Iron Transport-related Genes Research Articles

Glyphosate and aminomethylphosphonic acid impact on redox status and biotransformation in fish and the mitigating effects of diet supplementation.

Fish reared under seminatural conditions can be challenged by exposure to herbicides. Farming facilities relying on the surrounding area's water quality can be affected by glyphosate and aminomethylphosphonic acid (AMPA) contamination. This review summarizes findings on how glyphosate and AMPA in the amounts registered in surface waterbodies affect redox status and biotransformation in fish and covers the aspect of diet supplementation for oxidative stress relief. Environmentally relevant concentrations of glyphosate and AMPA can alter the transcription and catalytic activities of antioxidant enzymes, decrease the content of reduced glutathione, and increase the accumulation of lipid peroxidation products, all of which are signs of a redox imbalance. Glyphosate has been shown to affect complex I in the mitochondrial respiratory chain and dysregulate iron transport-related genes, causing redox disturbance. Relatively high but environmentally realistic glyphosate concentrations can initiate the induction of cytochrome P450 biotransformation enzymes, alter the regulation of ABC exporters, and cause the inhibition of the redox-sensitive Nrf2 signaling pathway. Studies on reducing herbicide toxicity through dietary supplementation are a promising area of research. Natural functional supplements have been proven to have great potential for mitigating glyphosate-induced oxidative stress and thereby improving fish health, which in turn means maintaining productivity in fish farms that use natural water. However, data on the effects of AMPA on fish are scarce, and studies on the alleviation of its toxicity in fish are lacking. Considering the variety of AMPA contamination routes, one cannot underestimate the need for further research.

The new CFEM protein CgCsa required for Fe3+ homeostasis regulates the growth, development, and pathogenicity of Colletotrichum gloeosporioides

Secreted common fungal extracellular membrane (CFEM) domain proteins have been implicated in multiple biological functions in fungi. However, it is still largely unknown whether the ferric iron (Fe3+), as an important trace element, was involved with the biological function of CFEM proteins. In this study, a new CFEM protein CgCsa, with high expression levels at the early inoculation stage on peppers by Colletotrichum gloeosporioides was investigated. Deletion of the targeted gene CgCsa revealed multiple biological roles in hyphal growth restriction, highly reduced conidial yield, delayed conidial germination, abnormal appressorium with elongated bud tubes, and significantly reduced virulence of C. gloeosporioides. Moreover, in CgCsa mutants, the expression levels of four cell wall synthesis-related genes were downregulated, and cell membrane permeability and electrical conductivity were increased. Compared to the wild-type, the CgCsa mutants downregulated expressions of iron transport-related genes, in addition, its three-dimensional structure was capable binding with iron. Increase in the Fe3+ concentration in the culture medium partially recovered the functions of ΔCgCsa mutant. This is probably the first report to show the association between CgCsa and iron homeostasis in C. gloeosporioides. The results suggest an alternative pathway for controlling plant fungal diseases by deplete their trace elements.

A GATA-type transcription factor SreA affects manganese susceptibility by regulating the expression of iron uptake-related genes

SreA has been identified as a GATA-type transcription factor that represses iron uptake to avoid iron excess during iron sufficiency. However, knowledge about whether SreA also affects the homeostasis of other divalent metal ions is limited. In this study, by screening Aspergillus fumigatus transcription factor deletion mutant libraries, we demonstrate that the sreA deletion mutant shows the greatest tolerance to MnCl2 among the tested divalent metal ions. Fe and Mn stimuli are able to enhance the expression of SreA with the different time-dependent manner, while the expression of SreA contributes to Mn2+ tolerance. Lack of SreA results in abnormally increased expression of a series of siderophore biosynthesis genes and iron transport-related genes, especially under MnCl2 treatment. Further mechanistic exploration indicated that lack of SreA exacerbates abnormal iron uptake, and iron excess inhibits cellular Mn content; thus, deletion of sreA results in Mn tolerance. Thus, findings in this study have demonstrated a new unexplored function for the transcription factor SreA in regulation of the Mn2+ tolerance.

