficient cohort. TP53 and LRP1 mRNA expressions were significantly lower in IDAP [p=0.002 & p=0.000]. Maternal serum ferritin had a significant positive correlation with placental expression of IREB2 (r=0.519; p=0.027) and LRP1 (r=0.470; p=0.049). At the protein level, placental GDF15 protein was significantly elevated in IDAP [p=0.04]. Placental GDF15 and ferroportin (FPN) was positively correlated with fetal ferritin (r=0.626; p=0.017 & r=0.552; p=0.041). Even when the maternal iron stores are low, the fetus gets the priority according to the hierarchy of iron usage. In IDAP, we observed a higher expression of placental ferroportin and transferrin receptor which probably leads to the increased efflux of iron towards fetus. Elevated placental GDF15 and ferroportin may imply that they play a major role in accomplishing iron trafficking and has to be further explored. Disclosures No relevant conflicts of interest to declare.
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Luscieti, Sara, Pietro Pilo Boyl, Bruno Galy, Lucia Gutierrez, Maya Shvartsman, M. Puertos Morales, Matthias W. Hentze, Walter Witke, and Mayka Sanchez. "Profilin2 Is Controlled By the Iron Regulatory Proteins and Modulates Iron Homeostasis." Blood 124, no. 21 (December 6, 2014): 749. http://dx.doi.org/10.1182/blood.v124.21.749.749.
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Abstract Objective: The IRPs/IRE regulatory network plays a central role in the control of cellular iron homeostasis. Using a high throughput approach, we have previously identified novel IRP1 and IRP2 interacting mRNAs. Among the identified mRNAs, we studied more in depth Profilin2 (Pfn2), a protein involved in endocytosis and neurotransmitters release. The aim of this work is to characterize Pfn2 as a novel IRPs target mRNA and study its role in iron homeostasis. Materials and Methods: Mouse and human Pfn2 mRNAs were tested by non-radioactive competitive electrophoretic mobility shift assays (EMSA) for the binding to IRP1 and IRP2. To test the responsiveness of Pfn2 to IRP activity, Pfn2 mRNA levels were analyzed in mice with intestinal IRP1 and IRP2 deficiency. The labile iron pool (LIP) was measured in HeLa and Hepa1-6 cell lines with transient or stable overexpression of Pfn2. Tissues derived from Pfn2 knock-out mice were analyzed for iron content, measured by atomic absorption or colorimetric assay, and for mRNA and protein levels of iron-related genes. Results: Combination of EMSA experiments and bioinformatic analyses allowed the identification of a novel and conserved 3’UTR iron responsive element in Pfn2 mRNA with an atypical hexanucleotide apical loop (AAGUGG). Pfn2 mRNA levels were significantly reduced (~20-25%) in duodenal samples from mice with IRP1 and IRP2 intestinal specific ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Overexpression of Pfn2 cDNA in HeLa and Hepa1-6 cells reduces LIP levels compared to control cells. Finally, analysis of Pfn2 KO mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus and midbrain) together with an hepatic iron deficiency with ferritin reduction. Conclusion: Our results indicate that Pfn2 is controlled by the IRP regulatory system in vivo and that Pfn2 modulates iron homeostasis in cell lines and mice. Funding: Work supported by grant SAF2012-40106 from Spanish Secretary of Research, Development and Innovation (MINECO) and grant CIVP16A1857 “Ayudas a proyectos de Investigación en Ciéncias de la Vida - Fundación Ramón Areces” to M.S. M.S. held a research contract under the Ramón y Cajal program from the Spanish Ministry of Science and Innovation (RYC-2008-02352). Disclosures No relevant conflicts of interest to declare.
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Pieper, Rembert, C. R. Fisher, Moo-Jin Suh, S. T. Huang, P. Parmar, and S. M. Payne. "Analysis of the Proteome of Intracellular Shigella flexneri Reveals Pathways Important for Intracellular Growth." Infection and Immunity 81, no. 12 (October 7, 2013): 4635–48. http://dx.doi.org/10.1128/iai.00975-13.
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ABSTRACTGlobal proteomic analysis was performed withShigella flexneristrain 2457T in association with three distinct growth environments:S. flexnerigrowing in broth (in vitro),S. flexnerigrowing within epithelial cell cytoplasm (intracellular), andS. flexnerithat were cultured with, but did not invade, Henle cells (extracellular). Compared toin vitroand extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread ofS. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in theS. flexneritransition from the extra- to the intracellular milieu.
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Petrak, Jiri, Denisa Myslivcova, Petr Man, Radek Cmejla, Jana Cmejlova, Daniel Vyoral, Milan Elleder, and Christopher D. Vulpe. "Proteomic analysis of hepatic iron overload in mice suggests dysregulation of urea cycle, impairment of fatty acid oxidation, and changes in the methylation cycle." American Journal of Physiology-Gastrointestinal and Liver Physiology 292, no. 6 (June 2007): G1490—G1498. http://dx.doi.org/10.1152/ajpgi.00455.2006.
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Liver iron overload can be found in hereditary hemochromatosis, chronic liver diseases such as alcoholic liver disease, and chronic viral hepatitis or secondary to repeated blood transfusions. The excess iron promotes liver damage, including fibrosis, cirrhosis, and hepatocellular carcinoma. Despite significant research effort, we remain largely ignorant of the cellular consequences of liver iron overload and the cellular processes that result in the observed pathological changes. In addition, the variability in outcome and the compensatory response that likely modulates the effect of increased iron levels are not understood. To provide insight into these critical questions, we undertook a study to determine the consequences of iron overload on protein levels in liver using a proteomic approach. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we studied hepatic iron overload induced by carbonyl iron-rich diet in mice and identified 30 liver proteins whose quantity changes in condition of excess liver iron. Among the identified proteins were enzymes involved in several important metabolic pathways, namely the urea cycle, fatty acid oxidation, and the methylation cycle. This pattern of changes likely reflects compensatory and pathological changes associated with liver iron overload and provides a window into these processes.
