Iron Uptake Transport Research Articles

Influence of Divalent and Trivalent Sources of Iron on Growth, Iron Uptake-Transport and Photosynthetic Pigments in Rice (Oryza sativa L.) Cultivars During the Vegetative Stage

ABSTRACT Rice (Oryza sativa L.) is the most significant staple food globally. It can cultivate in a wide range of environments. Iron (Fe) deficiency is an important abiotic stress in crop production. To deal with Fe deficiency, plants have evolved mechanisms for Fe uptake. The study aims to examine the abilities of the cultivars – Biga İncisi, Osmancık-97, Hamzadere, Ronaldo, and Edirne – to utilize both non-chelated (FeSO4·7 H2O) and chelated forms (Fe(II)-EDTA and Fe(III)-EDTA) of Fe, which exist in divalent and trivalent states, along with their physiological responses to these different forms of Fe. All cultivars were grown in a sand culture under Fe-deficient (Fe:0) and -sufficient conditions (45 µM Fe). Roots of all rice cultivars grown under Fe deficiency conditions exhibited both phytosiderophore (PS) release and ferric chelate reductase (FCR) activity. These results indicate that all cultivars can employ a Combined Strategy (Strategy I/II) mechanism for Fe uptake. Nevertheless, the efficiency of rice plants in utilizing Strategy I and Strategy II mechanisms for Fe uptake varied depending on the cultivars. Hamzadere exhibited higher PS production, whereas Ronaldo displayed higher FCR activity. The application of divalent and trivalent Fe sources significantly increased the dry weight of shoots, Fe uptake by shoots and roots, and the pigment content of leaves in the rice cultivars. For other cultivars except Hamzadere and Edirne, the utilization rate of Fe(II)-EDTA was higher than that of Fe(III)-EDTA and FeSO4.7 H2O. The cultivar Osmancık-97 had the highest utilization rate (21.30%) of Fe(II)-EDTA.

Gallium Uncouples Iron Metabolism to Enhance Glioblastoma Radiosensitivity.

Gallium-based therapy has been considered a potentially effective cancer therapy for decades and has recently re-emerged as a novel therapeutic strategy for the management of glioblastoma tumors. Gallium targets the iron-dependent phenotype associated with aggressive tumors by mimicking iron in circulation and gaining intracellular access through transferrin-receptor-mediated endocytosis. Mechanistically, it is believed that gallium inhibits critical iron-dependent enzymes like ribonucleotide reductase and NADH dehydrogenase (electron transport chain complex I) by replacing iron and removing the ability to transfer electrons through the protein secondary structure. However, information regarding the effects of gallium on cellular iron metabolism is limited. As mitochondrial iron metabolism serves as a central hub of the iron metabolic network, the goal of this study was to investigate the effects of gallium on mitochondrial iron metabolism in glioblastoma cells. Here, it has been discovered that gallium nitrate can induce mitochondrial iron depletion, which is associated with the induction of DNA damage. Moreover, the generation of gallium-resistant cell lines reveals a highly unstable phenotype characterized by impaired colony formation associated with a significant decrease in mitochondrial iron content and loss of the mitochondrial iron uptake transporter, mitoferrin-1. Moreover, gallium-resistant cell lines are significantly more sensitive to radiation and have an impaired ability to repair any sublethal damage and to survive potentially lethal radiation damage when left for 24 h following radiation. These results support the hypothesis that gallium can disrupt mitochondrial iron metabolism and serve as a potential radiosensitizer.

Abstract PO4-28-05: Prolactin drives labile iron transfer from macrophages to breast cancer cells through CD44