Cadmium adsorption in leaf cell walls prevents redistribution to silique in Arabidopsis thaliana ecotypes Jm-1 and Kyo-0

Cadmium (Cd) redistribution from leaf to silique determined seed Cd content in Arabidopsis, but the control mechanism remains largely unknown. Here, we found that the Cd concentrations in the leaves, stems, and siliques of Arabidopsis thaliana (A. thaliana) ecotype Jm-1 were higher than in the ecotype Kyo-0. The Cd concentrations in the leaves’ cell walls (CW) were 39% lower in Jm-1 than in Kyo-0, while the concentrations in the CW of the stem and silique were 14% and 42% higher in Jm-1. The Cyclohexane Diamine Tetraacetic Acid (CDTA)-pectin and hemicellulose in Kyo-0 had higher Cd concentrations than those of Jm-1. The pectin methylesterase (PME) activity was 19% higher in Kyo-0 than in Jm-1, and the expression levels of PME1, PME2, PME12, and PME25 were upregulated in Kyo-0 after Cd treatment. In addition, the Cd and Fe concentrations in the phloem was 36% and 73% higher in Jm-1 than in Kyo-0 respectively. The expression of iron transport-related genes showed that only YSL3 and ZIP11 had significant differences between the two ecotypes after Cd treatment. Kyo-0 accumulated less Cd than Jm-1 in the silique, which may be because (1) the activity of PME that is mainly regulated by PME1, PME2, PME12, and PME25 was higher in Kyo-0 leaves, leading to more Cd chelation in the pectin of the CWs, and (2) the expression of YSL3 was 2 times lower in Kyo-0 leaves， which regulate the transport of Cd in the phloem, thus reducing the transport of Cd to the silique.

Relevance of FeoAB system in Rhodanobacter sp. B2A1Ga4 resistance to heavy metals, aluminium, gallium, and indium.

Aluminium (Al), gallium (Ga), and indium (In) are metals widely used in diverse applications in industry, which consequently result in a source of environmental contamination. In this study, strain Rhodanobacter sp. B2A1Ga4, highly resistant to Al, Ga, and In, was studied to reveal the main effects of these metals on the strain and the bacterial mechanisms linked to the ability to cope with them. An indium-sensitive mutant obtained by random transposon mutagenesis has the feoA gene interrupted. This gene together with the feoB gene is part of the feo operon which encodes a ferrous uptake system (FeoAB). The mutant strain exhibited higher oxidative stress supported by a high concentration of reactive oxygen species (ROS) and low levels of reduced glutathione (GSH) in the presence of metals. The iron supplementation of the growth medium reverted the growth inhibition of the mutant strain caused by Ga and In, significantly reduced the ROS amounts in mutant cells grown in all conditions, and increased its GSH/total glutathione ratio to values similar to those of the native strain. Moreover, the mutant strain when submitted to In increased the production of siderophores. The genome sequence analysis of strain B2A1Ga4 showed a large number of genes encoding putative proteins involved in iron uptake from the cell surface to the cytoplasm. Understanding the bacteria-metal interactions linked to resistance to high-tech metals is relevant to future application of microorganisms in bioremediation and/or biorecovery processes of these metals. KEY POINTS: • The disruption of FeoAB system compromises the bacterial resistance to Al, Ga, and In. • The iron acquisition in Rhodanobacter sp. B2A1Ga4 controls the oxidative stress. • Genome mining of strain B2A1Ga4 reveals several iron transport related genes.

1266. Characterization of Shifts in Minimum Inhibitory Concentrations During Treatment with Cefiderocol or Comparators in the Phase 3 CREDIBLE-CR and APEKS-NP Studies