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Yu, Chunxiao, Ryan McClure, Kathleen Nudel, Nadine Daou, and Caroline Attardo Genco. "Characterization of the Neisseria gonorrhoeae Iron and Fur Regulatory Network." Journal of Bacteriology 198, no. 16 (May 31, 2016): 2180–91. http://dx.doi.org/10.1128/jb.00166-16.
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ABSTRACTTheNeisseria gonorrhoeaeferricuptakeregulator (Fur) protein controls expression of iron homeostasis genes in response to intracellular iron levels. In this study, using transcriptome sequencing (RNA-seq) analysis of anN. gonorrhoeaefurstrain, we defined the gonococcal Fur and iron regulons and characterized Fur-controlled expression of an ArsR-like DNA binding protein. We observed that 158 genes (8% of the genome) showed differential expression in response to iron in anN. gonorrhoeaewild-type orfurstrain, while 54 genes exhibited differential expression in response to Fur. The Fur regulon was extended to additional regulators, including NrrF and 13 other small RNAs (sRNAs), and two transcriptional factors. One transcriptional factor, coding for an ArsR-like regulator (ArsR), exhibited increased expression under iron-replete conditions in the wild-type strain but showed decreased expression across iron conditions in thefurstrain, an effect that was reversed in afur-complemented strain. Fur was shown to bind to the promoter region of thearsRgene downstream of a predicted σ70promoter region. Electrophoretic mobility shift assay (EMSA) analysis confirmed binding of the ArsR protein to thenorBpromoter region, and sequence analysis identified two additional putative targets, NGO1411 and NGO1646. A gonococcalarsRstrain demonstrated decreased survival in human endocervical epithelial cells compared to that of the wild-type andarsR-complemented strains, suggesting that the ArsR regulon includes genes required for survival in host cells. Collectively, these results demonstrate that theN. gonorrhoeaeFur functions as a global regulatory protein to repress or activate expression of a large repertoire of genes, including additional transcriptional regulatory proteins.IMPORTANCEGene regulation in bacteria in response to environmental stimuli, including iron, is of paramount importance to both bacterial replication and, in the case of pathogenic bacteria, successful infection. Bacterial DNA binding proteins are a common mechanism utilized by pathogens to control gene expression under various environmental conditions. Here, we show that the DNA binding protein Fur, expressed by the human pathogenNeisseria gonorrhoeae, controls the expression of a large repertoire of genes and extends this regulon by controlling expression of additional DNA binding proteins. One of these proteins, an ArsR-like regulator, was required forN. gonorrhoeaesurvival within host cells. These results show that the Fur regulon extends to additional regulatory proteins, which together contribute to gonococcal mechanisms of pathogenesis.
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Ciuraszkiewicz, Justyna, Mariusz Olczak, and Wiesław Watorek. "Isolation and characterisation of crocodile and python ovotransferrins." Acta Biochimica Polonica 54, no. 1 (March 9, 2007): 175–82. http://dx.doi.org/10.18388/abp.2007_3284.
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Transferrins play a major role in iron homeostasis and metabolism. In vertebrates, these proteins are synthesised in the liver and dispersed within the organism by the bloodstream. In oviparous vertebrates additional expression is observed in the oviduct and the synthesised protein is deposited in egg white as ovotransferrin. Most research on ovotransferrin has been performed on the chicken protein. There is a limited amount of information on other bird transferrins, and until our previous paper on red-eared turtle protein there was no data on the isolation, sequencing and biochemical properties of reptilian ovotransferrins. Recently our laboratory deposited ten new sequences of reptilian transferrins in the EMBL database. A comparative analysis of these sequences indicates a possibility of different mechanisms of iron release among crocodile and snake transferrin. In the present paper we follow with the purification and analysis of the basic biochemical properties of two crocodile (Crocodilus niloticus, C. rhombifer) and one snake (Python molurus bivittatus) ovotransferrins. The proteins were purified by anion exchange and hydrophobic chromatography, and their N-terminal amino-acid sequences, molecular mass and isoelectric points were determined. All three proteins are glycosylated and their N-glycan chromatographic profiles show the largest contribution of neutral oligosaccharides in crocodile and disialylated glycans in python ovotransferrin. The absorption spectra of iron-saturated transferrins were analysed. Iron release from these proteins is pH-dependent, showing a biphasic character in crocodile ovotransferrins and a monophasic type in the python protein. The reason for the different types of iron release is discussed.
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Topham, R., B. Cooper, S. Tesh, G. Godette, C. Bonaventura, and J. Bonaventura. "Isolation, purification and characterization of an iron-binding protein from the horseshoe crab (Limulus polyphemus)." Biochemical Journal 252, no. 1 (May 15, 1988): 151–57. http://dx.doi.org/10.1042/bj2520151.
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The presence of an iron-binding protein in the haemolymph of the horseshoe crab, Limulus polyphemus, was detected by gel filtration of 59Fe-labelled haemolymph. Lysis of amoebocytes did not change the amount of iron-binding protein in haemolymph samples. The protein was purified to homogeneity by ion-exchange chromatography. The molecular mass of the purified protein was estimated to be 282,000 +/- 10,000 Da by gel filtration and analytical ultracentrifugation. SDS/polyacrylamide-gel electrophoresis demonstrated that the protein is composed of ten subunits having a molecular mass of 28,000 +/- 2,000 Da. The purified, unlabelled protein efficiently sequestered 59Fe in the absence of haemolymph indicating that no other haemolymph factors are required for the incorporation of iron into the protein. No 59Fe was removed from the purified protein with EDTA or 2,2′-bipyridyl. Partial removal of 59Fe was achieved by dialysis with nitrilotriacetic acid or desferal. Analysis of the iron-loaded protein indicated that each subunit has the capacity to bind two iron atoms with high affinity. The isolation of an iron-binding protein from L. polyphemus supports the proposal that such proteins are an ancient evolutionary development not necessarily linked to the appearance of iron proteins which serve as oxygen carriers.
