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BACKGROUND: Cancer cells often have altered iron metabolism relative to non-malignant cells with increased transferrin receptor and ferritin expression. Targeting iron regulatory proteins as part of a cancer therapy regimen is currently being investigated in various malignancies. Anti-cancer therapies that exploit the differences in iron metabolism between malignant and non-malignant cells (e.g. pharmacological ascorbate and iron chelation therapy) have shown promise in various cancers, including glioblastoma, lung, and pancreas cancers. Non-invasive techniques that probe tissue iron metabolism may provide valuable information for the personalization of iron-based cancer therapies. T(2)* mapping is a clinically available MRI technique that assesses tissue iron content in the heart and liver. We aimed to investigate the capacity of T(2)* mapping to detect iron stores in soft tissue sarcomas (STS). METHODS: In this study, we evaluated T(2)* relaxation times ex vivo in five STS samples from subjects enrolled on a phase Ib/IIa clinical trial combining pharmacological ascorbate with neoadjuvant radiation therapy. Iron protein expression levels (ferritin, transferrin receptor, iron response protein 2) were evaluated by Western blot analysis. Bioinformatic data relating clinical outcomes in STS patients and iron protein expression levels were evaluated using the KMplotter database. RESULTS: There was a high level of inter-subject variability in the expression of iron protein and T(2)* relaxation times. We identified that T(2)* relaxation time is capable of accurately detecting ferritin-heavy chain expression (r = -0.96) in these samples. Bioinformatic data acquired from the KMplot database revealed that transferrin receptor and iron-responsive protein 2 may be negative prognostic markers while ferritin expression may be a positive prognostic marker in the management of STS. CONCLUSION: These data suggest that targeting iron regulatory proteins may provide a therapeutic approach to enhance STS management. Additionally, T(2)* mapping has the potential to be used a clinically accessible, non-invasive marker of STS iron regulatory protein expression and influence cancer therapy decisions that warrants further investigation. Level of Evidence: IV

Disturbance of the brain homeostasis, either directly via the formation of abnormal proteins or cerebral hypo-perfusion, or indirectly via peripheral inflammation, will activate microglia to synthesise a variety of pro-inflammatory agents which may lead to inflammation and cell death. The pro-inflammatory cytokines will induce changes in the iron proteins responsible for maintaining iron homeostasis, such that increased amounts of iron will be deposited in cells in the brain. The generation of reactive oxygen and nitrogen species, which is directly involved in the inflammatory process, can significantly affect iron metabolism via their interaction with iron-regulatory proteins (IRPs). This underlies the importance of ensuring that iron is maintained in a form that can be kept under control; hence, the elegant mechanisms which have become increasingly well understood for regulating iron homeostasis. Therapeutic approaches to minimise the toxicity of iron include N-acetyl cysteine, non-steroidal anti-inflammatory compounds and iron chelation.

Iron is mostly devoted to the hemoglobinization of erythrocytes for oxygen transport. Yet, emerging evidence points to a broader role for the metal in hematopoiesis, including the formation of the immune system. Iron availability in mammalian cells is controlled by iron-regulatory proteins (IRP)-1 and −2. We report that global disruption of both IRP1 and IRP2 in adult mice impairs neutrophil development and differentiation in the bone marrow, yielding immature neutrophils with abnormally high glycolytic and autophagic activity, resulting in neutropenia. IRPs promote neutrophil differentiation in a cell intrinsic manner by securing cellular iron supply together with transcriptional control of neutropoiesis to facilitate differentiation to fully mature neutrophils. Unlike neutrophils, monocyte count was not affected by IRP and iron deficiency, suggesting a lineage-specific effect of iron on myeloid output. This study unveils the previously unrecognized importance of IRPs and iron metabolism in the formation of a major branch of the innate immune system.

