Iron-containing Proteins Research Articles

Gene atlas of iron-containing proteins in Arabidopsis thaliana.

Iron (Fe) is an essential element for the development and physiology of plants, owing to its presence in numerous proteins involved in central biological processes. Here, we established an exhaustive, manually curated inventory of genes encoding Fe-containing proteins in Arabidopsis thaliana, and summarized their subcellular localization, spatiotemporal expression and evolutionary age. We have currently identified 1068 genes encoding potential Fe-containing proteins, including 204 iron-sulfur (Fe-S) proteins, 446 haem proteins and 330 non-Fe-S/non-haem Fe proteins (updates of this atlas are available at https://conf.arabidopsis.org/display/COM/Atlas+of+Fe+containing+proteins). A fourth class, containing 88 genes for which iron binding is uncertain, is indexed as 'unclear'. The proteins are distributed in diverse subcellular compartments with strong differences per category. Interestingly, analysis of the gene age index showed that most genes were acquired early in plant evolutionary history and have progressively gained regulatory elements, to support the complex organ-specific and development-specific functions necessitated by the emergence of terrestrial plants. With this gene atlas, we provide a valuable and updateable tool for the research community that supports the characterization of the molecular actors and mechanisms important for Fe metabolism in plants. This will also help in selecting relevant targets for breeding or biotechnological approaches aiming at Fe biofortification in crops.

Interaction mechanism of ferritin protein with chlorogenic acid and iron ion: The structure, iron redox, and polymerization evaluation

Ferritin is an iron-containing protein and functions in the maintenance of iron balance in organisms. Currently the interaction among ferritin, ion iron, and food bioactive compounds is still unclear. In this study, the mechanism underlying the interaction of ferritin, ion iron, and chlorogenic acid was investigated, as well as the effect of chlorogenic acid on the physicochemical properties of ferritin. The results showed that chlorogenic acid could interact with Fe(III) to form chlorogenic acid-Fe(III) complexes, which then bonded with ferritin via hydrogen bonds in the ferritin–chlorogenic acid-Fe(III) complexes. The chlorogenic acid showed a high efficiency in Fe(II) chelation and hydroxyl radical (•OH) capture, and could promote iron oxidation and iron release induced by ferritin. Chlorogenic acid could also effectively reduce the polymerization extent of ferritin induced by Fe(III) and Fe(II). This study elucidates the interactions of multiple components in foodstuffs by using a protein-metal-polyphenol model.

Perls' Prussian Blue Stains of Lung Tissue, Bronchoalveolar Lavage, and Sputum.

Perls' Prussian blue (PPB) stain recognizes Fe3+ associated with hemosiderin. The employment of this stain in clinical medicine and research has been extensive and novel applications continue to evolve. Ferruginous bodies are intracellular structures in lung tissue, bronchoalveolar lavage (BAL), and sputum that stain with PPB. Inhaled, insoluble, biopersistent particles and fibers are phagocytosed by lung macrophages and thought to be coated, either partially or completely, with an iron-containing protein at the interface forming a ferruginous body. These structures can be categorized as ferruginous bodies having either an inorganic or a carbonaceous core (e.g., asbestos and byssinotic bodies, respectively). In lung tissue, BAL, and sputum, the only cells that stain with PPB are macrophages. These are described as iron- and hemosiderin-laden macrophages and called either siderophages or sideromacrophages. Siderophages can be observed in the lung tissue, BAL, and sputum after various exposures and can also be associated with many different pulmonary and extrapulmonary diseases.

Hemoglobins from Scapharca subcrenata (Bivalvia: Arcidae) likely play an bactericidal role through their peroxidase activity.