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Xu, Piao, Ming Chen, Cui Lai, Guangming Zeng, Danlian Huang, Han Wang, Xiaomin Gong, et al. "Effects of typical engineered nanomaterials on 4-nonylphenol degradation in river sediment: based on bacterial community and function analysis." Environmental Science: Nano 6, no. 7 (2019): 2171–84. http://dx.doi.org/10.1039/c9en00371a.
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Iron oxide nanoparticles incorporation promoted 4-nonylphenol degradation, taking advantage of their good biocompatibility and stimulation in iron reducing microbes and expression of iron-regulated proteins.
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Rahman, Md Shahedur, Chayon Biswas, Polash Kumar Biswas, Md Ashraful Kader, S. M. Nur Alam, Christian Sonne, and Ki-Hyun Kim. "In Silico Analysis of the Antigenic Properties of Iron-Regulated Proteins against Neisseria meningitidis." Applied Sciences 10, no. 17 (September 3, 2020): 6113. http://dx.doi.org/10.3390/app10176113.
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Neisseria meningitidis is a commensal pathogen that causes infectious cerebrospinal disease in people of all ages. The multivariate role of six disease-causing polysaccharide serotypes is found to play a crucial role in developing vaccines (or general treatment strategies) to treat this emerging pathogen. Iron is a crucial transition metal for N. meningitidis. Proteomic analysis data could be valuable for vaccine design. Here, we conduct a comparative study using computational bioinformatic tools to identify the most effective iron-regulated outer membrane proteins (OMPs) as immunogenic targets for a potential vaccine against N. meningitidis. The basic properties of N. meningitidis OMPs are explored for flexibility, solubility, hydrophilicity, beta-turns, and overall antigenic probability. Results of our study suggest that iron-regulated OMPs are flexible and soluble in water with high densities of conformational B-cell epitopes. As such, they can be recommended as a novel candidate for a vaccine against N. meningitidis both in vitro and in vivo.
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Ishikawa, Takahiko, Yoshimitsu Mizunoe, Shun-ichiro Kawabata, Akemi Takade, Mine Harada, Sun Nyunt Wai, and Shin-ichi Yoshida. "The Iron-Binding Protein Dps Confers Hydrogen Peroxide Stress Resistance to Campylobacter jejuni." Journal of Bacteriology 185, no. 3 (February 1, 2003): 1010–17. http://dx.doi.org/10.1128/jb.185.3.1010-1017.2003.
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ABSTRACT We identified and characterized the iron-binding protein Dps from Campylobacter jejuni. Electron microscopic analysis of this protein revealed a spherical structure of 8.5 nm in diameter, with an electron-dense core similar to those of other proteins of the Dps (DNA-binding protein from starved cells) family. Cloning and sequencing of the Dps-encoding gene (dps) revealed that a 450-bp open reading frame (ORF) encoded a protein of 150 amino acids with a calculated molecular mass of 17,332 Da. Amino acid sequence comparison indicated a high similarity between C. jejuni Dps and other Dps family proteins. In C. jejuni Dps, there are iron-binding motifs, as reported in other Dps family proteins. C. jejuni Dps bound up to 40 atoms of iron per monomer, whereas it did not appear to bind DNA. An isogenic dps-deficient mutant was more vulnerable to hydrogen peroxide than its parental strain, as judged by growth inhibition tests. The iron chelator Desferal restored the resistance of the Dps-deficient mutant to hydrogen peroxide, suggesting that this iron-binding protein prevented generation of hydroxyl radicals via the Fenton reaction. Dps was constitutively expressed during both exponential and stationary phase, and no induction was observed when the cells were exposed to H2O2 or grown under iron-supplemented or iron-restricted conditions. On the basis of these data, we propose that this iron-binding protein in C. jejuni plays an important role in protection against hydrogen peroxide stress by sequestering intracellular free iron and is expressed constitutively to cope with the harmful effect of hydrogen peroxide stress on this microaerophilic organism without delay.
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Banerjee, Sharmistha, Ashok Kumar Nandyala, Podili Raviprasad, Niyaz Ahmed, and Seyed E. Hasnain. "Iron-Dependent RNA-Binding Activity of Mycobacterium tuberculosis Aconitase." Journal of Bacteriology 189, no. 11 (March 23, 2007): 4046–52. http://dx.doi.org/10.1128/jb.00026-07.
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ABSTRACT Cellular iron levels are closely monitored by iron regulatory and sensor proteins of Mycobacterium tuberculosis for survival inside macrophages. One such class of proteins systematically studied in eukaryotes and reported in a few prokaryotes are the iron-responsive proteins (IRPs). These IRPs bind to iron-responsive elements (IREs) present at untranslated regions (UTRs) of mRNAs and are responsible for posttranscriptional regulation of the expression of proteins involved in iron homeostasis. Amino acid sequence analysis of M. tuberculosis aconitase (Acn), a tricarboxylic acid (TCA) cycle enzyme, showed the presence of the conserved residues of the IRP class of proteins. We demonstrate that M. tuberculosis Acn is bifunctional. It is a monomeric protein that is enzymatically active in converting isocitrate to cis-aconitate at a broad pH range of 7 to 10 (optimum, pH 8). As evident from gel retardation assays, M. tuberculosis Acn also behaves like an IRP by binding to known mammalian IRE-like sequences and to predicted IRE-like sequences present at the 3′ UTR of thioredoxin (trxC) and the 5′ UTR of the iron-dependent repressor and activator (ideR) of M. tuberculosis. M. tuberculosis Acn when reactivated with Fe2+ functions as a TCA cycle enzyme, but upon iron depletion by a specific iron chelator, it behaves like an IRP, binding to the selected IREs in vitro. Since iron is required for the Acn activity and inhibits the RNA-binding activity of Acn, the two activities of M. tuberculosis Acn are mutually exclusive. Our results demonstrate the bifunctional nature of M. tuberculosis Acn, pointing to its likely role in iron homeostasis.
