Iron-replete Cells Research Articles

Human IRP1 Translocates to the Nucleus in a Cell-Specific and Iron-Dependent Manner.

Iron regulatory protein 1 (IRP1) is a bifunctional protein with mutually exclusive RNA-binding or enzymatic activities that depend on the presence of a 4Fe-4S cluster. While IRP1 is a well-established cytosolic protein, work in a Drosophila model suggested that it may also exhibit nuclear localization. Herein, we addressed whether mammalian IRP1 can likewise translocate to the nucleus. We utilized primary cells and tissues from wild type and Irp1−/− mice, as well as human cell lines and tissue biopsy sections. IRP1 subcellular localization was analyzed by Western blotting, immunofluorescence and immunohistochemistry. We did not detect presence of nuclear IRP1 in wild type mouse embryonic fibroblasts (MEFs), primary hepatocytes or whole mouse liver. However, we observed IRP1-positive nuclei in human liver but not ovary sections. Biochemical fractionation studies revealed presence of IRP1 in the nucleus of human Huh7 and HepG2 hepatoma cells, but not HeLa cervical cancer cells. Importantly, nuclear IRP1 was only evident in iron-replete cells and disappeared following pharmacological iron chelation. These data provide the first experimental evidence for nuclear IRP1 expression in mammals, which appears to be species- and cell-specific. Furthermore, they suggest that the nuclear translocation of IRP1 is mediated by an iron-dependent mechanism.

Interactive effects of iron and light limitation on the molecular physiology of the Southern Ocean diatom Fragilariopsis kerguelensis

AbstractThe polar diatom Fragilariopsis kerguelensis is ubiquitous in Southern Ocean waters and is a major responder to iron fertilization, encountering large gradients in iron concentrations and light availability. We performed a comparative transcriptomic analysis of F. kerguelensis grown under varying iron and light conditions in order to investigate the molecular underpinnings that may explain its physiological response to iron and light limitation. Low iron reduced growth rates more than low light, but there was not an additive effect of low iron and low light on growth rate. Low iron treatments (saturating and low light) had the largest transcriptomic response; however, expression patterns were more similar in low light treatments (high and low iron). Under iron and light limitation, carbon fixation and amino acid, ribosome, and sulfur metabolisms were overrepresented relative to the control (iron replete, saturating light). Transcripts of genes encoding for the proteins aquaporin, proteorhodopsin, plastocyanin, and flavodoxin were overrepresented to the greatest extent in the low iron/low light treatment, indicating there may be an additive effect of iron/light colimitation at the molecular level. Iron and light replete cells demonstrated increased expression of genes encoding for the proteins ferritin, carbonic anhydrase, and numerous iron‐dependent proteins relative to the growth‐limiting treatments. This transcriptome analysis reveals mechanisms that may underpin the ecological success of this diatom in low iron and light environments, highlighting the important role of diversified photosynthetic isoforms, iron acquisition, unique detoxification mechanisms of reactive oxygen species, and metabolic shifts in amino acid recycling and carbon metabolism.

Aberrant Responsiveness of Erythropoiesis to Iron Deficiency in Polycythemia Vera