Hemoglobin (Hb) is an iron-containing respiratory protein present in all vertebrates and some invertebrates. The blood clam Scapharca subcrenata is one of the few invertebrates that have Hb-containing red hemocytes. In this study, we purified Hb (Ss-Hb), including Ss-HbI and Ss-HbII, from S. subcrenata hemocytes using gel chromatography with a recovery rate of 70.71%, and then characterized their peroxidase activities. Both Ss-Hbs possessed peroxidase activity with high affinity to the substrates guaiacol and H2O2. Moreover, both Ss-Hbs had structural similarities, such as type b heme, proximal histidine (His), distal His, and heme pocket arginine (Arg), with other peroxidases. The optimal peroxidase activity of both Ss-Hbs was at pH5 and 35°C, but this was inhibited in the presence of Cu2+ and Fe2+. Ss-Hbs produced [Formula: see text] in the presence of H2O2. β-phenylethylamine, a substrate of peroxidase, increased the [Formula: see text] generation, while Cu2+, an inhibitor of peroxidase, inhibited this reaction. These results indicated that the peroxidase cycle of Ss-Hb was involved in the production of [Formula: see text] . A large amount of [Formula: see text] may be generated by the peroxidase cycle if the substrate is sufficient. During the incubation of Ss-Hbs with Bacillus subtilis, it was speculated that trace H2O2, probably from autoxidation of Ss-Hbs or generated by B. subtilis, started the peroxidase cycle of Ss-Hb. and produced a large amount of [Formula: see text] in the presence of sufficient substrate in the culture medium. It is therefore reasonable to assume that Ss-Hbs played an antibacterial role owing to their peroxidase activity, which produced [Formula: see text] .

Free Rather Than Total Iron Content Is Critically Linked to the Fur Physiology in Shewanella oneidensis.

Ferric uptake regulator (Fur) is a transcriptional regulator playing a central role in iron homeostasis of many bacteria, and Fur inactivation commonly results in pleiotropic phenotypes. In Shewanella oneidensis, a representative of dissimilatory metal-reducing γ-proteobacteria capable of respiring a variety of chemicals as electron acceptors (EAs), Fur loss substantially impairs respiration. However, to date the mechanism underlying the physiological phenomenon remains obscure. This investigation reveals that Fur loss compromises activity of iron proteins requiring biosynthetic processes for their iron cofactors, heme in particular. We then show that S. oneidensis Fur is critical for maintaining heme homeostasis by affecting both its biosynthesis and decomposition of the molecule. Intriguingly, the abundance of iron-containing proteins controlled by H2O2-responding regulator OxyR increases in the fur mutant because the Fur loss activates OxyR. By comparing suppression of membrane-impermeable, membrane-permeable, and intracellular-only iron chelators on heme deficiency and elevated H2O2 resistance, our data suggest that the elevation of the free iron content by the Fur loss is likely to be the predominant factor for the Fur physiology. Overall, these results provide circumstantial evidence that Fur inactivation disturbs bacterial iron homeostasis by altering transcription of its regulon members, through which many physiological processes, such as respiration and oxidative stress response, are transformed.

FusB Energizes Import across the Outer Membrane through Direct Interaction with Its Ferredoxin Substrate.

Phytopathogenic Pectobacterium spp. import ferredoxin into the periplasm for proteolytic processing and iron release via the ferredoxin uptake system. Although the ferredoxin receptor FusA and the processing protease FusC have been identified, the mechanistic basis of ferredoxin import is poorly understood. In this work, we demonstrate that protein translocation across the outer membrane is dependent on the TonB-like protein FusB. In contrast to the loss of FusC, loss of FusB or FusA abolishes ferredoxin transport to the periplasm, demonstrating that FusA and FusB work in concert to transport ferredoxin across the outer membrane. In addition to an interaction with the "TonB box" region of FusA, FusB also forms a complex with the ferredoxin substrate, with complex formation required for substrate transport. These data suggest that ferredoxin transport requires energy transduction from the cytoplasmic membrane via FusB both for removal of the FusA plug domain and for substrate translocation through the FusA barrel.IMPORTANCE The ability to acquire iron is key to the ability of bacteria to cause infection. Plant-pathogenic Pectobacterium spp. are able to acquire iron from plants by transporting the iron-containing protein ferredoxin into the cell from proteolytic processing. In this work, we show that the TonB-like protein FusB plays a key role in transporting ferredoxin across the bacterial outer membrane by directly energizing its transport into the cell. The direct interaction of the TonB-like protein with substrate is unprecedented and explains the requirement for the system-specific TonB homologue in the ferredoxin uptake system. Since multiple genes encoding TonB-like proteins are commonly found in the genomes of Gram-negative bacteria, this may be a common mechanism for the uptake of atypical substrates via TonB-dependent receptors.