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McDonald, Cameron J., Daniel F. Wallace, Lesa Ostini, and V. Nathan Subramaniam. "Parenteral vs. oral iron: influence on hepcidin signaling pathways through analysis of Hfe/Tfr2-null mice." American Journal of Physiology-Gastrointestinal and Liver Physiology 306, no. 2 (January 15, 2014): G132—G139. http://dx.doi.org/10.1152/ajpgi.00256.2013.
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Treatment for iron deficiency anemia can involve iron supplementation via dietary or parenteral routes that result in different cellular iron distributions. The effect of the administered iron on the iron regulatory system and hepcidin in the liver has not been well studied. Hepcidin, the liver-expressed central iron-regulatory peptide, is itself regulated through the bone morphogenetic protein (BMP)/SMAD signaling pathway. Specifically, Bmp6 expression is upregulated in response to iron and induces hepcidin through phosphorylation of Smad1/5/8. The hemochromatosis-associated proteins Hfe and transferrin receptor 2 (Tfr2) are known upstream regulators of hepcidin, although their precise roles are still unclear. To investigate the mechanisms of this regulation and the roles of the Hfe and Tfr2, we subjected wild-type, Hfe−/−, Tfr2−/−, and Hfe−/−/ Tfr2−/− mice to iron loading via dietary or parenteral routes. Systematic analysis demonstrated that Tfr2 is required for effective upregulation of Bmp6 in response to hepatocyte iron, but not nonparenchymal iron. Hfe is not required for Bmp6 upregulation, regardless of iron localization, but rather, is required for efficient downstream transmission of the regulatory signal. Our results demonstrate that Hfe and Tfr2 play separate roles in the regulatory responses to iron compartmentalized in different cell types and further elucidates the regulatory mechanisms controlling iron homeostasis.
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Dopson, Mark, Craig Baker-Austin, and Philip L. Bond. "Analysis of differential protein expression during growth states of Ferroplasma strains and insights into electron transport for iron oxidation." Microbiology 151, no. 12 (December 1, 2005): 4127–37. http://dx.doi.org/10.1099/mic.0.28362-0.
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To investigate the metabolic biochemistry of iron-oxidizing extreme acidophiles, a proteomic analysis of chemomixotrophic and chemo-organotrophic growth, as well as protein expression in the absence of organic carbon, was carried out in Ferroplasma species. Electron transport chain inhibitor studies, spectrophotometric analysis and proteomic results suggest that oxidation of ferrous iron may be mediated by the blue copper-haem protein sulfocyanin and the derived electron passes to a cbb 3 terminal electron acceptor. Despite previous suggestions of a putative carbon dioxide fixation pathway, no up-regulation of proteins typically associated with carbon dioxide fixation was evident during incubation in the absence of organic carbon. Although a lack of known carbon dioxide fixation proteins does not constitute proof, the results suggest that these strains are not autotrophic. Proteins putatively involved in central metabolic pathways, a probable sugar permease and flavoproteins were up-regulated during chemo-organotrophic growth in comparison to the protein complement during chemomixotrophic growth. These results reflect a higher energy demand to be derived from the organic carbon during chemo-organotrophic growth. Proteins with suggested function as central metabolic enzymes were expressed at higher levels during chemomixotrophic growth by Ferroplasma acidiphilum YT compared to ‘Ferroplasma acidarmanus’ Fer1. This study addresses some of the biochemical and bioenergetic questions fundamental for survival of these organisms in extreme acid-leaching environments.
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Schryvers, Anthony B., and B. Craig Lee. "Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae." Canadian Journal of Microbiology 35, no. 3 (March 1, 1989): 409–15. http://dx.doi.org/10.1139/m89-063.
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Intact cells of several bacterial species were tested for their ability to bind human transferrin and lactoferrin by a solid-phase binding assay using horseradish peroxidase conjugated transferrin and lactoferrin. The ability to bind lactoferrin was detected in all isolates of Neisseria and Branhamella catarrhalis but not in isolates of Escherichia coli or Pseudomonas aeruginosa. Transferrin-binding activity was similarly detected in most isolates of Neisseria and Branhamella but not in E. coli or P. aeruginosa. The expression of transferrin- and lactoferrin-binding activity was induced by addition of ethylenediamine di-o-phenylacetic acid and reversed by excess FeCl3, indicating regulation by the level of available iron in the medium. The transferrin receptor was specific for human transferrin and the lactoferrin receptor had a high degree of specificity for human lactoferrin in all species tested. The transferrin- and lactoferrin-binding proteins were identified after affinity isolation using biotinylated human transferrin or lactoferrin and streptavidin–agarose. The lactoferrin-binding protein was identified as a 105-kilodalton protein in all species tested. Affinity isolation with biotinylated transferrin yielded two or more proteins in all species tested. A high molecular mass protein was observed in all isolates, and was of similar size (approximately 98 kilodaltons) in all species of Neisseria but was larger (105 kilodaltons) in B. catarrhalis.Key words: iron, Neisseria, transferrin, lactoferrin, receptor.
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Santander, Javier, Greg Golden, Soo-Young Wanda, and Roy Curtiss. "Fur-Regulated Iron Uptake System of Edwardsiella ictaluri and Its Influence on Pathogenesis and Immunogenicity in the Catfish Host." Infection and Immunity 80, no. 8 (May 21, 2012): 2689–703. http://dx.doi.org/10.1128/iai.00013-12.