Polycythemia vera (PV) presents with iron deficiency at diagnosis, and the mainstay of treatment, i.e. therapeutic phlebotomy, worsens iron deficiency. Iron deficiency is associated with anemia and symptoms of cognitive impairment and fatigue even in the absence of anemia, and patients with low risk PV often suffer from iron deficiency related symptoms How iron deficiency develops in PV patients prior to phlebotomy is not well understood. We previously demonstrated that PV patients exhibit a greater extent of iron deficiency relative to wild type JAK2 patients with other causes of erythrocytosis [Ginzburg Leuk 2018]. We hypothesize that mutated JAK2 leads to aberrant insensitivity of erythropoiesis to iron deficiency. To explore this hypothesis, we first analyzed serum data from iron deficient PV patients (n=14) and blood donors (n=15), normalized for age (51 vs. 42 years, P=0.12) and serum ferritin concentration (22 vs. 22 ng/ml, P=0.95); our data demonstrate that PV patients have significantly lower hepcidin, transferrin saturation, MCV, a higher HCT, and a trend toward higher erythroferrone (ERFE) relative to controls (Fig 1a-1e). Secondly, CD34+ cells were isolated from mononuclear cells and plated with erythropoietin and either 100% or 10% transferrin saturation to mimic iron replete and iron deficient conditions, respectively; differentiation and proliferation were analyzed using flow cytometry. These experiments revealed that although glycophorin A (GPA) and CD36+ cells were decreased in iron deficient relative to iron replete control cells, PV cells continue to proliferate irrespective of iron status (Fig 1f). In addition, only iron deficient control, not PV, cells demonstrated an erythroid lineage specific decrease in proliferation relative to iron replete cells (Fig 1g), demonstrating that iron restriction in PV does not limit erythroid differentiation or proliferation in vitro. Thirdly, we transplanted JAK2 V617F (PV) and wild type (WT) cells into recipient females and placed mice on 35ppm (iron replete (IR)) or 2.5ppm (iron deficient (ID)) diets. IR PV mice exhibited the expected erythrocytosis and decrease in MCV and MCH relative to WT controls (Fig 2a-2d). WT mice on an ID diet exhibited decreased MCV, MCH, and RET-He while PV mice had decreased RBC counts with an increased MCHC (Fig 2a, 2c-2f). These findings demonstrate altered iron regulation in PV erythroblasts with a preference for decreasing RBC count, rather than cellular Hb production, in iron deficiency in vivo. No differences were found in the total number of bone marrow erythroblasts in PV mice relative to WT mice on IR or ID diets. IR and ID PV mice demonstrated splenomegaly relative to WT controls. In addition, IR PV erythroblasts expressed significantly more ERFE relative to WT controls with decreased ERFE expression in ID PV erythroblasts (Fig 2g). Similarly, liver hepcidin expression was lower in IR PV relative to WT controls, but was restored in ID PV mice (Fig 2h), the later likely a response to decreased ERFE expression (Fig 2g); no changes are observed in liver iron concentration in IR relative to ID PV mice. Furthermore, the expected decrease in pSTAT is observed only in ID WT (Fig 2i), not ID PV (Fig 2j) erythroblasts. Lastly, TfR2 protein expression was increased in IR PV relative to WT controls (Fig 2k) and decreased only in ID WT (Fig 2l) but not ID PV (Fig 2m) erythroblasts. Since TfR2 degradation is enhanced during iron deficiency [Khalil JEM 2018] and TfR2 enables iron delivery for mitochondrial heme synthesis [Khalil Blood Adv 2017], persistently increased TfR2 in PV erythroblasts may explain why cellular Hb synthesis (i.e. MCH and RET-He) remains unaffected by iron deficiency in PV mice. Taken together, these findings demonstrate that in vitro iron deficiency fails to limit differentiation and proliferation in PV erythroblasts and enhances STAT5 signaling in ID PV erythroblasts in vivo but results in decreased circulating RBCs in PV mice. In addition, decreased erythroblast ERFE expression in ID PV mice results in increased hepcidin to limit erythroblast iron availability, but persistently increased TfR2 concentration enables mitochondrial iron delivery for Hb synthesis despite cellular iron deficiency. Our studies provide novel mechanistic insights into the dysregulation of iron utilization for erythropoiesis in PV. Disclosures Levine: Prelude Therapeutics: Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Research Funding; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Novartis: Consultancy; Gilead: Consultancy; Lilly: Honoraria; Loxo: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Isoplexis: Membership on an entity's Board of Directors or advisory committees. Hoffman:Merus: Research Funding. Ginzburg:La Jolla Pharma: Membership on an entity's Board of Directors or advisory committees.