A set of common-acting iron-responsive 5′untranslated region (5′UTR) motifs can fold into RNA stem loops that appear significant to the biology of cognitive declines of Parkinson's disease dementia (PDD), Lewy body dementia (LDD), and Alzheimer's disease (AD). Neurodegenerative diseases exhibit perturbations of iron homeostasis in defined brain subregions over characteristic time intervals of progression. While misfolding of Aβ from the amyloid-precursor-protein (APP), alpha-synuclein, prion protein (PrP) each cause neuropathic protein inclusions in the brain subregions, iron-responsive-like element (IRE-like) RNA stem–loops reside in their transcripts. APP and αsyn have a role in iron transport while gene duplications elevate the expression of their products to cause rare familial cases of AD and PDD. Of note, IRE-like sequences are responsive to excesses of brain iron in a potential feedback loop to accelerate neuronal ferroptosis and cognitive declines as well as amyloidosis. This pathogenic feedback is consistent with the translational control of the iron storage protein ferritin. We discuss how the IRE-like RNA motifs in the 5′UTRs of APP, alpha-synuclein and PrP mRNAs represent uniquely folded drug targets for therapies to prevent perturbed iron homeostasis that accelerates AD, PD, PD dementia (PDD) and Lewy body dementia, thus preventing cognitive deficits. Inhibition of alpha-synuclein translation is an option to block manganese toxicity associated with early childhood cognitive problems and manganism while Pb toxicity is epigenetically associated with attention deficit and later-stage AD. Pathologies of heavy metal toxicity centered on an embargo of iron export may be treated with activators of APP and ferritin and inhibitors of alpha-synuclein translation.

The recently solved crystal structures of the human cysteine desulfurase NFS1, in complex with the LYR protein ISD11, the acyl carrier protein ACP and the main scaffold ISCU, have shed light on the molecular interactions that govern initial cluster assembly on ISCU. Here, we aim to highlight recent insights into iron-sulfur (Fe-S) cluster biogenesis in mammalian cells that have arisen from the crystal structures of the core iron sulfur cluster (ISC) assembly complex. We will also discuss how iron sulfur clusters are delivered to recipient proteins, and the challenges that remain in dissecting the pathways that deliver clusters to numerous Fe-S recipient proteins in both the mitochondrial matrix and cytosolic compartments of mammalian cells.

Background During the recombinant protein expression, most heterologous proteins expressed in E. coli cell factories are generated as insoluble and inactive aggregates, which prohibit E. coli from being employed as an expression host despite its numerous advantages and ease of use. The yeast mitochondrial aconitase protein, which has a tendency to aggregate when expressed in E. coli cells in the absence of heterologous chaperones GroEL/ES was utilised as a model to investigate how the modulation of physiological stimuli in the host cell can increase protein solubility. The presence of folding modulators such as exogenous molecular chaperones or osmolytes, as well as process variables such as incubation temperature, inducer concentrations, growth media are all important for cellular folding and are investigated in this study. This study also investigated how the cell's stress response system activates and protects the proteins from aggregation. Results The cells exposed to osmolytes plus a pre-induction heat shock showed a substantial increase in recombinant aconitase activity when combined with modulation of process conditions. The concomitant GroEL/ES expression further assists the folding of these soluble aggregates and increases the functional protein molecules in the cytoplasm of the recombinant E. coli cells. Conclusions The recombinant E. coli cells enduring physiological stress provide a cytosolic environment for the enhancement in the solubility and activity of the recombinant proteins. GroEL/ES-expressing cells not only aided in the folding of recombinant proteins, but also had an effect on the physiology of the expression host. The improvement in the specific growth rate and aconitase production during chaperone GroEL/ES co-expression is attributed to the reduction in overall cellular stress caused by the expression host's aggregation-prone recombinant protein expression. Graphical Abstract