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ABSTRACTThe ability of bacterial pathogens to take up iron from the host during infection is necessary for their multiplication within the host. However, host high-affinity iron binding proteins limit levels of free iron in fluids and tissues. To overcome this deficiency of iron during infection, bacterial pathogens have developed iron uptake systems that are upregulated in the absence of iron, typically tightly controlled by the ferric uptake regulator (Fur) protein. The iron uptake system ofEdwardsiella ictaluri, a host-restricted pathogen of channel catfish (Ictalurus punctatus) and the main pathogen of this fish in aquaculture, is unknown. Here we describe theE. ictaluriFur protein, the iron uptake machinery controlled by Fur, and the effects offurgene deletion on virulence and immunogenicity in the fish host. Analysis of theE. ictaluriFur protein shows that it lacks the N-terminal region found in the majority of pathogen-encoded Fur proteins. However, it is fully functional in regulated genes encoding iron uptake proteins.E. ictalurigrown under iron-limited conditions upregulates an outer membrane protein (HemR) that shows heme-hemoglobin transport activity and is tightly regulated by Fur.In vivostudies showed that anE. ictaluriΔfurmutant is attenuated and immune protective in zebrafish (Danio rerio) and catfish (Ictalurus punctatus), triggering systemic immunity. We conclude that anE. ictaluriΔfurmutant could be an effective component of an immersion-oral vaccine for the catfish industry.
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Bender, Sara J., Dawn M. Moran, Matthew R. McIlvin, Hong Zheng, John P. McCrow, Jonathan Badger, Giacomo R. DiTullio, Andrew E. Allen, and Mak A. Saito. "Colony formation in <i>Phaeocystis antarctica</i>: connecting molecular mechanisms with iron biogeochemistry." Biogeosciences 15, no. 16 (August 21, 2018): 4923–42. http://dx.doi.org/10.5194/bg-15-4923-2018.
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Abstract. Phaeocystis antarctica is an important phytoplankter of the Ross Sea where it dominates the early season bloom after sea ice retreat and is a major contributor to carbon export. The factors that influence Phaeocystis colony formation and the resultant Ross Sea bloom initiation have been of great scientific interest, yet there is little known about the underlying mechanisms responsible for these phenomena. Here, we present laboratory and field studies on Phaeocystis antarctica grown under multiple iron conditions using a coupled proteomic and transcriptomic approach. P. antarctica had a lower iron limitation threshold than a Ross Sea diatom Chaetoceros sp., and at increased iron nutrition (> 120 pM Fe') a shift from flagellate cells to a majority of colonial cells in P. antarctica was observed, implying a role for iron as a trigger for colony formation. Proteome analysis revealed an extensive and coordinated shift in proteome structure linked to iron availability and life cycle transitions with 327 and 436 proteins measured as significantly different between low and high iron in strains 1871 and 1374, respectively. The enzymes flavodoxin and plastocyanin that can functionally replace iron metalloenzymes were observed at low iron treatments consistent with cellular iron-sparing strategies, with plastocyanin having a larger dynamic range. The numerous isoforms of the putative iron-starvation-induced protein (ISIP) group (ISIP2A and ISIP3) had abundance patterns coinciding with that of either low or high iron (and coincident flagellate or the colonial cell types in strain 1871), implying that there may be specific iron acquisition systems for each life cycle type. The proteome analysis also revealed numerous structural proteins associated with each cell type: within flagellate cells actin and tubulin from flagella and haptonema structures as well as a suite of calcium-binding proteins with EF domains were observed. In the colony-dominated samples a variety of structural proteins were observed that are also often found in multicellular organisms including spondins, lectins, fibrillins, and glycoproteins with von Willebrand domains. A large number of proteins of unknown function were identified that became abundant at either high or low iron availability. These results were compared to the first metaproteomic analysis of a Ross Sea Phaeocystis bloom to connect the mechanistic information to the in situ ecology and biogeochemistry. Proteins associated with both flagellate and colonial cells were observed in the bloom sample consistent with the need for both cell types within a growing bloom. Bacterial iron storage and B12 biosynthesis proteins were also observed consistent with chemical synergies within the colony microbiome to cope with the biogeochemical conditions. Together these responses reveal a complex, highly coordinated effort by P. antarctica to regulate its phenotype at the molecular level in response to iron and provide a window into the biology, ecology, and biogeochemistry of this group.
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Pozzi, Cecilia, Flavio Di Pisa, Caterina Bernacchioni, Silvia Ciambellotti, Paola Turano, and Stefano Mangani. "Iron binding to human heavy-chain ferritin." Acta Crystallographica Section D Biological Crystallography 71, no. 9 (August 25, 2015): 1909–20. http://dx.doi.org/10.1107/s1399004715013073.
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Maxi-ferritins are ubiquitous iron-storage proteins with a common cage architecture made up of 24 identical subunits of five α-helices that drive iron biomineralization through catalytic iron(II) oxidation occurring at oxidoreductase sites (OS). Structures of iron-bound human H ferritin were solved at high resolution by freezing ferritin crystals at different time intervals after exposure to a ferrous salt. Multiple binding sites were identified that define the iron path from the entry ion channels to the oxidoreductase sites. Similar data are available for another vertebrate ferritin: the M protein fromRana catesbeiana. A comparative analysis of the iron sites in the two proteins identifies new reaction intermediates and underlines clear differences in the pattern of ligands that define the additional iron sites that precede the oxidoreductase binding sites along this path. Stopped-flow kinetics assays revealed that human H ferritin has different levels of activity compared with itsR. catesbeianacounterpart. The role of the different pattern of transient iron-binding sites in the OS is discussed with respect to the observed differences in activity across the species.
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Yu, Rong-Hua, and Anthony B. Schryvers. "Bacterial lactoferrin receptors: insights from characterizing theMoraxella bovisreceptors." Biochemistry and Cell Biology 80, no. 1 (February 1, 2002): 81–90. http://dx.doi.org/10.1139/o01-235.