Abstract Cancer cells are “ferrophilic” and exhibit great dependence on iron. While iron metabolism is closely related to tumor development, the source of iron and the mechanisms cancer cells adopt to actively acquire iron is not well understood. In the present study, we discovered a novel function of the lactating hormone, prolactin, in the regulation of iron transport. In breast cancer cells using mouse triple negative breast cancer cell lines EO771 and Py230 for demonstration, prolactin stimulation increased intracellular labile iron pool. Analyses of gene expression involved in iron transport revealed dramatic induction of CD44, a surface receptor for iron bound hyaluronan. Other genes involved in iron uptake including transferrin receptor (TFRC), CD163 and divalent metal transporter 1 (DMT1) were not changed. The utilization of neutralizing antibodies against CD44 significantly blocked prolactin mediated labile iron pool accumulation in breast cancer cells. In contrast to what was found in breast cancer cells, prolactin had the opposite impact on immune macrophages. Prolactin treatment increased ferroportin (FPN I, iron exporter) but had no influence on all iron uptake transporters leading to net iron release by macrophages. To further determine if the iron released by macrophages can be a direct source of iron utilized by breast cancer cells, we conducted co-culture work of macrophages with pre-stained labile iron pool and breast cancer cells without any iron staining and found a gradual increase of fluorescent iron pool developed in breast cancer cells over time. In conclusion, our work presents a multidisciplinary regulation of iron transport among prolactin, macrophages, and cancer cells. The novel regulatory role of prolactin to drive iron flow can provide new information on fine-tuning immune responses in tumor microenvironment and potentially benefit the development of novel therapeutics. Citation Format: Reagan Farrell, Nicholas Pascuzzi, Yi-Ling Chen, Miguel Torres, Mary Kim, Lauren Gollahon, Kuan-Hui Chen. Prolactin drives labile iron transfer from macrophages to breast cancer cells through CD44 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-05.

Reduced expression of bZIP19 and bZIP23 increases zinc and cadmium accumulation in Arabidopsis halleri.

Zinc is an essential micronutrient for all living organisms. When challenged by zinc-limiting conditions, Arabidopsis thaliana plants use a strategy centered on two transcription factors, bZIP19 and bZIP23, to enhance the expression of several zinc transporters to improve their zinc uptake capacity. In the zinc and cadmium hyperaccumulator plant Arabidopsis halleri, highly efficient root-to-shoot zinc translocation results in constitutive local zinc deficiency in roots and in constitutive high expression of zinc deficiency-responsive ZIP genes, supposedly boosting zinc uptake and accumulation. Here, to disrupt this process and to analyze the functions of AhbZIP19, AhbZIP23 and their target genes in hyperaccumulation, the genes encoding both transcriptional factors were knocked down using artificial microRNAs (amiRNA). Although AhbZIP19, AhbZIP23, and their ZIP target genes were downregulated, amiRNA lines surprisingly accumulated more zinc and cadmium compared to control lines in both roots and shoot driving to shoot toxicity symptoms. These observations suggested the existence of a substitute metal uptake machinery in A. halleri to maintain hyperaccumulation. We propose that the iron uptake transporter AhIRT1 participates in this alternative pathway in A. halleri.

A FtsZ Inhibitor That Can Utilize Siderophore-Ferric Iron Uptake Transporter Systems for Activity against Gram-Negative Bacterial Pathogens.

The global threat of multidrug-resistant Gram-negative bacterial pathogens necessitates the development of new and effective antibiotics. FtsZ is an essential and highly conserved cytoskeletal protein that is an appealing antibacterial target for new antimicrobial therapeutics. However, the effectiveness of FtsZ inhibitors against Gram-negative species has been limited due in part to poor intracellular accumulation. To address this limitation, we have designed a FtsZ inhibitor (RUP4) that incorporates a chlorocatechol siderophore functionality that can chelate ferric iron (Fe3+) and utilizes endogenous siderophore uptake pathways to facilitate entry into Gram-negative pathogens. We show that RUP4 is active against both Klebsiella pneumoniae and Acinetobacter baumannii, with this activity being dependent on direct Fe3+ chelation and enhanced under Fe3+-limiting conditions. Genetic deletion studies in K. pneumoniae reveal that RUP4 gains entry through the FepA and CirA outer membrane transporters and the FhuBC inner membrane transporter. We also show that RUP4 exhibits bactericidal synergy against K. pneumoniae when combined with select antibiotics, with the strongest synergy observed with PBP2-targeting β-lactams or MreB inhibitors. In the aggregate, our studies indicate that incorporation of Fe3+-chelating moieties into FtsZ inhibitors is an appealing design strategy for enhancing activity against Gram-negative pathogens of global clinical significance.