BackgroundCefiderocol (CFDC) is a novel siderophore cephalosporin developed to treat serious carbapenem-resistant (CR) Gram-negative (GN) infections.MethodsIn CREDIBLE-CR (NCT02714595), adults with serious infections caused by CR GN pathogens received CFDC 2 g, q8h, 3-h infusion, or best available therapy (BAT). In APEKS-NP (NCT03032380), adults with nosocomial pneumonia received CFDC or high-dose, extended-infusion meropenem (each 2 g, q8h, 3-h infusion). All treatments were given for 7‒14 days (extendable to 21 days). Biospecimens were collected before the first dose of study drug and at subsequent visits for assessments, and minimum inhibitory concentrations (MIC) to various antibiotics, including CFDC and carbapenems, were determined. Isolates with an increased MIC were evaluated by RT-PCR or whole genome sequencing (WGS) for CFDC resistance-related genes or mutations. Results for genetically related isolates with an elevated MIC during therapy are shown.ResultsOn-therapy ≥4-fold CFDC MIC increase was found in 12 out of 106 (CREDIBLE-CR; Table 1) and 7 out of 159 (APEKS-NP; Table 2) isolates, respectively. For most isolates, CFDC MIC increased by 4–8-fold but remained ≤4 µg/mL. Specific mutations which could explain CFDC MIC increases were found in only 3 isolates. Mutations in iron-transport related genes were not identified. Mutation in CFDC target gene PBP-3 was identified in 1 A. baumannii isolate. Class-C enzyme mutation was observed in 2 isolates (CREDIBLE-CR: PDC-30 in P. aeruginosa; APEKS-NP: ACT-17 in E. cloacae), although the contribution to CFDC MIC increase has not been confirmed. In the BAT arm in CREDIBLE-CR, 6 out of 46 isolates had ≥4-fold MIC increase; all post-treatment isolates were resistant to BAT agents (Table 1), although WGS was not conducted for these isolates. In the meropenem arm in APEKS-NP, 5 out of 164 isolates had ≥4-fold MIC increase (Table 2).Table 1. MIC changes in CREDIBLE-CR Table 2. MIC changes in APEKS-NP ConclusionAmong isolates with ≥4-fold MIC increase during CFDC treatment, actual CFDC MIC values remained relatively low for most isolates. Frequency of MIC increase in BAT or meropenem arms was similar to that of CFDC, but the magnitude was greater. Acquisition of contributory mechanism has not been identified except for the mutation in PBP 3 and some β-lactamases.DisclosuresMiki Takemura, MSc, Shionogi & Co., Ltd. (Employee) Yoshinori Yamano, PhD, Shionogi & Co., Ltd. (Employee) Yuko Matsunaga, MD, Shionogi Inc. (Employee) Mari Ariyasu, BPharm, Shionogi & Co., Ltd. (Employee) Roger Echols, MD, Shionogi Inc. (Consultant) Tsutae Den Nagata, MD, Shionogi & Co., Ltd. (Employee)

Cadmium induces iron deficiency anemia through the suppression of iron transport in the duodenum

Cadmium (Cd) is an environmental contaminant that triggers toxic effects in various tissues such as the kidney, liver, and lung. Cd can also cause abnormal iron metabolism, leading to anemia. Iron homeostasis is regulated by intestinal absorption. However, whether Cd affects the iron absorption pathway is unclear. We aimed to elucidate the relationship between the intestinal iron transporter system and Cd-induced iron deficiency anemia. C57BL/6J female and male mice, 129/Sv female mice, and DBA/2 female mice were given a single oral dose of CdCl2 by gavage. After 3 or 24 h, Cd decreased serum iron concentrations and inhibited the expression of iron transport-related genes in the duodenum. In particular, Cd decreased the levels of divalent metal transporter 1 and ferroportin 1 in the duodenum. In addition, human colon carcinoma Caco-2 cells were treated with CdCl2. After 72 h, Cd decreased the expression of iron transport-related factors in Caco-2 cells with a pattern similar to that seen in the murine duodenum. These findings suggest that Cd inhibits iron absorption through direct suppression of iron transport in duodenal enterocytes and contributes to abnormal iron metabolism.

Comparative accumulation and transcriptomic analysis of juvenile Marsupenaeus japonicus under cadmium or copper exposure