As lung cancer remains the leading cause of cancer deaths globally, characterizing the tumor molecular profiles is crucial to tailoring treatments for individuals at advanced stages. Cancer cells exhibit strong dependence on iron for their proliferation, and several iron-regulatory proteins have been proposed as either oncogenes or tumor suppressive genes. This study aims to evaluate the prospective therapeutic and prognostic values of the sideroflexin (SFXN) gene family, whose functions involve mitochondrial iron metabolism, in lung adenocarcinoma (LUAD). Differential expression analysis using TIMER and UALCAN tools was first employed to compare SFXNs expression levels between normal and LUAD tissues. Next, SFXNs' prognostic values, biological significance, and potential as immunotherapy candidates were examined from GEPIA, cBioPortal, MetaCore, Cytoscape, and TIMER databases. It was found that all members of SFXN family, except SFXN3, were differentially expressed in LUAD compared to normal samples and within different stages of LUAD. Survival analysis then revealed SFXN1 to be related to worse overall survival outcome in patients with LUAD. Furthermore, several correlations between expression of SFXN1 and immune infiltration cells were discovered. To conclude, our study provides evidence of SFXN family gene's relevance to the prognosis and immunotherapeutic targets of LUAD.

22 Pags.- 8 Fgis.- 1 Tabl. © The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License., IRON-REGULATED TRANSPORTER1 (IRT1) is the root high-affinity ferrous iron (Fe) uptake system and indispensable for the completion of the life cycle of Arabidopsis thaliana without vigorous Fe supplementation. Here we provide evidence supporting a second role of IRT1 in root-to-shoot partitioning of Fe. We show that irt1 mutants overaccumulate Fe in roots, most prominently in the cortex of the differentiation zone in irt1-2, compared to the wild type. Shoots of irt1-2 are severely Fe-deficient according to Fe content and marker transcripts, as expected. We generated irt1-2 lines producing IRT1 mutant variants carrying single amino-acid substitutions of key residues in transmembrane helices IV and V, Ser206 and His232, which are required for transport activity in yeast. Root short-term 55Fe uptake rates were uninformative concerning IRT1-mediated transport. Overall irt1-like concentrations of the secondary substrate Mn suggested that the transgenic Arabidopsis lines also remain incapable of IRT1-mediated root Fe uptake. Yet, IRT1S206A partially complements rosette dwarfing and leaf chlorosis of irt1-2, as well as root-to-shoot Fe partitioning and gene expression defects of irt1-2, all of which are fully complemented by wild-type IRT1. Taken together, these results suggest a regulatory function for IRT1 in root-to-shoot Fe partitioning that does not require Fe transport activity of IRT1. Among the genes of which transcript levels are partially dependent on IRT1, we identify MYB DOMAIN PROTEIN10, MYB DOMAIN PROTEIN72 and NICOTIANAMINE SYNTHASE4 as candidates for effecting IRT1-dependent Fe mobilization in roots. Understanding the biological functions of IRT1 will help to improve Fe nutrition and the nutritional quality of agricultural crops., This work was supported by Deutsche Forschungsgemeinschaft grants Kr1967/3-3and 15-1, European Union FOOD-CT-2006-016253 and ERC-AdGLEAP EXTREME (788380) to UK, Ruhr University Bochum, European Union fellowship PIIF-GA-2008-219457 to MJH and Araid-Ibercaja-young scientist grant 2010, as well as Juan de la Cierva(MICINN) and JAE-DOC (CSIC) funding, to MB.

Eukaryotic initiation factor (eIF) 4G plays an important role in assembling the initiation complex required for ribosome binding to mRNA and promote translation. Translation of ferritin IRE mRNAs is regulated by iron through iron responsive elements (IREs) and iron regulatory protein (IRP). The noncoding IRE stem-loop (30-nt) structure control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. High cellular iron concentrations promote IRE RNA binding to ribosome and initiation factors, and allow synthesis of ferritin.Eukaryotic initiation factor eIF4G increases the translation of ferritin through binding to stem loop structure of iron responsive elements mRNA in the 5'-untranslated region. Our translation experiment demonstrated that exogenous addition of eIF4G selectively enhanced the translation of ferritin IRE RNA in depleted WG lysate. However, eIF4G facilitates capped IRE RNA translation significantly higher than uncapped IRE RNA translation. Addition of iron with eIF4G to depleted WG lysate significantly enhanced translation for both IRE mRNA (capped and uncapped), confirming the contribution of eIF4G and iron as a potent enhancer of ferritin IRE mRNA translation. Fluorescence data revealed that ferritin IRE strongly interacts to eIF4G (eIF4G specifically interacts with ferritin IRE RNA and promotes eIF4G-dependent translation.