Direct interaction of small non-coding RNAs CjNC140 and CjNC110 optimizes expression of key pathogenic phenotypes of Campylobacter jejuni.

Small non-coding RNAs (sRNAs) are important players in modulating gene expression in bacterial pathogens, but their functions are largely undetermined in Campylobacter jejuni, an important cause of foodborne gastroenteritis in humans. In this study, we elucidated the functions of sRNA CjNC140 and its interaction with CjNC110, a previously characterized sRNA involved in the regulation of several virulence phenotypes of C. jejuni. Inactivation of CjNC140 increased motility, autoagglutination, L-methionine concentration, autoinducer-2 production, hydrogen peroxide resistance, and early chicken colonization, indicating a primarily inhibitory role of CjNC140 for these phenotypes. Apart from motility, all these effects directly contrasted the previously demonstrated positive regulation by CjNC110, suggesting that CjNC110 and CjNC140 operate in an opposite manner to modulate physiologic processes in C. jejuni. RNAseq and northern blotting further demonstrated that expression of CjNC140 increased in the absence of CjNC110, while expression of CjNC110 decreased in the absence of CjNC140, suggesting a possibility of their direct interaction. Indeed, electrophoretic mobility shift assay demonstrated a direct binding between the two sRNAs via GA- (CjNC110) and CU- (CjNC140) rich stem-loops. Additionally, RNAseq and follow-up experiments identified that CjNC140 positively regulates p19, which encodes a key iron uptake transporter in Campylobacter. Furthermore, computational analysis revealed both CjNC140 and CjNC110 are highly conserved in C. jejuni, and the predicted secondary structures support CjNC140 as a functional homolog of the iron regulatory sRNA, RyhB. These findings establish CjNC140 and CjNC110 as a key checks-and- balances mechanism in maintaining homeostasis of gene expression and optimizing phenotypes critical for C. jejuni pathobiology. IMPORTANCE Gene regulation is critical to all aspects of pathogenesis of bacterial disease, and small non-coding RNAs (sRNAs) represent a new frontier in gene regulation of bacteria. In Campylobacter jejuni, the role of sRNAs remains largely unexplored. Here, we investigate the role of two highly conserved sRNAs, CjNC110 and CjNC140, and demonstrate that CjNC140 displays a primarily inhibitory role in contrast to a primarily activating role for CjNC110 for several key virulence-associated phenotypes. Our results also revealed that the sRNA regulatory pathway is intertwined with the iron uptake system, another virulence mechanism critical for in vivo colonization. These findings open a new direction for understanding C. jejuni pathobiology and identify potential targets for intervention for this major foodborne pathogen.

Non-mitochondrial aconitase regulates the expression of iron-uptake genes by controlling the RNA turnover process in fission yeast.

Aconitase, a highly conserved protein across all domains of life, functions in converting citrate to isocitrate in the tricarboxylic acid cycle. Cytosolic aconitase is also known to act as an iron regulatory protein in mammals, binding to the RNA hairpin structures known as iron-responsive elements within the untranslated regions of specific RNAs. Aconitase-2 (Aco2) in fission yeast is a fusion protein consisting of an aconitase and a mitochondrial ribosomal protein, bL21, residing not only in mitochondria but also in cytosol and the nucleus. To investigate the role of Aco2 in the nucleus and cytoplasm of fission yeast, we analyzed the transcriptome of aco2ΔN mutant that is deleted of nuclear localization signal (NLS). RNA sequencing revealed that the aco2ΔN mutation caused increase in mRNAs encoding iron uptake transporters, such as Str1, Str3, and Shu1. The half-lives of mRNAs for these genes were found to be significantly longer in the aco2ΔN mutant than the wild-type strain, suggesting the role of Aco2 in mRNA turnover. The three conserved cysteines required for the catalytic activity of aconitase were not necessary for this role. The UV cross-linking RNA immunoprecipitation analysis revealed that Aco2 directly bound to the mRNAs of iron uptake transporters. Aco2-mediated degradation of iron-uptake mRNAs appears to utilize exoribonuclease pathway that involves Rrp6 as evidenced by genetic interactions. These results reveal a novel role of non-mitochondrial aconitase protein in the mRNA turnover in fission yeast to fine-tune iron homeostasis, independent of regulation by transcriptional repressor Fep1.