Sensing Changes in Cellular Iron Metabolism: Regulation of IRP1 by FBXL5 and Cytosolic Iron‐Sulfur Cluster Assembly

Iron is a micronutrient essential for the viability of nearly all cells, but in excess, iron can overwhelm antioxidant defense mechanisms resulting in damage of cellular structures and cell death. Therefore, cellular iron levels must be maintained within narrow limits to prevent iron deficiency and iron toxicity. In order to maintain optimal iron levels, mammalian cells utilize Iron Regulatory Proteins (IRP). IRP1 and IRP2 are iron‐regulated RNA binding proteins that control the fate of mRNAs encoding proteins important for maintaining cellular iron homeostasis. For example, in iron replete cells IRP RNA binding is inactivated resulting in decreased iron uptake by transferrin receptor 1 and increased iron storage by ferritin. IRP1 RNA binding is inactivated primarily by the insertion of a [4Fe–4S] cluster by the cytosolic iron‐sulfur cluster assembly (CIA) system converting it into the cytosolic isoform of aconitase (c‐acon). IRP1 can also be inactivated by the iron‐dependent E3 ligase FBXL5 that also targets IRP2. Since, human diseases associated with impaired Fe‐S cluster assembly can disrupt cellular iron metabolism, we sought to understand the mechanism by which CIA and FBXL5 regulate IRP1. I will discuss a model for how CIA and FBXL5 collaborate to control the level of IRP1 RNA binding activity and optimize iron metabolism to meet cellular needs for proliferation.Support or Funding InformationThis work was supported by National Institutes of Health Grant RO1 DK66600, United States Department of Agriculture Hatch Project WIS01667, and the Iron Metabolism Research Fund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The photosynthetic cytochrome c 550 from the diatom Phaeodactylum tricornutum.

The photosynthetic cytochrome c 550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c 550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c 550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c 550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c 550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c 550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c 550 decreases up to about 45% as compared to iron-replete cells, pointing to an iron-regulated synthesis. Oxidized cytochrome c 550 has been characterized using continuous wave EPR and pulse techniques, including HYSCORE, and the obtained results have been interpreted in terms of the electrostatic charge distribution in the surroundings of the heme centre.

Iron Deficiency Induces a Partial Inhibition of the Photosynthetic Electron Transport and a High Sensitivity to Light in the Diatom Phaeodactylum tricornutum

Iron limitation is the major factor controlling phytoplankton growth in vast regions of the contemporary oceans. In this study, a combination of thermoluminescence (TL), chlorophyll fluorescence, and P700 absorbance measurements have been used to elucidate the effects of iron deficiency in the photosynthetic electron transport of the marine diatom P. tricornutum. TL was used to determine the effects of iron deficiency on photosystem II (PSII) activity. Excitation of iron-replete P. tricornutum cells with single turn-over flashes induced the appearance of TL glow curves with two components with different peaks of temperature and contributions to the total signal intensity: the B band (23°C, 63%), and the AG band (40°C, 37%). Iron limitation did not significantly alter these bands, but induced a decrease of the total TL signal. Far red excitation did not increase the amount of the AG band in iron-limited cells, as observed for iron-replete cells. The effect of iron deficiency on the photosystem I (PSI) activity was also examined by measuring the changes in P700 redox state during illumination. The electron donation to PSI was substantially reduced in iron-deficient cells. This could be related with the important decline on cytochrome c6 content observed in these cells. Iron deficiency also induced a marked increase in light sensitivity in P. tricornutum cells. A drastic increase in the level of peroxidation of chloroplast lipids was detected in iron-deficient cells even when grown under standard conditions at low light intensity. Illumination with a light intensity of 300 μE m-2 s-1 during different time periods caused a dramatic disappearance in TL signal in cells grown under low iron concentration, this treatment not affecting to the signal in iron-replete cells. The results of this work suggest that iron deficiency induces partial blocking of the electron transfer between PSII and PSI, due to a lower concentration of the electron donor cytochrome c6. This decreased electron transfer may induce the over-reduction of the plastoquinone pool and consequently the appearance of acceptor side photoinhibition in PSII even at low light intensities. The functionality of chlororespiratory electron transfer pathway under iron restricted conditions is also discussed.