Participation of two sRNA RyhB homologs from the fish pathogen Yersinia ruckeri in bacterial physiology

Small noncoding RNAs (sRNAs) are important regulators of gene expression and physiology in bacteria. RyhB is an iron-responsive sRNA well characterized in Escherichia coli and conserved in other Enterobacteriaceae. In this study, we identified and characterized two RyhB homologs (named RyhB-1 and RyhB-2) in the fish pathogen Yersinia ruckeri. We found that, as in other Enterobacteriaceae, both RyhB-1 and RyhB-2 are induced under iron starvation, repressed by the Fur regulator, and depend on Hfq for stability. Despite these similarities in expression, the mutant strains of Y. ruckeri lacking RyhB-1 (ΔryhB-1) or RyhB-2 (ΔryhB-2) exhibited differential phenotypes. In comparison with the wild type, the ΔryhB-1 strain showed a hypermotile phenotype, reduced biofilm formation, increased replication rate, faster growth, and increased ATP levels in bacterial cultures. By contrast, in salmon cell cultures, the ΔryhB-1 strain exhibited an increased survival. On the other hand, the ΔryhB-2 strain was non-motile and showed augmented biofilm formation as compared to the wild type. The expression of a subset of RyhB conserved targets, selected from different bacterial species, was analyzed by quantitative RT-PCR in wild type, ΔryhB-1, ΔryhB-2, and ΔryhB-1 ΔryhB-2 strains cultured in iron-depleted media. RyhB-1 negatively affected the expression of most analyzed genes (sodB, acnA, sdhC, bfr, fliF, among others), whose functions are related to metabolism and motility, involving iron-containing proteins. Among the genes analyzed, only sdhC and bfr appeared as targets for RyhB-2. Taken together, these results indicate that Y. ruckeri RyhB homologs participate in the modulation of the bacterial physiology with non-redundant roles.

Iron Regulatory Mechanisms in Saccharomyces cerevisiae.

Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox cofactor in many cellular processes. However, excess iron can damage cells since it promotes the generation of reactive oxygen species. The budding yeast Saccharomyces cerevisiae has been used as a model organism to study the adaptation of eukaryotic cells to changes in iron availability. Upon iron deficiency, yeast utilizes two transcription factors, Aft1 and Aft2, to activate the expression of a set of genes known as the iron regulon, which are implicated in iron uptake, recycling and mobilization. Moreover, Aft1 and Aft2 activate the expression of Cth2, an mRNA-binding protein that limits the expression of genes encoding for iron-containing proteins or that participate in iron-using processes. Cth2 contributes to prioritize iron utilization in particular pathways over other highly iron-consuming and non-essential processes including mitochondrial respiration. Recent studies have revealed that iron deficiency also alters many other metabolic routes including amino acid and lipid synthesis, the mitochondrial retrograde response, transcription, translation and deoxyribonucleotide synthesis; and activates the DNA damage and general stress responses. At high iron levels, the yeast Yap5, Msn2, and Msn4 transcription factors activate the expression of a vacuolar iron importer called Ccc1, which is the most important high-iron protecting factor devoted to detoxify excess cytosolic iron that is stored into the vacuole for its mobilization upon scarcity. The complete sequencing and annotation of many yeast genomes is starting to unveil the diversity and evolution of the iron homeostasis network in this species.

Reactive Oxygen Species: Participation in Cellular Processes and Progression of Pathology

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are not only side products of chemical reactions, but participants in various cellular processes as well. ROS and RNS are involved in the defense against pathogenic microorganisms (H2O2, HOCl, ONOO–, $${\text{O}}_{2}^{{\centerdot - }},$$ and OH•), fertilization (H2O2), cell division $$\left( {{\text{O}}_{2}^{{\centerdot - }}} \right),$$ apoptosis (H2O2), regeneration (H2O2), coordination of a direction of the cellular movement, regulation of the vascular tone (NO•), etc. A balance between the production and the removal of ROS and RNS results in an intracellular homeostasis, whereas their overproduction causes cell damage and most probably leads to changes in the cellular metabolism. ROS and RNS can act as intracellular messengers, i.e., change the intracellular oxidative–reductive state and/or structure and function of a protein by means of a modification of amino acid residues (mainly cysteines), and the red-ox state of a number of proteins can affect the cellular metabolism. Hydrogen peroxide is the main form of ROS which participates in oxidative–reductive transduction of signals in eukaryotes. Alterations in antioxidant systems contribute to aging and a development of the age-related diseases. Primarily, aging is associated with an increased level of oxidative stress, various types of macromolecular changes, and an accumulation of DNA damage. Ageing can, to some extent, be a consequence of disorders in the proteostasis regulation and changes in the proteome functioning, because proteins are responsible for most of the cellular functions. Moreover, not all the cellular proteins can be resynthesized due to the age-related DNA damage. Thus, reactive oxygen and nitrogen species that are permanently generated in an organism are important participants in regulatory mechanisms in a cell, but also a reason for several pathological states, including cancers. ROSs are known to regulate the metabolism of signal molecules which are necessary for the cell cycle. Moreover, ROSs are able to change the activity of the iron-containing proteins. The aging that is associated with an ineffective functioning of the antioxidant defense is connected with the oxidative stress, various changes in cellular structures and macromolecules, an accumulation of metabolic products which can have a negative effect, the DNA damage (for example, owing to mistakes during a replication by DNA polymerases), and disorders in functioning of reparation systems.