Waterborne metals may be hazardous to aquatic organisms and trigger stress responses. The present study aimed to assess the effect of exposure to 100 μg/L cadmium (Cd) or copper (Cu) for 48 h on juvenile Marsupenaeus japonicus, in terms of bioaccumulation and the whole body transcriptome. The results demonstrated that Cu accumulation in M. japonicas was much higher than that of Cd. Meanwhile, transcriptome analysis identified 1802 and 2670 differentially expressed genes (DEGs) after 48 h exposure to 100 μg/L Cd and Cu, respectively. Among them, 851 DEGs responded to both metals. Cd and Cu stress shared genes were related to the cytoskeleton, immunity, antioxidation, and detoxification. Metallothionein 1 (MT1) was specifically induced in the Cd-stress response, while glycometabolism, heat shock protein 90 (HSP90), metallothionein 2 (MT2), apoptosis, and iron transport-related genes were changed specifically in response to Cu stress. In addition, real-time PCR was used to verify the expression patterns of 28 randomly selected DEGs. The sequencing and real-time PCR results were consistent. Moreover, based on the number of significantly modulated genes and their expression levels, we deduced that Cu acts as a stronger stress inducer than Cd in M. japonicus. The identified Cd and Cu stress related genes and pathways will provide new insights into the common and different molecular mechanisms underlying Cd and Cu toxicity effects in M. japonicus.

Gut Microbial Composition and Function Are Altered in Patients with Early Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation of the joints and extra-articular manifestations. Recent studies have shown that microorganisms affect RA pathogenesis. However, few studies have examined the microbial distribution of early RA patients, particularly female patients. In the present study, we investigated the gut microbiome profile and microbial functions in early RA female patients, including preclinical and clinically apparent RA cases. Changes in microbiological diversity, composition, and function in each group were analyzed using quantitative insights into microbial ecology (QIIME) and phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt). The results revealed the dysbiosis due to decreased diversity in the early RA patients compared with healthy subjects. There were significant differences in the microbial distribution of various taxa from phylum to genus levels between healthy subjects and early RA patients. Phylum Bacteroidetes was enriched in early RA patients, while Actinobacteria, including the genus Collinsella, was enriched in healthy subjects. Functional analysis based on clusters of orthologous groups revealed that the genes related to the biosynthesis of menaquinone, known to be derived from gram-positive bacteria, were enriched in healthy subjects, while iron transport-related genes were enriched in early RA patients. Genes related to the biosynthesis of lipopolysaccharide, the gram-negative bacterial endotoxin, were enriched in clinically apparent RA patients. The obvious differences in microbial diversity, taxa, and associated functions of the gut microbiota between healthy subjects and early RA patients highlight the involvement of the gut microbiome in the early stages of RA.

Comparative transcriptome combined with metabolomic and physiological analyses revealed ROS-mediated redox signaling affecting rice growth and cellular iron homeostasis under varying pH conditions

Mechanisms by which soil pH affects rice growth await further elucidation. We have used a Systems Biology approach to elucidate the nature of the damage caused by extreme pH to plant growth and iron homeostasis, and the adaptive plant responses elicited. Optimum pH for rice growth was pH 6. Comparative transcriptome analysis revealed that 83% of 1318 DEGs were down-regulated at pH 4, while 73% among of 1168 DEGs were up-regulated at pH 8. GO enrichment analysis showed significant enhancement of oxidation-reduction and oxidative stress responses. Environmental pH regulated cellular oxidation-reduction processes and metabolic pathways controlling rice growth. Additionally, pH affected cellular iron-homeostasis by regulating root apoplastic iron deposition. Low pH enhanced iron mobilization from root apoplast and accumulation in plant tissues, and down-regulated iron transport related genes to prevent iron toxicity. Conversely,high pH induced blockage of iron mobilization from root apoplast. Rhizosphere pH affected aerenchyma formation and exodermis-apoplastic barriers under control of ROS, already weakened and enhanced by low pH and high pH, respectively. ROS-mediated redox signaling plays an important role in regulating rice growth under varying pH conditions. Cellular iron homeostasis was disturbed through regulation of iron plaque formation and apoplastic iron mobilization in rice roots under acidic and alkaline conditions.

Sucrose is involved in the regulation of iron deficiency responses in rice (Oryza sativa L.)