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Moraxella bovis is the causative agent of infectious conjunctivitis in cattle. Moraxella bovis isolates were shown to specifically bind bovine lactoferrin (bLf) and bovine transferrin (bTf) and to use these proteins as a source of iron to support the growth of iron-limited cells. Affinity isolation experiments with immobilized bTf yielded two proteins readily resolved by SDS-PAGE analysis, whereas only a single band of approximately 100 kDa was detected when immobilized bLf was used as the affinity ligand. Using a novel cloning strategy, regions containing the genes encoding the lactoferrin (Lf) and transferrin (Tf) receptor proteins were isolated and sequenced, demonstrating that they both consisted of two genes, with the tbpB or lbpB gene preceding the tbpA or lbpA gene. The cloned lbp genes were used to generate isogenic mutants deficient in lactoferrin binding protein A and (or) B, and the resulting strains were tested in growth and binding assays. The isogenic mutants were deficient in their use of bLf for growth and had substantially diminished bLf binding capability. The predicted amino acid sequence from the segment encoding Lf binding protein B revealed an internal amino acid homology suggesting it is a bi-lobed protein, with a C-lobe enriched in acidic amino acids, but without the evident clustering observed in Lf-binding proteins from other species.Key words: outer membrane protein, iron-binding protein, lactoferrin, receptor, iron, transport, specificity.
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Puchalski, A., R. Urban-Chmiel, M. Dec, and A. Wernicki. "An electrophoretic characterization of iron-transporting proteins in Mannheimia haemolytica A1." Polish Journal of Veterinary Sciences 16, no. 3 (September 1, 2013): 527–32. http://dx.doi.org/10.2478/pjvs-2013-0073.
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AbstractIron-regulated outer membrane proteins (IROMPs) in Mannheimia haemolytica A1, which function as a receptor for complexes containing iron ions, are induced by iron deficiency in the growth environment of the bacteria. Densitometric analysis of SDS-PAGE separation showed expression of IROMPs of 71, 77, and 100 kDa in the case of bacteria grown in a medium with 2,2-dipyridyl. The electrophoregrams obtained in 2-DE separations confirmed the presence of protein fractions with these molecular weights and isoelectric points ranging from 5.4 to 6.4. The results of the study also confirmed the ability of M. haemolytica A1 proteins involved in iron uptake to induce a protective immune response. In Western blot with serum from convalescent calves naturally infected with M. haemolytica A1, distinct reactions were obtained for IROMPs of 71, 77, and 100 kDa.
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Zimbler, Daniel L., Brock A. Arivett, Amber C. Beckett, Sharon M. Menke, and Luis A. Actis. "Functional Features of TonB Energy Transduction Systems of Acinetobacter baumannii." Infection and Immunity 81, no. 9 (July 1, 2013): 3382–94. http://dx.doi.org/10.1128/iai.00540-13.
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ABSTRACTAcinetobacter baumanniiis an opportunistic pathogen that causes severe nosocomial infections. Strain ATCC 19606Tutilizes the siderophore acinetobactin to acquire iron under iron-limiting conditions encountered in the host. Accordingly, the genome of this strain has threetonBgenes encoding proteins for energy transduction functions needed for the active transport of nutrients, including iron, through the outer membrane. Phylogenetic analysis indicates that thesetonBgenes, which are present in the genomes of all sequencedA. baumanniistrains, were acquired from different sources. Two of these genes occur as components oftonB-exbB-exbDoperons and one as a monocistronic copy; all are actively transcribed in ATCC 19606T. The abilities of components of these TonB systems to complement the growth defect ofEscherichia coliW3110 mutants KP1344 (tonB) and RA1051 (exbBD) under iron-chelated conditions further support the roles of these TonB systems in iron acquisition. Mutagenesis analysis of ATCC 19606TtonB1(subscripted numbers represent different copies of genes or proteins) andtonB2supports this hypothesis: their inactivation results in growth defects in iron-chelated media, without affecting acinetobactin biosynthesis or the production of the acinetobactin outer membrane receptor protein BauA.In vivoassays usingGalleria mellonellashow that each TonB protein is involved in, but not essential for, bacterial virulence in this infection model. Furthermore, we observed that TonB2plays a role in the ability of bacteria to bind to fibronectin and to adhere to A549 cells by uncharacterized mechanisms. Taken together, these results indicate thatA. baumanniiATCC 19606Tproduces three independent TonB proteins, which appear to provide the energy-transducing functions needed for iron acquisition and cellular processes that play a role in the virulence of this pathogen.
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Łęcka, Monika, Artur Słomka, Katarzyna Albrecht, Ewa Żekanowska, Michał Romiszewski, and Jan Styczyński. "Unbalance in Iron Metabolism in Childhood Leukemia Converges with Treatment Intensity: Biochemical and Clinical Analysis." Cancers 13, no. 12 (June 17, 2021): 3029. http://dx.doi.org/10.3390/cancers13123029.
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Objective: The aim of this study was to evaluate non-transferrin-bound iron (NTBI) and labile plasma iron (LPI) levels and other parameters of iron metabolism in children undergoing therapy for acute leukemia or after hematopoietic cell transplantation (HCT), in the context of iron overload. Patients: A total number of 85 children were prospectively included into four groups: controls, acute leukemia de novo, acute leukemia after intensive treatment, and after HCT. Methods: The following iron metabolism parameters were analyzed: (1) parameters measuring functional and storage iron pools: NTBI, LPI, iron, transferrin, total iron-binding capacity, ferritin, ferritin heavy and light chains; (2) proteins regulating iron absorption and its release from tissue stores: hepcidin, soluble hemojuvelin, soluble ferroportin-1; (3) proteins regulating the erythropoietic activity of bone marrow: erythroferrone, erythropoietin, soluble transferrin receptor. Results: Intensive treatment of leukemia in children was associated with the presence of serum NTBI and LPI, which was the highest in the HCT group followed by the acute leukemia after treatment and de novo groups. In patients after HCT, the most significant changes were found in NTBI, LPI, iron, ferritin, hepcidin, and ferroportin-1 levels. Conclusions: The occurrence of NTBI and LPI in the circulation and the intensification of disturbances in iron metabolism were associated with the intensity of the anti-leukemic treatment.