The Presence of the IsiA-PSI Supercomplex Leads to Enhanced Photosystem I Electron Throughput in Iron-Starved Cells of Synechococcus sp. PCC 7002.

Photosystem I (PS I) is highly demanding of iron, requiring 12 atoms in the bound FX, FB, and FA iron-sulfur clusters and two atoms in the mobile acceptor protein ferredoxin. When grown under iron-limiting conditions, certain cyanobacteria express IsiA, a peripheral chlorophyll a antenna protein, and IsiB, a flavodoxin that substitutes for ferredoxin. The IsiA protein forms single and double rings around PS I, presumably to increase the optical cross-section so as to compensate for fewer PS I complexes. Previous studies have shown that IsiA serves as an efficient light-harvesting structure ( Andrizhievskaya , G. G. ; et al. Biochim. Biophys. Acta 2002 , 1556 , 262 - 272 ); however, few, if any, studies have been carried out to show that the increased optical cross-section leads to an enhanced rate of electron transfer through PS I. Here, we report a more rapid transient accumulation of the A1(-) phyllosemiquinone anion radical by EPR spectroscopy in dark-adapted iron-depleted cells than in iron-replete cells after a block of intense light. A derivative-shaped optical signal in the light-minus-dark difference spectrum of PS I from an electrochromic bandshift of a carotenoid located near the A1 phylloquinones is enhanced in iron-depleted wild-type cells and in an iron-depleted isiB deletion strain, which lacks flavodoxin, but is greatly diminished in an iron-depleted isiA deletion strain, which lacks IsiA and flavodoxin. These findings indicate that the transient accumulation of electrons on A1 occurs more rapidly in the IsiA/PS I supercomplex than in the PS I complex alone. Thus, the increased absorption cross-section from the IsiA proteins translates directly to an enhanced rate of electron transfer through PS I.

F-box and Leucine-rich Repeat Protein 5 (FBXL5) Is Required for Maintenance of Cellular and Systemic Iron Homeostasis

Maintenance of cellular iron homeostasis requires post-transcriptional regulation of iron metabolism genes by iron regulatory protein 2 (IRP2). The hemerythrin-like domain of F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, senses iron and oxygen availability and facilitates IRP2 degradation in iron replete cells. Disruption of the ubiquitously expressed murine Fbxl5 gene results in a failure to sense increased cellular iron availability, accompanied by constitutive IRP2 accumulation and misexpression of IRP2 target genes. FBXL5-null mice die during embryogenesis, although viability is restored by simultaneous deletion of the IRP2, but not IRP1, gene. Mice containing a single functional Fbxl5 allele behave like their wild type littermates when fed an iron-sufficient diet. However, unlike wild type mice that manifest decreased hematocrit and hemoglobin levels when fed a low-iron diet, Fbxl5 heterozygotes maintain normal hematologic values due to increased iron absorption. The responsiveness of IRP2 to low iron is specifically enhanced in the duodena of the heterozygotes and is accompanied by increased expression of the divalent metal transporter-1. These results confirm the role of FBXL5 in the in vivo maintenance of cellular and systemic iron homeostasis and reveal a privileged role for the intestine in their regulation by virtue of its unique FBXL5 iron sensitivity.

Systems and trans-system level analysis identifies conserved iron deficiency responses in the plant lineage.

We surveyed the iron nutrition-responsive transcriptome of Chlamydomonas reinhardtii using RNA-Seq methodology. Presumed primary targets were identified in comparisons between visually asymptomatic iron-deficient versus iron-replete cells. This includes the known components of high-affinity iron uptake as well as candidates for distributive iron transport in C. reinhardtii. Comparison of growth-inhibited iron-limited versus iron-replete cells revealed changes in the expression of genes in chloroplastic oxidative stress response pathways, among hundreds of other genes. The output from the transcriptome was validated at multiple levels: by quantitative RT-PCR for assessing the data analysis pipeline, by quantitative proteomics for assessing the impact of changes in RNA abundance on the proteome, and by cross-species comparison for identifying conserved or universal response pathways. In addition, we assessed the functional importance of three target genes, Vitamin C 2 (VTC2), monodehydroascorbate reductase 1 (MDAR1), and conserved in the green lineage and diatoms 27 (CGLD27), by biochemistry or reverse genetics. VTC2 and MDAR1, which are key enzymes in de novo ascorbate synthesis and ascorbate recycling, respectively, are likely responsible for the 10-fold increase in ascorbate content of iron-limited cells. CGLD27/At5g67370 is a highly conserved, presumed chloroplast-localized pioneer protein and is important for growth of Arabidopsis thaliana in low iron.