Sucrose signaling pathways were rapidly induced in response to early iron deficiency in rice plants, and the change of sucrose contents in plants was essential for the activation of iron deficiency responses. Sucrose is the main product of photosynthesis in plants, and it functions not only as an energy metabolite but also a signal molecule. However, a few studies have examined the involvement of sucrose in mediating iron deficiency responses in rice. In this study, we found that the decrease in photosynthesis and total chlorophyll concentration (SPAD values) in leaves occurred at a very early stage under iron deficiency. In addition, the sucrose was increased in leaves but decreased in roots of rice plants under iron deficiency, and also the sucrose transporter (SUT) encoded genes' expression levels in leaves were all inhibited, including OsSUT1, OsSUT2, OsSUT3, OsSUT4, and OsSUT5. The carbohydrate distribution was changed under iron deficiency and sucrose might be involved in the iron deficiency responses of rice plants. Furthermore, exogenous application of sucrose or dark treatment experiments were used to test the hypothesis; we found that the increased endogenous sucrose would cause the repression of iron acquisition-related genes in roots, while further stimulated iron transport-related genes in leaves. Compared to the exogenous application of sucrose, the dark treatment had the opposite effects. All the above results highlighted the important role of sucrose in regulating the responses of rice plants to iron deficiency.

Knockdown of copper-transporting ATPase 1 (Atp7a) impairs iron flux in fully-differentiated rat (IEC-6) and human (Caco-2) intestinal epithelial cells

Intestinal iron absorption is highly regulated since no mechanism for iron excretion exists. We previously demonstrated that expression of an intestinal copper transporter (Atp7a) increases in parallel with genes encoding iron transporters in the rat duodenal epithelium during iron deprivation (Am. J. Physiol.: Gastrointest. Liver Physiol., 2005, 288, G964-G971). This led us to postulate that Atp7a may influence intestinal iron flux. Therefore, to test the hypothesis that Atp7a is required for optimal iron transport, we silenced Atp7a in rat IEC-6 and human Caco-2 cells. Iron transport was subsequently quantified in fully-differentiated cells plated on collagen-coated, transwell inserts. Interestingly, 59Fe uptake and efflux were impaired in both cell lines by Atp7a silencing. Concurrent changes in the expression of key iron transport-related genes were also noted in IEC-6 cells. Expression of Dmt1 (the iron importer), Dcytb (an apical membrane ferrireductase) and Fpn1 (the iron exporter) was decreased in Atp7a knockdown (KD) cells. Paradoxically, cell-surface ferrireductase activity increased (>5-fold) in Atp7a KD cells despite decreased Dcytb mRNA expression. Moreover, increased expression (>10-fold) of hephaestin (an iron oxidase involved in iron efflux) was associated with increased ferroxidase activity in KD cells. Increases in ferrireductase and ferroxidase activity may be compensatory responses to increase iron flux. In summary, in these reductionist models of the mammalian intestinal epithelium, Atp7a KD altered expression of iron transporters and impaired iron flux. Since Atp7a is a copper transporter, it is a logical supposition that perturbations in intracellular copper homeostasis underlie the noted biologic changes in these cell lines.

ATP7a Silencing Impairs Iron Transport in Fully‐Differentiated Rat Intestinal Epithelial (IEC‐6) Cells

Intestinal iron absorption is tightly regulated since no active excretory pathways for iron exist in mammals. Iron homeostasis isintertwined withthat of copper (Cu), but specific molecular interactions are yet to be defined. To further evaluate iron‐copper interplay in the mammalian intestine, low, control, or high‐iron diets (3, 50and 10,000 ppm, respectively) were fed to weanling rats ad libitum for 5 weeks. Rats fed the low‐iron diet developed iron‐deficiency anemia, and genes involved in duodenal Fe homeostasis increased significantly as compared to the other groups. Interestingly, genes encoding proteins involved in Cu homeostasis were also upregulated in the low Fe group, including copper‐transporting ATPase 1 (ATP7a) and metallothionein Low iron Control iron High iron Hemoglobin 2.8 (0.29)**** 13.09 (0.64) 13.14 (0.64) Hematocrit (%) 14.05 (2.58)**** 50.61 (4.78) 50.47 (1.15) Atp7a mRNA 6.34 (2.69)**** 1.03 (0.28) 2.18 (1.31) Metallothionein 1a 12.59 (15.81)* 1.85 (2.40) 1.30 (0.94) Dmt1 mRNA 179.89 (84.5)**** 1.07 (0.42) 1.67 (0.88) Dcytb mRNA 6939.76 (4386.76)**** 1.70 (1.88) 17.56 (21.17) Ferroportin1 mRNA 3.95 (1.55)**** 1.15 (0.62) 1.66 (1.13) Transferrin receptor 1 mRNA 10.67 (2.83)**** 1.09 (0.47) 0.71 (0.25) We thus hypothesized that ATP7a function is important for intestinal iron metabolism. To test this postulate, we blocked ATP7a expression in rat IEC‐6 cells by shRNA‐mediated gene silencing. Notably, ATP7a knock down (KD) impaired Fe uptake and efflux in fully‐differentiated cells. Moreover, ATP7a KD decreased doudenal cytochrome b and ferroportin 1 mRNA expression, while conversely, hephaestin (Heph) gene expression increased significantlyimageConsistent with Heph induction, ferroxidase (FOX) activity in total cell lysates increased in the KD cellsimageThese observations demonstrate that ATP7a activity is necessary for iron absorption in IEC‐6 cells. Altered expression of iron transport‐related genes in the KD cells could be the underlying molecular mechanism by which ATP7A alters iron flux. Furthermore, increased Heph expression and FOX activity may be a compensatory response to increase iron transport.