Maintaining iron homeostasis is fundamental for almost all living being, and its deregulation correlates with severe and debilitating pathologies. The process is made more complicated by the omnipresence of iron and by its role as a fundamental component of a number of crucial metallo proteins. The response to modifications in the amount of the free iron pool is performed via the inhibition of ferritin translation by sequestering consensus messenger RNA (mRNA) sequences. In turn this is regulated by the iron-sensitive conformational equilibrium between aconitase and IRP, mediated by the presence of an iron-sulfur cluster. In this contribution we analyze by full-atom molecular dynamics simulation, the factors leading to both the interaction with mRNA, and the conformational transition. Furthermore, the role of the iron-sulfur cluster in driving the conformational transition is assessed by obtaining the related free energy profile via enhanced sampling molecular dynamics simulations.

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.

Iron responsive elements (IREs) are mRNA stem-loop targets for translational control by the two iron regulatory proteins IRP1 and IRP2. They are found in the untranslated regions (UTRs) of genes that code for proteins involved in iron metabolism. There are ten “classic” IRE types that define the conserved secondary and tertiary structure elements necessary for proper IRP binding, and there are 83 published “IRE-like” sequences, most of which depart from the established IRE model. Here are structurally-guided discussions regarding the essential features of an IRE and what is important for IRE family membership.

Survival of the Acinetobacter baumannii inside host requires different micronutrients such as iron, but their bioavailability is limited because of nutritional immunity created by host. A. baumannii has to develop mechanisms to acquire nutrient iron during infection. The present study is an attempt to identify membrane proteins involved in iron sequestration mechanism of A. baumannii using two-dimensional electrophoresis and LC-MS/MS analysis. The identified iron-regulated membrane protein (IRMP) of A. baumannii was used for its interaction studies with different siderophores, and designing of the inhibitor against A. baumannii targeting this IRMP. Membrane proteomic results identified over-expression of four membrane proteins (Fhu-E receptor, ferric-acinetobactin receptor, ferrienterochelin receptor, and ferric siderophore receptor) under iron-limited condition. A. baumannii produces siderophores that have good interaction with the FhuE receptor. Result also showed that FhuE receptor has interaction with siderophores produced by other bacteria. Interaction of FhuE receptor and siderophores helps in iron sequestration and survival of Acinetobacter under nutritional immunity imposed by the host. Hence it becomes essential to find a potential inhibitor for the FhuE receptor that can inhibit the survival of A. baumannii in the host. In-silico screening, and molecular mechanics studies identified ZINC03794794 and ZINC01530652 as a likely lead to design inhibitor against the FhuE receptor of A. baumannii. The designed inhibitor is experimentally validated for its antibacterial activity on the A. baumannii. Therefore, designed inhibitor interferes with the iron acquisition mechanism of Acinetobacter hence may prove useful for preventing infection caused by A. baumannii by limiting nutrient availability.

Interaction of iron responsive elements (IRE) mRNA with the translational machinery is an early step critical in the initiation of protein synthesis. To investigate the binding specificity of IRE mRNA for eIF4F, kinetic rates for the eIF4F·IRE RNA interactions were determined and correlated with the translational efficiency. The observed rate of eIF4F·FRT IRE RNA interactions was 2-fold greater as compared to eIF4F·ACO2 IRE RNA binding. Addition of iron enhanced the association rates and lowered the dissociation rates for the eIF4F binding to both IRE RNAs, with having higher preferential binding to the FRT IRE RNA. The binding rates of both eIF4F·IRE RNA complexes correlated with the enhancement of protein synthesis in vitro. Presence of iron and eIF4F in the depleted WGE significantly enhanced translation for both IRE RNAs. This suggests that iron promotes translation by enhancing the binding rates of the eIF4F∙IRE RNA complex. eIF4F·IRE RNA binding is temperature-dependent; raising the temperature from 5 to 25°C, enhanced the binding rates of eIF4F·FRT IRE (4-fold) and eIF4F·ACO2 IRE (5-fold). Presence of Fe2+ caused reduction in the activation energy for the binding of FRT IRE and ACO2 IRE to eIF4F, suggesting a more stable platform for initiating protein synthesis. In the presence of iron, lowered energy barrier has leads to the faster association rate and slower rate of dissociation for the protein-RNA complex, thus favoring efficient protein synthesis. Our results correlate well with the observed translational efficiency of IRE RNA, thereby suggesting that the presence of iron leads to a rapid, favorable, and stable complex formation that directs regulatory system to respond efficiently to cellular iron levels.