The Structural Basis of Iron Sensing by the Human F‐box Protein FBXL5

Iron is an essential chemical element for all forms of life. It serves as a cofactor for many proteins and enzymes involved in oxygen transport, energy metabolism and DNA synthesis[1, 2]. Mammalian cells need to maintain a sufficient amount of iron to support the synthesis of proteins that require iron as a co-factor. Iron is imported into the cells through the circulating iron transporter transferrin[3, 4]. Iron-loaded transferrin binds to cell surface transferrin receptor, resulting in the endocytosis of transferrin and delivery of the iron cargo into the cells[4]. Excess iron in the cells is stored in the cytosolic protein ferritin[4]. Iron regulatory proteins IRP1 and IRP2 maintain the homeostasis of iron through post-translational regulation of the expression of transferrin receptor and ferritin[2, 5]. In iron-replete cells IRP2 is ubiquitinated and degraded by the proteasome. The F-box and leucine-rich repeat containing protein FBXL5 serves as a cytosolic iron sensor that regulates the ubiquitination of IRP2 by the SKP1-CUL1-FBXL5 (SCF) E3 ubiquitin ligase complex[6, 7]. FBXL5 also plays critical roles in sensing oxygen in the cytosol[6, 7]. Knock out of FBXL5 in mice result in embryonic mortality due to excess accumulation of iron[8].

Distinct Mechanisms of Ferritin Delivery to Lysosomes in Iron-Depleted and Iron-Replete Cells

Ferritin is a cytosolic protein that stores excess iron, thereby protecting cells from iron toxicity. Ferritin-stored iron is believed to be utilized when cells become iron deficient; however, the mechanisms underlying the extraction of iron from ferritin have yet to be fully elucidated. Here, we demonstrate that ferritin is degraded in the lysosome under iron-depleted conditions and that the acidic environment of the lysosome is crucial for iron extraction from ferritin and utilization by cells. Ferritin was targeted for degradation in the lysosome even under iron-replete conditions in primary cells; however, the mechanisms underlying lysosomal targeting of ferritin were distinct under depleted and replete conditions. In iron-depleted cells, ferritin was targeted to the lysosome via a mechanism that involved autophagy. In contrast, lysosomal targeting of ferritin in iron-replete cells did not involve autophagy. The autophagy-independent pathway of ferritin delivery to lysosomes was deficient in several cancer-derived cells, and cancer-derived cell lines are more resistant to iron toxicity than primary cells. Collectively, these results suggest that ferritin trafficking may be differentially regulated by cell type and that loss of ferritin delivery to the lysosome under iron-replete conditions may be related to oncogenic cellular transformation.

Redox control of iron regulatory protein 2 stability

Iron regulatory protein 2 (IRP2) is a critical switch for cellular and systemic iron homeostasis. In iron-deficient or hypoxic cells, IRP2 binds to mRNAs containing iron responsive elements (IREs) and regulates their expression. Iron promotes proteasomal degradation of IRP2 via the F-box protein FBXL5. Here, we explored the effects of oxygen and cellular redox status on IRP2 stability. We show that iron-dependent decay of tetracycline-inducible IRP2 proceeds efficiently under mild hypoxic conditions (3% oxygen) but is compromised in severe hypoxia (0.1% oxygen). A treatment of cells with exogenous H 2O 2 protects IRP2 against iron and increases its IRE-binding activity. IRP2 is also stabilized during menadione-induced oxidative stress. These data demonstrate that the degradation of IRP2 in iron-replete cells is not only oxygen-dependent but also sensitive to redox perturbations.