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Hoffmann, Tamara, Alexandra Schütz, Margot Brosius, Andrea Völker, Uwe Völker, and Erhard Bremer. "High-Salinity-Induced Iron Limitation in Bacillus subtilis." Journal of Bacteriology 184, no. 3 (February 1, 2002): 718–27. http://dx.doi.org/10.1128/jb.184.3.718-727.2002.
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ABSTRACT Proteome analysis of Bacillus subtilis cells grown at low and high salinity revealed the induction of 16 protein spots and the repression of 2 protein spots, respectively. Most of these protein spots were identified by mass spectrometry. Four of the 16 high-salinity-induced proteins corresponded to DhbA, DhbB, DhbC, and DhbE, enzymes that are involved in the synthesis of 2,3-dihydroxybenzoate (DHB) and its modification and esterification to the iron siderophore bacillibactin. These proteins are encoded by the dhbACEBF operon, which is negatively controlled by the central iron regulatory protein Fur and is derepressed upon iron limitation. We found that iron limitation and high salinity derepressed dhb expression to a similar extent and that both led to the accumulation of comparable amounts of DHB in the culture supernatant. DHB production increased linearly with the degree of salinity of the growth medium but could still be reduced by an excess of iron. Such an excess of iron also partially reversed the growth defect exhibited by salt-stressed B. subtilis cultures. Taken together, these findings strongly suggest that B. subtilis cells grown at high salinity experience iron limitation. In support of this notion, we found that the expression of several genes and operons encoding putative iron uptake systems was increased upon salt stress. The unexpected finding that high-salinity stress has an iron limitation component might be of special ecophysiological importance for the growth of B. subtilis in natural settings, in which bioavailable iron is usually scarce.
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Dubbels, Bradley L., Alan A. DiSpirito, John D. Morton, Jeremy D. Semrau, J. N. E. Neto, and Dennis A. Bazylinski. "Evidence for a copper-dependent iron transport system in the marine, magnetotactic bacterium strain MV-1." Microbiology 150, no. 9 (September 1, 2004): 2931–45. http://dx.doi.org/10.1099/mic.0.27233-0.
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Cells of the magnetotactic marine vibrio, strain MV-1, produce magnetite-containing magnetosomes when grown anaerobically or microaerobically. Stable, spontaneous, non-magnetotactic mutants were regularly observed when cells of MV-1 were cultured on solid media incubated under anaerobic or microaerobic conditions. Randomly amplified polymorphic DNA analysis showed that these mutants are not all genetically identical. Cellular iron content of one non-magnetotactic mutant strain, designated MV-1nm1, grown anaerobically, was ∼20- to 80-fold less than the iron content of wild-type (wt) MV-1 for the same iron concentrations, indicating that MV-1nm1 is deficient in some form of iron uptake. Comparative protein profiles of the two strains showed that MV-1nm1 did not produce several proteins produced by wt MV-1. To understand the potential roles of these proteins in iron transport better, one of these proteins was purified and characterized. This protein, a homodimer with an apparent subunit mass of about 19 kDa, was an iron-regulated, periplasmic protein (p19). Two potential ‘copper-handling’ motifs (MXM/MX2M) are present in the amino acid sequence of p19, and the native protein binds copper in a 1 : 1 ratio. The structural gene for p19, chpA (copper handling protein) and two other putative genes upstream of chpA were cloned and sequenced. These putative genes encode a protein similar to the iron permease, Ftr1, from the yeast Saccharomyces cerevisiae, and a ferredoxin-like protein of unknown function. A periplasmic, copper-containing, iron(II) oxidase was also purified from wt MV-1 and MV-1nm1. This enzyme, like p19, was regulated by media iron concentration and contained four copper atoms per molecule of enzyme. It is hypothesized that ChpA, the iron permease and the iron(II) oxidase might have analogous functions for the three components of the S. cerevisiae copper-dependent high-affinity iron uptake system (Ctr1, Ftr1 and Fet3, respectively), and that strain MV-1 may have a similar iron uptake system. However, iron(II) oxidase purified from both wt MV-1 and MV-1nm1 displayed comparable iron oxidase activities using O2 as the electron acceptor, indicating that ChpA does not supply the multi-copper iron(II) oxidase with copper.
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Leong, S. A., Z. An, B. Mei, J. McEvoy, Q. Zhao, and J. Markley. "Sequences and proteins required for iron-regulated expression ofsid1ofUstilago maydis." Canadian Journal of Botany 73, S1 (December 31, 1995): 140–47. http://dx.doi.org/10.1139/b95-237.
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The molecular biology of the high affinity, siderophore-mediated iron uptake system of the basidiomycete fungus Ustilago maydis is under investigation. Ustilago maydis produces two cyclic peptide siderophores, ferrichrome and ferrichrome A. Biosynthesis of both siderophores is initiated by ornithine-N5-oxygenase, the product of sid1. sid1 mRNA accumulates only during growth under iron starvation conditions in wild-type cells or constitutively in urbs1 mutants, urbs1 encodes a 100-kDa protein with putative Zn finger domains that share sequence identity with those of the GATA family of transcription factors. The promoter region of sid1 was defined by deletion analysis of a 3.0-kb region 5′ to the translational start of sid1 using the Escherichia coli GUS gene as a reporter. Three regions were defined by this analysis to be critical to expression of sid1. These include (i) a 306-bp region containing two GATA sequences and mapping 2.4 kb from the start of translation; (ii) a 439-bp region immediately 5′ to the start of transcription; and (iii) a region encompassing the first intron of sid1. Deletion of the GATA sequences resulted in deregulated expression of sid1, while elimination of the latter two sequences ablated expression of the gene under all circumstances. Current efforts are focused on determining whether Urbsl interacts directly with the sid1 promoter via the GATA sequences and whether this interaction is dependent upon iron. Key words: GATA, transcription factor, siderophore, ferrichrome, iron, Urbs1.