Porphyromonas gingivalis outside the oral cavity.

Porphyromonas gingivalis, a Gram-negative anaerobic bacillus present in periodontal disease, is considered one of the major pathogens in periodontitis. A literature search for English original studies, case series and review articles published up to December 2019 was performed using the MEDLINE, PubMed and GoogleScholar databases, with the search terms "Porphyromonas gingivalis" AND the potentially associated condition or systemic disease Abstracts and full text articles were used to make a review of published research literature on P. gingivalis outside the oral cavity. The main points of interest of this narrative review were: (i) a potential direct action of the bacterium and not the systemic effects of the inflammatory acute-phase response induced by the periodontitis, (ii) the presence of the bacterium (viable or not) in the organ, or (iii) the presence of its virulence factors. Virulence factors (gingipains, capsule, fimbriae, hemagglutinins, lipopolysaccharide, hemolysin, iron uptake transporters, toxic outer membrane blebs/vesicles, and DNA) associated with P. gingivalis can deregulate certain functions in humans, particularly host immune systems, and cause various local and systemic pathologies. The most recent studies linking P. gingivalis to systemic diseases were discussed, remembering particularly the molecular mechanisms involved in different infections, including cerebral, cardiovascular, pulmonary, bone, digestive and peri-natal infections. Recent involvement of P. gingivalis in neurological diseases has been demonstrated. P. gingivalis modulates cellular homeostasis and increases markers of inflammation. It is also a factor in the oxidative stress involved in beta-amyloid production.

A multifaceted cellular damage repair and prevention pathway promotes high-level tolerance to β-lactam antibiotics.

Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two-component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high-level β-lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that β-lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the ∆vxrAB mutant. Mutating major iron uptake systems dramatically increases ∆vxrAB tolerance to β-lactams. We propose that VxrAB reduces antibiotic-induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against β-lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganism's ability to counteract diverse antibiotic-induced stresses promotes high-level antibiotic tolerance and highlights the complex secondary responses elicited by antibiotics.

Targeting Iron Metabolism for Colorectal Cancer Treatment

Hypoxia is a key feature of most solid tumors including colorectal cancer (CRC). Hypoxia‐inducible factors (HIFs) are the major transcription factors that respond to hypoxia and are essential for tumor survival and growth. However, the main downstream pathways of HIF‐mediated tumor growth are not clear. We show that HIF‐mediated dysregulation of intratumoral intestinal iron homeostasis through induction of the iron uptake transporter divalent metal transporter 1 (DMT1) and a ferrireductase six‐transmembrane epithelial antigen of prostate 4 (STEAP4) is a major mechanism leading to CRC. Decreasing intratumoral iron by low iron diet or germ‐line Dmt1 knockout inhibits, whereas genetic overexpression of STEAP4 promotes CRC progression. If this is a druggable pathway and could serve as a therapeutic method for CRC is not known. We generated a mouse model with a temporal and conditional disruption of Dmt1 gene in the intestine (VilERT2 Dmt1F/F) by crossing Dmt1F/F mice with Villin‐CreERT2 transgenic mice, carrying a tamoxifen‐inducible Cre. Colorectal tumors were first established in this mouse model by one dose of azoxymethane (AOM) and three cycles of 1.5% dextran sulfate sodium (DSS), and then mice were treated with three daily doses of 100 mg/kg tamoxifen to disrupt Dmt1 in established colorectal tumor epithelium. One month later tumor number, tumor size at 2–3 mm and 3–4 mm, and tumor burden were significantly reduced in VilERT2 Dmt1F/F mice compared to their littermate control (Dmt1F/F). Consistent with the findings in human CRC, the gene expression of Dmt1 was increased in the tumor tissues compared to normal adjacent tissues in littermate control mice, and significantly decreased in both normal and tumor tissues in VilERT2 Dmt1F/F mice. Immunofluorescence staining revealed that cell proliferation, as determined by bromodeoxyuridine (BrdU) and Ki67 staining, was not changed, but cell apoptosis assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 (cCasp3) staining was increased in colorectal tumor tissues from VilERT2 Dmt1F/F mice compared to control mice. In addition, tumor vascularization and macrophage infiltration were not altered. These results demonstrate that Dmt1 is critical for tumor growth in the AOM‐DSS‐induced mouse colitis‐tumor model, and targeting iron metabolism is a feasible strategy for CRC treatment.Support or Funding InformationX.X. was supported by NIH Grant 1K01DK114390‐01 and a Research Scholar Award from the American Gastroenterological Association. Y.M.S. was supported by NIH Grants CA148828 and DK095201.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cell death induced by mitochondrial complex I inhibition is mediated by Iron Regulatory Protein 1