Iron regulatory proteins (IRPs) are iron-responsive RNA binding proteins that dictate changes in cellular iron metabolism in animal cells by controlling the fate of mRNAs containing iron responsive elements (IREs). IRPs have broader physiological roles as some targeted mRNAs encode proteins with functions beyond iron metabolism suggesting hierarchical regulation of IRP-targeted mRNAs. We observe that the translational regulation of IRP-targeted mRNAs encoding iron storage (L- and H-ferritins) and export (ferroportin) proteins have different set-points of iron responsiveness compared to that for the TCA cycle enzyme mitochondrial aconitase. The ferritins and ferroportin mRNA were largely translationally repressed in the liver of rats fed a normal diet whereas mitochondrial aconitase mRNA is primarily polysome bound. Consequently, acute iron overload increases polysome association of H- and L-ferritin and ferroportin mRNAs while mitochondrial aconitase mRNA showed little stimulation. Conversely, mitochondrial aconitase mRNA is most responsive in iron deficiency. These differences in regulation were associated with a faster off-rate of IRP1 for the IRE of mitochondrial aconitase in comparison to that of L-ferritin. Thus, hierarchical control of mRNA translation by IRPs involves selective control of cellular functions acting at different states of cellular iron status and that are critical for adaptations to iron deficiency or prevention of iron toxicity.

Molluscs biomineralize structures that vary in composition, form, and function, prompting questions about the genetic mechanisms responsible for their production and the evolution of these mechanisms. Chitons (Mollusca, Polyplacophora) are a promising system for studies of biomineralization because they build a range of calcified structures including shell plates and spine- or scale-like sclerites. Chitons also harden the calcified teeth of their rasp-like radula with a coat of iron (as magnetite). Here we present the genome of the West Indian fuzzy chiton Acanthopleura granulata, the first from any aculiferan mollusc. The A. granulata genome contains homologs of many genes associated with biomineralization in conchiferan molluscs. We expected chitons to lack genes previously identified from pathways conchiferans use to make biominerals like calcite and nacre because chitons do not use these materials in their shells. Surprisingly, the A. granulata genome has homologs of many of these genes, suggesting that the ancestral mollusc may have had a more diverse biomineralization toolkit than expected. The A. granulata genome has features that may be specialized for iron biomineralization, including a higher proportion of genes regulated directly by iron than other molluscs. A. granulata also produces two isoforms of soma-like ferritin: one is regulated by iron and similar in sequence to the soma-like ferritins of other molluscs, and the other is constitutively translated and is not found in other molluscs. The A. granulata genome is a resource for future studies of molluscan evolution and biomineralization.