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Frazer, David M., Christopher D. Vulpe, Andrew T. McKie, Sarah J. Wilkins, Deborah Trinder, Geoffrey J. Cleghorn, and Gregory J. Anderson. "Cloning and gastrointestinal expression of rat hephaestin: relationship to other iron transport proteins." American Journal of Physiology-Gastrointestinal and Liver Physiology 281, no. 4 (October 1, 2001): G931—G939. http://dx.doi.org/10.1152/ajpgi.2001.281.4.g931.
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The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.
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Rondon, Michelle R., Katie S. Ballering, and Michael G. Thomas. "Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58." Microbiology 150, no. 11 (November 1, 2004): 3857–66. http://dx.doi.org/10.1099/mic.0.27319-0.
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Using the complete genome sequence from Agrobacterium tumefaciens C58, the authors identified a secondary metabolite gene cluster that encodes the biosynthesis of a metabolite with siderophore activity. Support for this conclusion came from genetic and regulatory analysis of the gene cluster, along with the purification of a metabolite from A. tumefaciens C58 with iron-chelating activity. Genetic analysis of mutant strains disrupted in this gene cluster showed that these strains grew more slowly than the wild-type strain in medium lacking iron. Additionally, the mutant strains failed to produce a chrome-azurol-S-reactive material in liquid or solid medium, and failed to produce the metabolite with iron-chelating characteristics that was identified in the wild-type strain. Addition of this purified metabolite to the growth medium of a mutant strain restored its ability to grow in iron-deficient medium. Furthermore, expression of this gene cluster was induced by growth under iron-limiting conditions, suggesting that expression of this gene cluster occurs when iron is scarce. These data are all consistent with the proposal that the proteins encoded by this gene cluster are involved in the production of a siderophore. Interestingly, these proteins show the highest level of amino acid similarity to proteins from a gene cluster found in the filamentous cyanobacterium Nostoc sp. PCC7120, rather than to known siderophore biosynthetic enzymes. Given these properties, it is proposed that the siderophore produced by A. tumefaciens C58 will have a unique chemical structure. Production of the siderophore was not required for virulence of A. tumefaciens when tested with a standard stem inoculation assay.
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Tonpitak, Walaiporn, Svenja Thiede, Winfried Oswald, Nina Baltes, and Gerald-F. Gerlach. "Actinobacillus pleuropneumoniae Iron Transport: a Set of exbBD Genes Is Transcriptionally Linked to the tbpB Gene and Required for Utilization of Transferrin-Bound Iron." Infection and Immunity 68, no. 3 (March 1, 2000): 1164–70. http://dx.doi.org/10.1128/iai.68.3.1164-1170.2000.
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ABSTRACT Upon iron restriction, Actinobacillus pleuropneumoniaehas been shown to express the transferrin-binding proteins TbpB and TbpA, both of which have been implied to be important virulence factors. In order to identify additional iron-regulated proteins, we cloned and analyzed the region upstream of the transferrin-binding protein genes in an A. pleuropneumoniae serotype 7 strain. We located immediately upstream of the tbpB gene two open reading frames which were 43% homologous to the neisserial ExbBD protein genes. By raising specific antibodies, we showed that ExbB is expressed under iron-limiting growth conditions only, and RT-PCR analysis revealed that the exbBD genes and thetbpB gene are transcribed on a single polycistronic mRNA. By constructing an isogenic and nonpolar exbBD mutant, we showed that the exbBD genes are required by A. pleuropneumoniae for utilization of transferrin-bound iron. Using PCR and Western blotting, we showed that the genetic organization found in A. pleuropneumoniae serotype 7 is similar in all 12A. pleuropneumoniae serotype reference strains.
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Beaumont, Fiona C., Ho Young Kang, Timothy J. Brickman, and Sandra K. Armstrong. "Identification and Characterization of alcR, a Gene Encoding an AraC-Like Regulator of Alcaligin Siderophore Biosynthesis and Transport in Bordetella pertussis andBordetella bronchiseptica." Journal of Bacteriology 180, no. 4 (February 15, 1998): 862–70. http://dx.doi.org/10.1128/jb.180.4.862-870.1998.
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ABSTRACT A Bordetella bronchiseptica iron transport mutant was isolated following an enrichment procedure based on streptonigrin resistance. The mutant displayed a growth defect on iron-restricted medium containing ferric alcaligin as the sole iron source. In addition to the apparent inability to acquire iron from the siderophore, the mutant failed to produce alcaligin as well as two known iron-regulated proteins, one of which is the AlcC alcaligin biosynthesis protein. A 1.6-kb KpnI-PstI Bordetella pertussis DNA fragment mapping downstream of the alcaligin biosynthesis genes alcABC restored both siderophore biosynthesis and expression of the iron-regulated proteins to the mutant. Nucleotide sequencing of this complementing 1.6-kb region identified an open reading frame predicted to encode a protein with strong similarity to members of the AraC family of transcriptional regulators, for which we propose the gene designation alcR. Primer extension analysis localized an iron-regulated transcription initiation site upstream of the alcR open reading frame and adjacent to sequences homologous to the consensus Fur repressor binding site. The AlcR protein was produced by using an Escherichia coli expression system and visualized in electrophoretic gels. In-frame alcR deletion mutants of B. pertussisand B. bronchiseptica were constructed, and the defined mutants exhibited the alcR mutant phenotype, characterized by the inability to produce and transport alcaligin and express the two iron-repressed proteins. The cloned alcR gene provided intrans restored these siderophore system activities to the mutants. Together, these results indicate that AlcR is involved in the regulation of Bordetella alcaligin biosynthesis and transport genes and is required for their full expression.