Iron deficiency attenuates protein synthesis stimulated by branched-chain amino acids and insulin in myotubes

Iron deficiency anemia indicates poor nutrition and is a public health problem. Iron deficiency is also associated with muscle weakness. However, the intracellular mechanisms by which iron deficiency induces muscle weakness are obscure. The purpose of the present study was to evaluate the effect of iron deficiency on protein synthesis in basal and branched-amino acids (BCAA)- and insulin-stimulated state in muscle cells. Differentiated C2C12 myotubes were incubated with an iron chelator, deferoxamine mesylate, and then stimulated with BCAA or insulin to activate protein synthesis. This iron deprivation resulted in a significant reduction in the abundance of iron-containing proteins, such as the mitochondrial complex 1 subunit protein, compared to control cells, but not of protein that does not contain iron, such as citrate synthase. Proteins involved in glucose utilization, such as glucose transpoter-1, hexokinase and AMP-activated protein kinase (AMPK), were upregulated under iron deficiency. Additionally, rates of BCAA- and insulin-stimulated protein synthesis, measured by puromycin incorporation, were lower in iron-deficient myotubes than in control cells. We suggest that low iron availability attenuates BCAA- and insulin-stimulated protein synthesis, possibly via activation of AMPK in myotubes. The present findings advance the understanding of the importance of iron to skeletal muscle protein synthesis and, thus, may contribute to the prevention of sarcopenia and frailty.

Genetic Redundancy in Iron and Manganese Transport in the Metabolically Versatile Bacterium Rhodopseudomonas palustris TIE-1.

The purple nonsulfur bacterium Rhodopseudomonas palustris TIE-1 can produce useful biochemicals such as bioplastics and biobutanol. Production of such biochemicals requires intracellular electron availability, which is governed by the availability and the transport of essential metals such as iron (Fe). Because of the distinct chemical properties of ferrous [Fe(II)] and ferric iron [Fe(III)], different systems are required for their transport and storage in bacteria. Although Fe(III) transport systems are well characterized, we know much less about Fe(II) transport systems except for the FeoAB system. Iron transporters can also import manganese (Mn). We studied Fe and Mn transport by five putative Fe transporters in TIE-1 under metal-replete, metal-depleted, oxic, and anoxic conditions. We observed that by overexpressing feoAB, efeU, and nramp1AB, the intracellular concentrations of Fe and Mn can be enhanced in TIE-1 under oxic and anoxic conditions, respectively. The deletion of a single gene/operon does not attenuate Fe or Mn uptake in TIE-1 regardless of the growth conditions used. This indicates that genetically dissimilar yet functionally redundant Fe transporters in TIE-1 can complement each other. Relative gene expression analysis shows that feoAB and efeU are expressed during Fe and Mn depletion under both oxic and anoxic conditions. The promoters of these transporter genes contain a combination of Fur and Fnr boxes, suggesting that their expression is regulated by both Fe and oxygen availability. The findings from this study will help us modulate intracellular Fe and Mn concentrations, ultimately improving TIE-1's ability to produce desirable biomolecules.IMPORTANCERhodopseudomonas palustris TIE-1 is a metabolically versatile bacterium that can use various electron donors, including Fe(II) and poised electrodes, for photoautotrophic growth. TIE-1 can produce useful biomolecules, such as biofuels and bioplastics, under various growth conditions. Production of such reduced biomolecules is controlled by intracellular electron availability, which, in turn, is mediated by various iron-containing proteins in the cell. Several putative Fe transporters exist in TIE-1's genome. Some of these transporters can also transport Mn, part of several important cellular enzymes. Therefore, understanding the ability to transport and respond to various levels of Fe and Mn under different conditions is important to improve TIE-1's ability to produce useful biomolecules. Our data suggest that by overexpressing Fe transporter genes via plasmid-based expression, we can increase the import of Fe and Mn in TIE-1. Future work will leverage these data to improve TIE-1 as an attractive microbial chassis and future biotechnological workhorse.