The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer. In addition to loss of tumor suppressor functions, mutations in TP53 promote cancer progression by altering cellular iron acquisition and metabolism. A newly identified role for TP53 in the coordination of iron homeostasis and cancer cell survival lies in the ability for TP53 to protect against ferroptosis, a form of iron-mediated cell death. The purpose of this study was to determine the extent to which TP53 mutation status affects the cellular response to ferroptosis induction. Using H1299 cells, which are null for TP53, we generated cell lines expressing either a tetracycline inducible wild-type (WT) TP53 gene, or a representative mutated TP53 gene from six exemplary &ldquo, hotspot&rdquo, mutations in the DNA binding domain (R273H, R248Q, R282W, R175H, G245S, and R249S). TP53 mutants (R273H, R248Q, R175H, G245S, and R249S) exhibited increased sensitivity ferroptosis compared to cells expressing WT TP53. As iron-mediated lipid peroxidation is critical for ferroptosis induction, we hypothesized that iron acquisition pathways would be upregulated in mutant TP53-expressing cells. However, only cells expressing the R248Q, R175H, and G245S TP53 mutation types exhibited statistically significant increases in spontaneous iron regulatory protein (IRP) RNA binding activity following ferroptosis activation. Moreover, changes in the expression of downstream IRP targets were inconsistent with the observed differences in sensitivity to ferroptosis. These findings reveal that canonical iron regulatory pathways are bypassed during ferroptotic cell death. These results also indicate that induction of ferroptosis may be an effective therapeutic approach for tumor cells expressing distinct TP53 mutation types.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in chronic bacterial lung infections and tissue damage. CF macrophages exhibit reduced bacterial killing and increased inflammatory signaling. Iron is elevated in the CF lung and is a critical nutrient for bacteria, including the common CF pathogen Pseudomonas aeruginosa (Pa). While macrophages are a key regulatory component of extracellular iron, iron metabolism has yet to be characterized in human CF macrophages. Secreted and total protein levels were analyzed in non-CF and F508del/F508del CF monocyte derived macrophages (MDMs) with and without clinically approved CFTR modulators ivacaftor/lumacaftor. CF macrophage transferrin receptor 1 (TfR1) was reduced with ivacaftor/lumacaftor treatment. When activated with LPS, CF macrophage expressed reduced ferroportin (Fpn). After the addition of exogenous iron, total iron was elevated in conditioned media from CF MDMs and reduced in conditioned media from ivacaftor/lumacaftor treated CF MDMs. Pa biofilm formation and viability were elevated in conditioned media from CF MDMs and biofilm formation was reduced in the presence of conditioned media from ivacaftor/lumacaftor treated CF MDMs. Defects in iron metabolism observed in this study may inform host–pathogen interactions between CF macrophages and Pa.

Despite its essential role in many biological processes, iron is toxic when in excess due to its propensity to generate reactive oxygen species. To prevent diseases associated with iron deficiency or iron loading, iron homeostasis must be tightly controlled. Intracellular iron content is regulated by the Iron Regulatory Element-Iron Regulatory Protein (IRE-IRP) system, whereas systemic iron availability is adjusted to body iron needs chiefly by the hepcidin-ferroportin (FPN) axis. Here, we aimed to review advances in the field that shed light on cell-type-specific regulatory mechanisms that control or modify systemic and local iron balance, and how shifts in cellular iron levels may affect specialized cell functions.

Iron is implicated in ocular diseases such as in age-related macular degeneration. Light is also considered as a pathological factor in this disease. Earlier, two studies reported the influence of constant light environment on the pattern of expressions of iron-handling proteins. Here, we aimed to see the influence of light in 12-h light–12-h dark (12L:12D) cycles on the expression of iron-handling proteins in chick retina. Chicks were exposed to 400 lx (control) and 5000 lx (experimental) light at 12L:12D cycles and sacrificed at variable timepoints. Retinal ferrous ion (Fe2+) level, ultrastructural changes, lipid peroxidation level, immunolocalization and expression patterns of iron-handling proteins were analysed after light exposure. Both total Fe2+ level (p = 0.0004) and lipid peroxidation (p = 0.002) significantly increased at 12-, 48- and 168-h timepoint (for Fe2+) and 48- and 168-h timepoint (for lipid peroxidation), and there were degenerative retinal changes after 168 h of light exposure. Intense light exposure led to an increase in the levels of transferrin and transferrin receptor-1 (at 168-h) and ferroportin-1, whereas the levels of ferritins, hephaestin, (at 24-, 48- and 168-h timepoint) and ceruloplasmin (at 168-h timepoint) were decreased. These changes in iron-handling proteins after light exposure are likely due to a disturbance in the iron storage pool evident from decreased ferritin levels, which would result in increased intracellular Fe2+ levels. To counteract this, Fe2+ is released into the extracellular space, an observation supported by increased expression of ferroportin-1. Ceruloplasmin was able to convert Fe2+ into Fe3+ until 48 h of light exposure, but its decreased expression with time (at 168-h timepoint) resulted in increased extracellular Fe2+ that might have caused oxidative stress and retinal cell damage.