Both immanently high active iron contents and increased root ferrous uptake in response to low iron stress contribute to the iron deficiency tolerance in Malus xiaojinensis

To better understand the mechanism of low-iron stress tolerance in Malus xiaojinensis, the differences in physiological parameters and gene expression between an iron deficiency-sensitive species, Malus baccata, and an iron deficiency-tolerant species, M. xiaojinensis were investigated under low-iron (4μM Fe) conditions. Under iron sufficient conditions, the expressions of iron uptake- and transport-related genes, i.e. FIT1, IRT1, CS1, FRD3 and NRMAP1, and the immanent leaf and root active iron contents were higher in M. xiaojinensis than those in M. baccata. However, on the first three days of low iron stress, the rhizospheric pH decreased and the root ferric chelate reductase (FCR) activity and the expression of ferrous uptake- and iron transport-related genes in the roots increased significantly only in M. xiaojinensis. Leaf chlorosis occurred on the 3rd and the 9th day after low-iron treatment in M. baccata and M. xiaojinensis, respectively. The expression of iron relocalization-related genes, such as NAS1, FRD3 and NRMAP3, increased after the 5th or 6th day of low iron stress in leaves of M. xiaojinensis, whereas the expression of NAS1, FRD3 and NRMAP3 in the leaves of M. baccata increased immediately after the onset of low iron treatment. Conclusively, the relative high active iron contents caused by the immanently active root ferrous uptake and the increased root ferrous uptake in response to low iron stress were the dominant mechanisms for the tolerance to iron deficiency in M. xiaojinensis.

Transcription Factors Sp1 and Hif2α Mediate Induction of the Copper-transporting ATPase (Atp7a) Gene in Intestinal Epithelial Cells during Hypoxia

Genes with G/C-rich promoters were up-regulated in the duodenal epithelium of iron-deficient rats including those encoding iron (e.g. Dmt1 and Dcytb) and copper (e.g. Atp7a and Mt1) metabolism-related proteins. It was shown previously that an intestinal copper transporter (Atp7a) was co-regulated with iron transport-related genes by a hypoxia-inducible transcription factor, Hif2α. In the current study, we sought to test the role of Sp1 in transcriptional regulation of Atp7a expression during iron deprivation/hypoxia. Initial studies in IEC-6 cells showed that mithramycin, an Sp1 inhibitor, reduced expression of Atp7a and iron transport-related genes (Dmt1, Dcytb, and Fpn1) and blocked their induction by CoCl2, a hypoxia mimetic. Consistent with this, overexpression of Sp1 increased endogenous Atp7a mRNA and protein expression and stimulated Atp7a, Dmt1, and Dcytb promoter activity. Site-directed mutagenesis and functional analysis of a basal Atp7a promoter construct revealed four functional Sp1 binding sites that were necessary for Hif2α-mediated induction of promoter activity. Furthermore, chromatin immunoprecipitation (ChIP) assays confirmed that Sp1 specifically interacts with the Atp7a promoter in IEC-6 cells and in rat duodenal enterocytes. This investigation has thus revealed a novel aspect of Atp7a gene regulation in which Sp1 may be necessary for the HIF-mediated induction of gene transcription during iron deficiency/hypoxia. Understanding regulation of Atp7a expression may help further clarify the physiological role of copper in the maintenance of iron homeostasis. Furthermore, this Sp1/Hif2α regulatory mechanism may have broader implications for understanding the genetic response of the intestinal epithelium to maintain whole-body iron homeostasis during states of deficiency.