Mitochondrial dysfunction and oxidative damage, often accompanied by elevated intracellular iron levels, are pathophysiological features in a number of neurodegenerative processes. The question arises as to whether iron dyshomeostasis is a consequence of mitochondrial dysfunction. Here we have evaluated the role of Iron Regulatory Protein 1 (IRP1) in the death of SH-SY5Y dopaminergic neuroblastoma cells subjected to mitochondria complex I inhibition. We found that complex I inhibition was associated with increased levels of transferrin receptor 1 (TfR1) and iron uptake transporter divalent metal transporter 1 (DMT1), and decreased levels of iron efflux transporter Ferroportin 1 (FPN1), together with increased 55Fe uptake activity and an increased cytoplasmic labile iron pool. Complex I inhibition also resulted in increased oxidative modifications and increased cysteine oxidation that were inhibited by the iron chelators desferoxamine, M30 and Q1. Silencing of IRP1 abolished the rotenone-induced increase in 55Fe uptake activity and it protected cells from death induced by complex I inhibition. IRP1 knockdown cells presented higher ferritin levels, a lower iron labile pool, increased resistance to cysteine oxidation and decreased oxidative modifications. These results support the concept that IRP1 is an oxidative stress biosensor that mediates iron accumulation and cell death when deregulated by mitochondrial dysfunction. IRP1 activation, secondary to mitochondrial dysfunction, may underlie the events leading to iron dyshomeostasis and neuronal death observed in neurodegenerative disorders with an iron accumulation component.

Molecular mechanisms of non‐transferrin‐bound and transferring‐bound iron uptake in primary hippocampal neurons

The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell-derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co-localize, indicating these two proteins may function in Fe(3+) reduction prior to Fe(2+) permeation. Zip8, DMT1, and Steap2 co-localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin-mediated iron assimilation. In brain interstitial fluid, transferring-bound iron (TBI) and non-transferrin-bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin-(59) Fe(3+)) and NTBI, whether presented as (59) Fe(2+) -citrate or (59) Fe(3+) -citrate; reductase-independent (59) Fe(2+) uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn(2+) inhibition of Fe(2+) uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of (59) Fe from TBI relies at least in part on an endocytosis-independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons. Analysis of the expression and localization of known iron uptake transporters demonstrated that Zip8 makes a major contribution to iron accumulation in primary cultures of rat embryonic hippocampal neurons. These cells exhibit uptake pathways for ferrous and ferric iron (non-transferrin-bound iron, NTBI in figure) and for transferrin-bound iron; the ferrireductases Steap2 and SDR2 support the uptake of ferric iron substrates. Zip8 and Steap2 are strongly expressed in the plasma membrane of both soma and processes, implying a crucial role in iron accumulation from NTBI and transferrin-bound iron (TBI) by hippocampal neurons.

The roles of mitoferrin-2 in the process of arsenic trioxide-induced cell damage in human gliomas.