In swine, even though the pregnant sows were with iron abundance, the inborn iron reserve of piglets was compromised. This indicates the insufficiency of molecular machinery involved in local placental iron flux. Here, we investigated the expression of iron regulatory proteins like hepcidin and ferroportin and also their association with iron reserve, inflammation and oxidative stress in placenta of full-term pregnant sows (n = 6). Amplification and sequencing of placental DNA confirmed the presence of hepcidin (MN579557) and ferroportin (MN565887) sequences and their 100% identity with existing GenBank data. Real-time amplification of placental mRNA revealed significant higher expression of hepcidin (p < .05) than ferroportin. Western blot analysis of placental tissues revealed specific bands for both hepcidin (~8 kDa) and ferroportin (~62 kDa) molecules. Immunohistochemistry revealed the immunoreactivity for both proteins in the cytoplasm and membrane of trophoblastic cells of the placenta. Hepcidin and ferroportin expressions were positively associated with placental non-haem iron reserve (p < .0001; p = .033), lipid peroxidation (p = .0060; p < .0001) and reactive oxygen species level (p = .0092; p = .0292). Hepcidin expression was positively associated with interleukin - 6 (p = .0002) and interferon gamma (p < .0001) expressions but ferroportin expression was negatively associated with interleukin-6 (p = .0005), interleukin-1β (p = .0226) and interferon gamma (p = .0059) expressions. This indicates hepcidin and ferroportin may have a role in controlling the local placental iron flux by acting as a molecular bridge between iron trafficking and inflammation.

Aims To characterize the 21-kDa iron-regulated cell wall protein in Mycobacterium smegmatis co-expressed with the siderophores mycobactin, exochelin and carboxymycobactin upon iron limitation. Methods and results Mycobacterium smegmatis, grown in the presence of 0·02 μg Fe ml-1 (low iron) produced high levels of all the three siderophores, which were repressed in bacteria supplemented with 8 μg Fe ml-1 (high iron). Exochelin, the major extracellular siderophore was the first to rise and was expressed at high levels during log phase of growth. Carboxymycobactin, a minor component in log phase iron-starved M. smegmatis continued to rise when cultured for longer periods, reaching levels greater than exochelin. Iron-starved bacteria expressed a 21-kDa iron-regulated protein (IrpA) that was identified as Clp protease subunit (MSMEG_3671) and characterized as a receptor for ferri-exochelin. Conclusions Ferri-exochelin is the preferred siderophore in M. smegmatis and this ferri-exochelin: IrpA machinery is absent in Mycobacterium tuberculosis. Significance and impact of the study Exochelin machinery is functional in M. smegmatis and the carboxymycobactin-mycobactin machinery is the sole iron uptake system in M. tuberculosis. The absence of the ferri-exochelin: IrpA system in the pathogen signifies the importance of the carboxymycobactin-mycobactin system machinery in M. tuberculosis.

The ability to regulate oxygen consumption evolved in ancestral animals and is intrinsically linked to iron metabolism. The iron pathways have been intensively studied in mammals, whereas data on distant invertebrates are limited. Sea sponges represent the oldest animal phylum and have unique structural plasticity and capacity to reaggregate after complete dissociation. We studied iron metabolic factors and their expression during reaggregation in the White Sea cold-water sponges Halichondria panicea and Halisarca dujardini. De novo transcriptomes were assembled using RNA-Seq data, and evolutionary trends were analyzed with bioinformatic tools. Differential expression during reaggregation was studied for H. dujardini. Enzymes of the heme biosynthesis pathway and transport globins, neuroglobin (NGB) and androglobin (ADGB), were identified in sponges. The globins mutate at higher evolutionary rates than the heme synthesis enzymes. Highly conserved iron-regulatory protein 1 (IRP1) presumably interacts with the iron-responsive elements (IREs) found in mRNAs of ferritin (FTH1) and a putative transferrin receptor NAALAD2. The reaggregation process is accompanied by increased expression of IRP1, the antiapoptotic factor BCL2, the inflammation factor NFκB (p65), FTH1 and NGB, as well as by an increase in mitochondrial density. Our data indicate a complex mechanism of iron regulation in sponge structural plasticity and help to better understand general mechanisms of morphogenetic processes in multicellular species.