Quantification of ferritin-bound iron in murine samples for Alzheimer’s disease studies using species-specific isotope dilution mass spectrometry

We have investigated species-specific isotope dilution mass spectrometry (IDMS) for the quantification of ferritin-bound iron in murine serum and brain. Therefore, fresh samples were analyzed using size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS). Isotopically labeled (57Fe)ferritin was used as calibrant for the quantification of ferritin-bound iron in murine samples. Assessment of the iron load of serum ferritin was impaired by concomitant iron-containing proteins of similar size and shape, which could not be separated by SEC nor centrifugal ultra-filtration. In contrast, ferritin was the main iron-containing protein in cytosolic extracts of murine brain, which showed a total ferritin-bound iron content of (1.05 ± 0.12) µg g−1 (n= 10; U, k= 2). The relative expanded uncertainty achieved for a mass fraction of ca. 1 µg g−1 ferritin-bound iron was 11% (Urel, k = 2). The relative expanded uncertainty of the iron mass fraction of the (57Fe)ferritin spike was 5.7% and represented the major contributing factor to the overall uncertainty. Statistical tests suggested no significant difference in ferritin-bound iron content between mouse brain hemispheres. The presented analytical tool provides low limits of quantification (2.2 ng g−1) and uncertainties (11%, Urel, k = 2), thus enables the quantification of ferritin-bound iron in murine brain extracts with high sensitivity and accuracy. Furthermore, this analytical workflow assures comparability of measurement results across research laboratories. This provides the basis for investigation of the iron loading of ferritin in brain tissue of healthy and Alzheimer’s disease mouse models, which may help answering the question if iron regulation is impaired in Alzheimer’s disease.

Iron and Chelation in Biochemistry and Medicine: New Approaches to Controlling Iron Metabolism and Treating Related Diseases.

Iron is essential for all living organisms. Many iron-containing proteins and metabolic pathways play a key role in almost all cellular and physiological functions. The diversity of the activity and function of iron and its associated pathologies is based on bond formation with adjacent ligands and the overall structure of the iron complex in proteins or with other biomolecules. The control of the metabolic pathways of iron absorption, utilization, recycling and excretion by iron-containing proteins ensures normal biologic and physiological activity. Abnormalities in iron-containing proteins, iron metabolic pathways and also other associated processes can lead to an array of diseases. These include iron deficiency, which affects more than a quarter of the world’s population; hemoglobinopathies, which are the most common of the genetic disorders and idiopathic hemochromatosis. Iron is the most common catalyst of free radical production and oxidative stress which are implicated in tissue damage in most pathologic conditions, cancer initiation and progression, neurodegeneration and many other diseases. The interaction of iron and iron-containing proteins with dietary and xenobiotic molecules, including drugs, may affect iron metabolic and disease processes. Deferiprone, deferoxamine, deferasirox and other chelating drugs can offer therapeutic solutions for most diseases associated with iron metabolism including iron overload and deficiency, neurodegeneration and cancer, the detoxification of xenobiotic metals and most diseases associated with free radical pathology.

Acireductone dioxygenase 1 (ADI1) is regulated by cellular iron by a mechanism involving the iron chaperone, PCBP1, with PCBP2 acting as a potential co-chaperone

The iron-containing protein, acireductone dioxygenase 1 (ADI1), is a dioxygenase important for polyamine synthesis and proliferation. Using differential proteomics, the studies herein demonstrated that ADI1 was significantly down-regulated by cellular iron depletion. This is important, since ADI1 contains a non-heme, iron-binding site critical for its activity. Examination of multiple human cell-types demonstrated a significant decrease in ADI1 mRNA and protein after incubation with iron chelators. The decrease in ADI1 after iron depletion was reversible upon incubation of cells with the iron salt, ferric ammonium citrate (FAC). A significant decrease in ADI1 mRNA levels was observed after 14 h of iron depletion. In contrast, the chelator-mediated reduction in ADI1 protein occurred earlier after 10 h of iron depletion, suggesting additional post-transcriptional regulation. The proteasome inhibitor, MG-132, prevented the iron chelator-mediated decrease in ADI1 expression, while the lysosomotropic agent, chloroquine, had no effect. These results suggest an iron-dependent, proteasome-mediated, degradation mechanism. Poly r(C)-binding protein (PCBPs) 1 and 2 act as iron delivery chaperones to other iron-containing dioxygenases and were shown herein for the first time to be regulated by iron levels. Silencing of PCBP1, but not PCBP2, led to loss of ADI1 expression. Confocal microscopy co-localization studies and proximity ligation assays both demonstrated decreased interaction of ADI1 with PCBP1 and PCBP2 under conditions of iron depletion using DFO. These data indicate PCBP1 and PCBP2 interact with ADI1, but only PCBP1 plays a role in ADI1 expression. In fact, PCBP2 appeared to play an accessory role, being involved as a potential co-chaperone.