Investigation of Iron Metabolism in Mice Expressing a Mutant Menke’s Copper Transporting ATPase (Atp7a) Protein with Diminished Activity (Brindled; MoBr/y)

During iron deficiency, perturbations in copper homeostasis have frequently been documented. Previous studies in iron-deprived rats demonstrated that enterocyte and hepatic copper levels increase and a copper transporter (the Menkes Copper ATPase; Atp7a) is induced in the duodenal epithelium in parallel to iron transport-related genes (e.g. Dmt1, Dcytb, Fpn1). Moreover, two ferroxidase proteins involved in iron homeostasis, hephaestin expressed in enterocytes and ceruloplasmin, produced and secreted into blood by the liver, are copper-dependent enzymes. We thus aimed to test the hypothesis that Atp7a function is important for the copper-related compensatory response of the intestinal epithelium to iron deficiency. Accordingly, iron homeostasis was studied for the first time in mice expressing a mutant Atp7a protein with minimal activity (Brindled [MoBr /y]). Mutant mice were rescued by perinatal copper injections, and, after a 7–8 week recovery period, were deprived of dietary iron for 3 weeks (along with WT littermates). Adult MoBr /y mice displayed copper-deficiency anemia but had normal iron status; in contrast, iron-deprived MoBr /y mice were iron deficient and more severely anemic with partial amelioration of the copper-deficient phenotype. Intestinal iron absorption in both genotypes (WT and MoBr /y) increased ∼3-fold when mice consumed a low-iron diet and ∼6-fold when mice were concurrently bled. WT mice exhibited no alterations in copper homeostasis in response to iron deprivation or phlebotomy. Conversely, upregulation of iron absorption was associated with increased enterocyte and liver copper levels and serum ferroxidase (ceruloplasmin) activity in MoBr /y mice, typifying the response to iron deprivation in many mammalian species. We thus speculate that a copper threshold exists that is necessary to allow appropriate regulate of iron absorption. In summary, MoBr /y mice were able to adequately regulate iron absorption, but unlike in WT mice, concurrent increases in enterocyte and liver copper levels and serum ferroxidase activity may have contributed to maintenance of iron homeostasis.

Regulation of iron transport related genes by boron in the marine bacterium Marinobacter algicola DG893

While there has been extensive interest in the use of boron isotope ratios as a surrogate of pH in paleoclimate studies in the context of climate change-related questions, the high (0.4 mM) concentration and the depth-independent (conservative or non-nutrient-like) concentration profile of this element have led to boron being neglected as a potentially biologically relevant element in the modern ocean. Here we report that boron affects the expression of a number of protein and genes in the "algal-associated" Gram-negative marine bacterium Marinobacter algicola DG893. Most intriguingly, a number of these proteins and genes are related to iron uptake. In a recent separate publication we have shown that boron regulates one such iron transport related protein, i.e. the periplasmic iron binding protein FbpA via a direct interaction of the metalloid with this protein. Here we show that a number of other iron uptake related genes are also affected by boron but in the opposite way i.e. they are up-regulated. We propose that the differential effect of boron on FbpA expression relative to other iron transport related genes is a result of an interaction between boron and the global iron regulatory protein Fur.