IsiA Is Required for the Formation of Photosystem I Supercomplexes and for Efficient State Transition in Synechocystis PCC 6803

Iron deficiency and other stress conditions strongly impact photosynthetic apparatus in photosynthetic organisms. Two novel chlorophyll (Chl)-containing supercomplexes (F4 and F5) in addition to the photosystem (PS) I trimers (F3) were observed by sucrose gradient ultracentrifugation in Synechocystis PCC 6803 under extensive iron starvation. 77K fluorescence and Western blot analyses of these supercomplexes revealed that they all contained IsiA. The F4 was identified as an IsiA-PSI-PSII supercomplex, while the F5 was assigned as an IsiA-PSI supercomplex. Deletion of isiA resulted in diminishing the PSI trimers (including the PSI trimers in iron-replete cells) and the two novel PSI supercomplexes (F4 and F5), and a significant reduction in the saturated whole-chain electron transport rate. However, the maximum PSII activities remained at levels similar to those of the wild type under various light conditions. The isiA- mutant was defective in state transition and sensitive to high light. The sensitivity of the isiA- mutant to high light was correlated with a higher level of membrane peroxidation. These results demonstrated that IsiA is required for the formation of PSI trimers and other higher complexes, and that IsiA is critical for efficient state transition.

Diatom elemental and morphological changes in response to iron limitation: a brief review with potential paleoceanographic applications

Diatoms are a major group of phytoplankton that account for approximately 40% of the ocean carbon fixation and the vast majority of biogenic silica production through the construction of their cell walls (termed frustules). These frustules accumulate and are partially preserved in the ocean sediments. Diatom growth and nutrient utilization in high-nitrate, low-chlorophyll regions of the world's oceans are mostly regulated by iron availability. Diatoms acclimate to iron limitation by decreasing cell size. The associated increase in surface area-to-volume ratio and decrease in diffusive boundary layer thickness may improve nutrient uptake kinetics. In parallel, cellular silicon (Si) contents are elevated in iron-limited diatoms relative to nitrogen (N) and carbon (C). Variations in degree of silicification and nutritional requirements of iron-limited diatoms have been hypothesized to account for higher cellular Si and/or lower cellular N and C, respectively. However, in some diatoms, frustule silicification does not significantly change when cells are iron-limited. Instead, changes in the Si-containing valve surface area relative to volume within these diatoms is hypothesized to be responsible for the variations in the cellular Si : N and Si : C ratios. In particular, some examined iron-limited pennate diatoms have reduced widths relative to their lengths (i.e. lower length-normalized widths, LNW) compared to iron-replete cells. In the pennate diatom Fragilariopsis kerguelensis, the mean LNWs of valves preserved in sediments throughout the Southern Ocean (a well-characterized iron-limited region) is positively correlated with satellite-derived, climatological net primary productivity in the overlying waters. Because of the specific morphological changes in pennate diatom frustules in response to iron availability, the valve morphometerics (e.g. LNWs) can potentially be used as a diagnostic tool for iron-limited diatom growth and relative changes in the Si : N (and Si : C) ratios in extant diatom assemblages as well as those preserved in the sediments.

Alterations in Helicobacter pylori outer membrane and outer membrane vesicle-associated lipopolysaccharides under iron-limiting growth conditions*