BackgroundAmong glioma treatment strategies, arsenic trioxide (As2O3) has shown efficacy as a therapeutic agent against human gliomas. However, the exact antitumor mechanism of action of As2O3 is still unclear. Mitochondria are considered to be the major source of intracellular reactive oxygen species (ROS), which are known to be associated with As2O3-induced cell damage. Therefore, we investigated whether mitoferrin-2, a mitochondrial iron uptake transporter, participates in As2O3-induced cell killing in human gliomas.MethodsHuman glioma cell lines were used to explore the mechanism of As2O3’s antitumor effects. First, expression of mitoferrin-2 was analyzed in glioma cells that were pretreated with As2O3. Changes in ROS production and apoptosis were assessed. Furthermore, cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).ResultsIn the present study we found that As2O3 induced ROS production and apoptosis in glioma cells. In addition, gene expression of mitoferrin-2, a mitochondrial iron uptake transporter, was increased 4 to 5 fold after exposure to As2O3 (5 μM) for 48 hours. Furthermore, apoptosis and cytotoxicity induced by As2O3 in glioma cells were decreased after silencing the mitoferrin-2 gene.ConclusionsOur findings indicated that mitoferrin-2 participates in mitochondrial ROS-dependent mechanisms underlying As2O3-mediated damage in glioma cells.

Transport proteins promoting Escherichia coli pathogenesis

Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies.

Ezetimibe Increases Hepatic Iron Levels in Mice Fed a High-Fat Diet

Accumulating evidence suggests that ezetimibe may be a promising agent for treatment of nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH). Phlebotomy and dietary iron restriction reduce serum transaminase in NAFLD/NASH patients. Recent studies have shown that a mutual effect exists between lipid metabolism and iron metabolism. Accordingly, we examined the effect of ezetimibe on iron metabolism in mice fed a high-fat diet with or without iron. We fed C57BL/6 mice the following diets for 12 weeks. Experiment 1 comprised [1] a control diet (C), [2] C plus ezetimibe (0.3 mg/day; 4 weeks) (CE), [3] a high-fat diet (H), and [4] H plus ezetimibe (HE). Experiment 2 comprised [1] C containing carbonyl iron (average; 22.4 mg/day; 6 weeks) (CI), [2] CI plus ezetimibe (CIE), [3] H containing carbonyl iron (HI), and [4] HI plus ezetimibe (HIE). Blood, livers, and duodenum were removed after 12 weeks. In experiment 1, the hepatic iron levels were higher in HE than H, whereas there was no difference between C and CE. Hepatic mRNA expression of transferrin receptor 1 and 2, ferritins, and hepcidin were increased more in CE than C, and more in HE than H. In the duodenum, divalent metal transporter 1, ferritin H, and hephaestin mRNA levels were increased in CE compared with C. In experiment 2, hepatic iron concentrations were higher in HIE than HI. Hepatic mRNA expression of ferritin L and hepcidin were increased in HIE compared with HI. In duodenum, ferritin L mRNA was increased in HIE compared with CIE. Ezetimibe induced hepatic iron uptake transporter expression in mice fed a high-fat diet, causing increased hepatic iron concentrations.

Quantitative Proteomic Analysis of Cell Wall and Plasma Membrane Fractions from Multidrug-Resistant Acinetobacter baumannii

Acinetobacter baumannii is a Gram-negative, nonmotile aerobic bacterium that has emerged as an important nosocomial pathogen. Multidrug-resistant (MDR) A. baumannii is difficult to treat with antibiotics, and treatment failure in infected patients is of great concern in clinical settings. To investigate proteome regulation in A. baumannii under antibiotic stress conditions, quantitative membrane proteomic analyses of a clinical MDR A. baumannii strain cultured in subminimal inhibitory concentrations of tetracycline and imipenem were performed using a combination of label-free (one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry) and label (isobaric tag for relative and absolute quantitation) approaches. In total, 484 proteins were identified, and 302 were classified as outer membrane, periplasmic, or plasma membrane proteins. The clinical A. baumannii strain DU202 responded specifically and induced different cell wall and membrane protein sets that provided resistance to the antibiotics. The induction of resistance-nodulation-cell division transporters and protein kinases, and the repression of outer membrane proteins were common responses in the presence of tetracycline and imipenem. Induction of a tetracycline resistant pump, ribosomal proteins, and iron-uptake transporters appeared to be dependent on tetracycline conditions, whereas β-lactamase and penicillin-binding proteins appeared to be dependent on imipenem conditions. These results suggest that combined liquid chromatography-based proteomic approaches can be used to identify cell wall and membrane proteins involved in the antibiotic resistance of A. baumannii.