Involvement of Sp1 in Transcriptional Regulation of Atp7a During Hypoxia

Genes with GC‐rich promoters were preferentially induced in the rodent intestine during iron deficiency. Moreover, promoters of iron homeostasis related genes (e.g. Dmt1, Dcytb) contain evolutionarily conserved Sp1 binding sites (GC‐rich sequences). The Menkes copper ATPase (Atp7a) gene was strongly induced during iron deficiency, and we previously demonstrated that Atp7a was co‐regulated with iron transport related genes by HIF2α. In this study, we sought to test the role of Sp‐like trans‐acting factors in transcriptional regulation of Atp7a. Phylogenetic footprinting revealed several conserved Sp‐like binding sites in the Atp7a promoter. These sites were mutated in a −224 bp promoter construct driving the luciferase reporter gene. Several of these sites participated in the induction of promoter activity in response to hypoxia in transfected rat intestinal epithelial (IEC‐6) cells. Moreover, phosphorylation of Sp1 increased in IEC‐6 cells exposed to hypoxia; Sp1 is known to be regulated by phosphorylation, which increases DNA binding and trans‐activation properties. Further studies will explore Sp factor interaction with the Atp7a promoter by chromatin immunoprecipitation (ChIP) assay in IEC‐6 cells and in intestine of iron deficient rats. Current data suggest that synergistic interactions between Sp1 (or a related factor) and HIF2α mediate induction of Atp7a gene expression during hypoxia.Grant Funding Source : 1R01 DK074867

EfeO-cupredoxins: major new members of the cupredoxin superfamily with roles in bacterial iron transport

The EfeUOB system of Escherichia coli is a tripartite, low pH, ferrous iron transporter. It resembles the high-affinity iron transporter (Ftr1p-Fet3p) of yeast in that EfeU is homologous to Ftr1p, an integral-membrane iron-permease. However, EfeUOB lacks an equivalent of the Fet3p component--the multicopper oxidase with three cupredoxin-like domains. EfeO and EfeB are periplasmic but their precise roles are unclear. EfeO consists primarily of a C-terminal peptidase-M75 domain with a conserved 'HxxE' motif potentially involved in metal binding. The smaller N-terminal domain (EfeO-N) is predicted to be cupredoxin (Cup) like, suggesting a previously unrecognised similarity between EfeO and Fet3p. Our structural modelling of the E. coli EfeO Cup domain identifies two potential metal-binding sites. Site I is predicted to bind Cu(2+) using three conserved residues (C41 and 103, and E66) and M101. Of these, only one (C103) is conserved in classical cupredoxins where it also acts as a Cu ligand. Site II most probably binds Fe(3+) and consists of four well conserved surface Glu residues. Phylogenetic analysis indicates that the EfeO-Cup domains form a novel Cup family, designated the 'EfeO-Cup' family. Structural modelling of two other representative EfeO-Cup domains indicates that different subfamilies employ distinct ligand sets at their proposed metal-binding sites. The ~100 efeO homologues in the bacterial sequence databases are all associated with various iron-transport related genes indicating a common role for EfeO-Cup proteins in iron transport, supporting a new copper-iron connection in biology.

Genomewide Expression Profiling of Cryptolepine-Induced Toxicity in Saccharomyces cerevisiae

We have used the budding yeast Saccharomyces cerevisiae to identify genes that may confer sensitivity in vivo to the antimalarial and cytotoxic agent cryptolepine. Five S. cerevisiae strains, with different genetic backgrounds in cell permeability and DNA damage repair mechanisms, were exposed to several concentrations of cryptolepine. Cryptolepine showed a relatively mild toxicity for wild-type strains, which was augmented by either increasing cell permeability (Deltaerg6 or ISE2 strains) or disrupting DNA damage repair (Deltarad52 strains). These results are compatible with the ability of cryptolepine to intercalate into DNA and thus promote DNA lesions. The effects of low concentrations of cryptolepine (20% and 40% inhibitory concentrations [IC(20) and IC(40)]) were analyzed by comparing the gene expression profiles of treated and untreated Deltaerg6 yeast cells. Significant changes in expression levels were observed for 349 genes (117 upregulated and 232 downregulated). General stress-related genes constituted the only recognizable functional cluster whose expression was increased upon cryptolepine treatment, making up about 20% of upregulated genes. In contrast, analysis of the characteristics of downregulated genes revealed a specific effect of cryptolepine on genes related to iron transport or acid phosphatases, as well as a significant proportion of genes related to cell wall components. The effects of cryptolepine on the transcription of iron transport-related genes were consistent with a loss of function of the iron sensor Aft1p, indicating a possible disruption of iron metabolism in S. cerevisiae. Since the interference of cryptolepine with iron metabolism is considered one of its putative antimalarial targets, this finding supports the utility of S. cerevisiae in drug-developing schemes.