Outer membrane vesicles (OMVs) shed from the gastroduodenal pathogen Helicobacter pylori have measurable effects on epithelial cell responses. The aim of this study was to determine the effect of iron availability, and its basis, on the extent and nature of lipopolysaccharide (LPS) produced on H. pylori OMVs and their parental bacterial cells. Electrophoretic, immunoblotting and structural analyses revealed that LPSs of bacterial cells grown under iron-limited conditions were notably shorter than those of bacteria and OMVs obtained from iron-replete conditions. Structural analysis and serological probing showed that LPSs of iron-replete cells and OMVs expressed O-chains of Lewis(x) with a terminal Lewis(y) unit, whereas Lewis(y) expression was notably reduced on bacteria and OMVs from iron-limiting conditions. Unlike the O-chain, the core oligosaccharide and lipid A moieties of iron-replete and iron-limited bacteria and their OMVs were similar. Quantitatively, shed OMVs from iron-replete bacteria were found to be LPSenriched, whereas shed OMVs from iron-limited bacteria had a significantly reduced content of LPS. These differences were linked to bacterial ATP levels. Since iron availability affects the extent and nature of LPS expressed by H. pylori, host iron status may contribute to H. pylori pathogenesis.

Excessive irradiance and antioxidant responses of an Antarctic marine diatom exposed to iron limitation and to dynamic irradiance

The synergistic effects of iron limitation and irradiance dynamics on growth, photosynthesis, antioxidant activity and excessive PAR (400–700 nm) and UV (280–400 nm) sensitivity were investigated for the Antarctic marine diatom Chaetoceros brevis. Iron-limited and iron-replete cultures were exposed to identical daily irradiance levels, supplied as dynamic (20–1350 μmol m −2 s −1) and constant (260 μmol m −2 s −1) irradiance. After acclimation, growth, maximal quantum yield of PSII ( F v/ F m), pigment composition, and the activities of the antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) were determined. Then, excessive irradiance sensitivity was assessed by monitoring pigment composition, F v/ F m and viability loss during a single excessive PAR and UV treatment. Iron limitation reduced growth rates, F v/ F m dynamics, and cellular pigments pools. Cellular pigment concentrations were higher under dynamic irradiance than under constant irradiance but this difference was less pronounced under iron limitation compared to iron-replete conditions. SOD and APX activities increased during dynamic irradiance under iron limitation, suggesting increased radical formation around PSII. Despite these physiological differences, no effects on growth were observed between constant and dynamic irradiance cultivation in iron-limited and iron-replete cells. The applied culturing conditions did not affect glutathione reductase activity in C. brevis. F v/ F m and xanthophyll de-epoxidation dynamics during excessive irradiance were not different for iron-limited and replete cells and viability loss was not found during excessive irradiance. This study revealed photoacclimation differences between iron-limited and iron-replete C. brevis cultures that did not affect growth rates and excessive irradiance sensitivity after acclimation to constant and dynamic irradiance.

FER1 and FER2 Encoding Two Ferritin Complexes in Chlamydomonas reinhardtii Chloroplasts Are Regulated by Iron

Two unlinked genes FER1 and FER2 encoding ferritin subunits were identified in the Chlamydomonas genome. An improved FER2 gene model, built on the basis of manual sequencing and incorporation of unplaced reads, indicated 49% identity between the ferritin subunits. Both FER1 and FER2 transcripts are increased in abundance as iron nutrition is decreased but the pattern for each gene is distinct. Using subunit-specific antibodies, we monitored expression at the protein level. In response to low iron, ferritin1 subunits and the ferritin1 complex are increased in parallel to the increase in FER1 mRNA. Nevertheless, the iron content of the ferritin1 complex is decreased. This suggests that increased expression results in increased capacity for iron binding in the chloroplast of iron-limited cells, which supports a role for ferritin1 as an iron buffer. On the other hand, ferritin2 abundance is decreased in iron-deprived cells, indicative of the operation of iron-nutrition-responsive regulation at the translational or post-translational level for FER2. Both ferritin subunits are plastid localized but ferritin1 is quantitatively recovered in soluble extracts of cells while ferritin2 is found in the particulate fraction. Partial purification of the ferritin1 complex indicates that the two ferritins are associated in distinct complexes and do not coassemble. The ratio of ferritin1 to ferritin2 is 70:1 in iron-replete cells, suggestive of a more dominant role of ferritin1 in iron homeostasis. The Volvox genome contains orthologs of each FER gene, indicating that the duplication of FER genes and potential diversification of function occurred prior to the divergence of species in the Volvocales.