Insights into how nucleotide-binding domains power ABC transport.

SummaryThe mechanism by which nucleotide-binding domains (NBDs) of ABC transporters power the transport of substrates across cell membranes is currently unclear. Here we report the crystal structure of an NBD, FbpC, from the Neisseria gonorrhoeae ferric iron uptake transporter with an unusual and substantial domain swap in the C-terminal regulatory domain. This entanglement suggests that FbpC is unable to open to the same extent as the homologous protein MalK. Using molecular dynamics we demonstrate that this is not the case: both NBDs open rapidly once ATP is removed. We conclude from this result that the closed structures of FbpC and MalK have higher free energies than their respective open states. This result has important implications for our understanding of the mechanism of power generation in ABC transporters, because the unwinding of this free energy ensures that the opening of these two NBDs is also powered.

Localization of Iron in Arabidopsis Seed Requires the Vacuolar Membrane Transporter VIT1

Iron deficiency is a major human nutritional problem wherever plant-based diets are common. Using synchrotron x-ray fluorescence microtomography to directly visualize iron in Arabidopsis seeds, we show that iron is localized primarily to the provascular strands of the embryo. This localization is completely abolished when the vacuolar iron uptake transporter VIT1 is disrupted. Vacuolar iron storage is also critical for seedling development because vit1-1 seedlings grow poorly when iron is limiting. We have uncovered a fundamental aspect of seed biology that will ultimately aid the development of nutrient-rich seed, benefiting both human health and agricultural productivity.

The Iron/Lead Transporter Superfamily of Fe3+/Pb2+ Uptake Systems

Oxidase-dependent ferrous iron uptake transporters of the OFeT family and lead uptake transporters of the PbrT family comprise the iron/lead transporter (ILT) superfamily (transporter classification No. 9.A.10). All sequenced homologues of the ILT superfamily were multiply aligned, and conserved motifs, including fully conserved acidic residues in putative transmembrane segments (TMSs) 1 and 4, previously implicated in heavy metal binding, were identified. Topological analyses confirmed the presence of 7 conserved TMSs in a 3 + 3 + 1 arrangement where the two 3 TMS elements are internally repeated. Phylogenetic analyses revealed the presence of several sequence divergent clusters of orthologous proteins that group roughly according to the phylogenes of the organisms of origin. The results serve to characterize and provide evolutionary insight into a novel superfamily of heavy metal uptake transporters.

1055 Health-related quality of life assessment in hereditary hemochromatosis

Mitochondrial dysfunction and oxidative damage, often accompanied by elevated intracellular iron levels, are pathophysiological features in a number of neurodegenerative processes. The question arises as to whether iron dyshomeostasis is a consequence of mitochondrial dysfunction. Here we have evaluated the role of Iron Regulatory Protein 1 (IRP1) in the death of SH-SY5Y dopaminergic neuroblastoma cells subjected to mitochondria complex I inhibition. We found that complex I inhibition was associated with increased levels of transferrin receptor 1 (TfR1) and iron uptake transporter divalent metal transporter 1 (DMT1), and decreased levels of iron efflux transporter Ferroportin 1 (FPN1), together with increased 55Fe uptake activity and an increased cytoplasmic labile iron pool. Complex I inhibition also resulted in increased oxidative modifications and increased cysteine oxidation that were inhibited by the iron chelators desferoxamine, M30 and Q1. Silencing of IRP1 abolished the rotenone-induced increase in 55Fe uptake activity and it protected cells from death induced by complex I inhibition. IRP1 knockdown cells presented higher ferritin levels, a lower iron labile pool, increased resistance to cysteine oxidation and decreased oxidative modifications. These results support the concept that IRP1 is an oxidative stress biosensor that mediates iron accumulation and cell death when deregulated by mitochondrial dysfunction. IRP1 activation, secondary to mitochondrial dysfunction, may underlie the events leading to iron dyshomeostasis and neuronal death observed in neurodegenerative disorders with an iron accumulation component.