Elizabethkingia anophelis: Physiologic and Transcriptomic Responses to Iron Stress.

In this study, we investigated the global gene expression responses of Elizabethkingia anophelis to iron fluxes in the midgut of female Anopheles stephensi mosquitoes fed sucrose or blood, and in iron-poor or iron-rich culture conditions. Of 3,686 transcripts revealed by RNAseq technology, 218 were upregulated while 112 were down-regulated under iron-poor conditions. Hemolysin gene expression was significantly repressed when cells were grown under iron-rich or high temperature (37°C) conditions. Furthermore, hemolysin gene expression was down-regulated after a blood meal, indicating that E. anophelis cells responded to excess iron and its associated physiological stress by limiting iron loading. By contrast, genes encoding respiratory chain proteins were up-regulated under iron-rich conditions, allowing these iron-containing proteins to chelate intracellular free iron. In vivo studies showed that growth of E. anophelis cells increased 3-fold in blood-fed mosquitoes over those in sucrose-fed ones. Deletion of siderophore synthesis genes led to impaired cell growth in both iron-rich and iron-poor media. Mutants showed more susceptibility to H2O2 toxicity and less biofilm formation than did wild-type cells. Mosquitoes with E. anophelis experimentally colonized in their guts produced more eggs than did those treated with erythromycin or left unmanipulated, as controls. Results reveal that E. anophelis bacteria respond to varying iron concentration in the mosquito gut, harvest iron while fending off iron-associated stress, contribute to lysis of red blood cells, and positively influence mosquito host fecundity.

The Availability of Iron Is Involved in the Murine Experimental Toxoplasma gondii Infection Outcome.

Iron is an important constituent of our environment, being necessary for both mammalian and pathogenic protozoa survival. Iron-containing proteins exert a wide range of biological processes such as biodegradation and biosynthesis, as well as immune function, fetal development, and physical and mental well-being. This work aimed to investigate the effect of iron deprivation in Toxoplasma gondii infection outcome. C57BL/6 mice were orally infected with T. gondii and treated with an iron chelator, deferoxamine, or supplemented with iron (ferrous sulfate), and the parasitism as well as immunological and histological parameters were analyzed. It was observed that the infection increased iron accumulation in the organs, as well as systemically, and deferoxamine treatment diminished the iron content in serum samples and intestine. The deferoxamine treatment decreased the parasitism and inflammatory alterations in the small intestine and lung. Additionally, they partially preserved the Paneth cells and decreased the intestinal dysbiosis. The ferrous sulfate supplementation, despite not significantly increasing the parasite load in the organs, increased the inflammatory alterations in the liver. Together, our results suggest that iron chelation, which is commonly used to treat iron overload, could be a promising medicine to control T. gondii proliferation, mainly in the small intestine, and consequently inflammation caused by infection.

A VIT‐like transporter facilitates iron transport into nodule symbiosomes for nitrogen fixation in soybean

Effective legume-rhizobia symbiosis depends on efficient nutrient exchange. Rhizobia need to synthesize iron-containing proteins for symbiotic nitrogen fixation (SNF) in nodules, which depends on host plant-mediated iron uptake into the symbiosome. We functionally investigated a pair of vacuolar iron transporter like (VTL) genes, GmVTL1a/b, in soybean (Glycine max) and evaluated their contributions to SNF, including investigations of gene expression patterns, subcellular localization, and mutant phenotypes. Though both GmVTL1a/b genes were specifically expressed in the fixation zone of the nodule, GmVTL1a was the lone member to be localized at the tonoplast of tobacco protoplasts, and shown to facilitate ferrous iron transport in yeast. GmVTL1a targets the symbiosome in infected cells, as verified by in situ immunostaining. Two vtl1 knockout mutants had lower iron concentrations in nodule cell sap and peribacteroid units than in wild-type plants, suggesting that GmVTL1 knockout inhibited iron import into symbiosomes. Furthermore, GmVTL1 knockout minimally affected soybean growth under nonsymbiotic conditions, but dramatically impaired nodule development and SNF activity under nitrogen-limited and rhizobia-inoculation conditions, which eventually led to growth retardation. Taken together, these results demonstrate that GmVTL1a is indispensable for SNF in nodules as a transporter of ferrous iron from the infected root cell cytosol to the symbiosome.