A role for lysosomes in the turnover of human iron regulatory protein 2

The two mammalian iron regulatory proteins, IRP1 and IRP2, are post-transcriptional regulators of cellular iron homeostasis. These cytosolic RNA-binding proteins control the synthesis of proteins involved in storage, transport, and utilization of iron. Whereas IRP1 levels remain nearly constant, IRP2 is rapidly degraded by the proteasome in iron-replete cells. In non iron-loaded H1299 human lung cancer cells, the decay of transfected hemagglutinin-tagged IRP2 was significantly antagonized by addition of not only proteasomal, but also lysosomal inhibitors. Similar results were obtained with IRP2 -Ins5, a molecular form lacking the specific IRP2 domain of 73 amino acids that is absent from IRP1. These data uncover an alternative, iron independent, mechanism of IRP2 degradation via the lysosomal pathway. Transfected IRP1 decayed slowly over several days and, in contrast to IRP2, was not further stabilized by proteasomal or lysosomal inhibitors. Experiments with an IRP1/IRP2 hybrid molecule and with IRP2 variants indicated that proteins lacking the C-terminus of IRP2 were insensitive to lysosomal inhibitors. Together with previous data obtained in the presence of iron excess, these results show that the parallel degradation pathways through lysosomes and the proteasome that are active on IRP2 under normal growth conditions are preferentially shifted to the proteasome when iron becomes plentiful.

Insights on Regulation and Function of the Iron Regulatory Protein 1 (IRP1)

Iron regulatory protein 1 (IRP1) controls the translation or stability of several mRNAs by binding to iron responsive elements (IREs) within their untranslated regions. Its activity is regulated by an unusual iron-sulfur cluster (ICS) switch. Thus, in iron-replete cells, IRP1 assembles a cubane [4Fe-4S] cluster that prevents RNA-binding activity and renders the protein to cytosolic aconitase. We show that wild type or mutant forms of IRP1 that fail to assemble a [4Fe-4S] cluster are sensitized for iron-dependent degradation by the ubiquitin-proteasome pathway. The regulation of IRP1 abundance poses an alternative mechanism to prevent accumulation of inappropriately high IRE-binding activity when the ICS assembly pathway is impaired. To study functional aspects of IRP1, we overexpressed wild type or mutant forms of the protein in human H1299 lung cancer cells in a tetracycline-inducible fashion, and analyzed how this affects cell growth. While the induction of IRP1 did not affect cell proliferation in culture, it dramatically reduced the capacity of the cells to form solid tumor xenografts in nude mice. These data provide a first link between IRP1 and cancer.

Human iron regulatory protein 2 is easily cleaved in its specific domain: consequences for the haem binding properties of the protein

Mammalian IRPs (iron regulatory proteins), IRP1 and IRP2, are cytosolic RNA-binding proteins that post-transcriptionally control the mRNA of proteins involved in storage, transport, and utilization of iron. In iron-replete cells, IRP2 undergoes degradation by the ubiquitin/proteasome pathway. Binding of haem to a 73aa-Domain (73-amino-acid domain) that is unique in IRP2 has been previously proposed as the initial iron-sensing mechanism. It is shown here that recombinant IRP2 and the 73aa-Domain are sensitive to proteolysis at the same site. NMR results suggest that the isolated 73aa-Domain is not structured. Iron-independent cleavage of IRP2 within the 73aa-Domain also occurs in lung cancer (H1299) cells. Haem interacts with a cysteine residue only in truncated forms of the 73aa-Domain, as shown by a series of complementary physicochemical approaches, including NMR, EPR and UV-visible absorption spectroscopy. In contrast, the cofactor is not ligated by the same residue in the full-length peptide or intact IRP2, although non-specific interaction occurs between these molecular forms and haem. Therefore it is unlikely that the iron-dependent degradation of IRP2 is mediated by haem binding to the intact 73aa-Domain, since the sequence resembling an HRM (haem-regulatory motif) in the 73aa-Domain does not provide an axial ligand of the cofactor unless this domain is cleaved.