Структура нейроглобина холодноводной губки Halisarca dujardinii

The iron-containing protein neuroglobin (Ngb) involved in the transport of oxygen is generally considered the precursor of all animal globins. In this report, we studied the structure of Ngb of the cold-water sponge Halisarca dujardinii. In sponges, the oldest multicellular organisms, the Ngb gene contains three introns. In contrast to human Ngb, its promoter contains a TATA-box, rather than CG-rich motifs. In sponges, Ngb consists of 169 amino acids showing rather low similarity with its mammalian orthologues. It lacks Glu and Arg residues in positions required for prevention of hypoxia-related apoptosis. Nevertheless, Ngb contains both proximal and distal conserved heme-biding histidines. The primary structure of H. dujardinii neuroglobin predicted by sequencing was confirmed by mass-spectrometry analysis of recombinant Ngb expressed in E. coli. The high level of Ngb expression in sponge tissues suggests its possible involvement in the gas metabolism and presumably in other key metabolic processes in H. dujardinii.

Characterisation of a mitochondrial iron transporter of the pathogen Trypanosoma brucei

Similar to higher eukaryotes, the protist parasite T. brucei harbours several iron-containing proteins that regulate DNA and protein processing, oxidative stress defence and mitochondrial respiration. The synthesis of these proteins occurs either in the cytoplasm or within the mitochondrion. For mitochondrial iron cluster protein synthesis, iron needs to be transported across the solute impermeable mitochondrial membrane. In T. brucei we previously identified 24 mitochondrial carrier proteins (TbMCPs) sharing conserved structural and functional features with those from higher eukaryotes. One of these carriers (TbMCP17) displayed high similarity with the iron carriers MRS3, MRS4 from yeast and mitoferrin from mammals, insects and plants. In the present study we demonstrated that TbMCP17 functions as an iron carrier by complementation studies using MRS3/4-deficient yeast. Depletion of TbMCP17 in procyclic form T. brucei resulted in growth deficiency, increased sensitivity to iron deprivation, and lowered mitochondrial iron content. Taken together our results suggest that TbMCP17 functions as a mitochondrial iron transporter in the parasite T. brucei.

Molecular Evolution and Functional Analysis of Rubredoxin-Like Proteins in Plants.

Rubredoxins are a class of iron-containing proteins that play an important role in the reduction of superoxide in some anaerobic bacteria and also act as electron carriers in many biochemical processes. Unlike the more widely studied about rubredoxin proteins in anaerobic bacteria, very few researches about the function of rubredoxins have been proceeded in plants. Previous studies indicated that rubredoxins in A. thaliana may play a critical role in responding to oxidative stress. In order to identify more rubredoxins in plants that maybe have similar functions as the rubredoxin-like protein of A. thaliana, we identified and analyzed plant rubredoxin proteins using bioinformatics-based methods. Totally, 66 candidate rubredoxin proteins were identified based on public databases, exhibiting lengths of 187–360 amino acids with molecular weights of 19.856–37.117 kDa. The results of subcellular localization showed that these candidate rubredoxins were localized to the chloroplast, which might be consistent with the fact that rubredoxins were predominantly expressed in leaves. Analyses of conserved motifs indicated that these candidate rubredoxins contained rubredoxin and PDZ domains. The expression patterns of rubredoxins in glycophyte and halophytic plant under salt/drought stress revealed that rubredoxin is one of the important stress response proteins. Finally, the coexpression network of rubredoxin in Arabidopsis thaliana under abiotic was extracted from ATTED-II to explore the function and regulation relationship of rubredoxin in Arabidopsis thaliana. Our results showed that putative rubredoxin proteins containing PDZ and rubredoxin domains, localized to the chloroplast, may act with other proteins in chloroplast to responses to abiotic stress in higher plants. These findings might provide value inference to promote the development of plant tolerance to some abiotic stresses and other economically important crops.