Post-transcriptional factors importantly contribute to the rapid and coordinated expression of the multiple genes required for the adaptation of living organisms to environmental stresses. In the model eukaryote Saccharomyces cerevisiae, a conserved mRNA-binding protein, known as Cth2, modulates the metabolic response to iron deficiency. Cth2 is a tandem zinc-finger (TZF)-containing protein that co-transcriptionally binds to adenine/uracil-rich elements (ARE) present in the 3′-untranslated region of iron-related mRNAs to promote their turnover. The nuclear binding of Cth2 to mRNAs via its TZFs is indispensable for its export to the cytoplasm. Although Cth2 nucleocytoplasmic transport is essential for its regulatory function, little is known about the recruitment of the mRNA degradation machinery. Here, we investigate the sequential assembly of mRNA decay factors during Cth2 shuttling. By using an enzymatic in vivo proximity assay called M-track, we show that Cth2 associates to the RNA helicase Dhh1 and the deadenylase Pop2/Caf1 before binding to its target mRNAs. The recruitment of Dhh1 to Cth2 requires the integrity of the Ccr4-Pop2 deadenylase complex, whereas the interaction between Cth2 and Pop2 needs Ccr4 but not Dhh1. M-track assays also show that Cth2-binding to ARE-containing mRNAs is necessary for the interaction between Cth2 and the exonuclease Xrn1. The importance of these interactions is highlighted by the specific growth defect in iron-deficient conditions displayed by cells lacking Dhh1, Pop2, Ccr4 or Xrn1. These results exemplify the stepwise process of assembly of different mRNA decay factors onto an mRNA-binding protein during the mechanism of post-transcriptional regulation., This work was supported by a post-doctoral “Juan de la Cierva-Formación” contract from the Spanish Ministry of Science, Innovation and Universities (MICINN) and the “Grupos Emergentes” GV/2017/063 grant from the Regional Government of Valencia to APG; and the MICINN grant BIO2017-87828-C2-1-P and European Union Funds (FEDER) to SP.

Iron regulatory proteins (IRPs) and iron regulatory element (IRE) systems are well known in the progression of neurodegenerative disorders by regulating iron related proteins. IRPs are also regulated by iron homeostasis. However, an increasing number of studies have suggested a close relationship between the IRPs/IRE system and non-iron-related neurodegenerative disorders. In this paper, we reviewed that the IRPs/IRE system is not only controlled by iron ions, but also regulated by such factors as post-translational modification, oxygen, nitric oxide (NO), heme, interleukin-1 (IL-1), and metal ions. In addition, by regulating the transcription of non-iron related proteins, the IRPs/IRE system functioned in oxidative metabolism, cell cycle regulation, abnormal proteins aggregation, and neuroinflammation. Finally, by emphasizing the multiple regulations of IRPs/IRE system and its potential relationship with non-iron metabolic neurodegenerative disorders, we provided new strategies for disease treatment targeting IRPs/IRE system.

Iron plays an essential role in various cellular metabolic processes of the body. Maintenance of cellular iron homeostasis is particularly important for keeping the normal functions of the cells. Ferroportin 1 (FPN1) is the currently only known iron exporter on the cell membrane. It has been indicated that the regulation of FPN1 in response to the alteration of iron level mainly involves two processes, posttranscriptional repression by iron regulatory proteins (IRPs) and posttranslational degradation by hepcidin, the major iron-sensing hormone. However, whether there is any communication between the two types of regulations or which one plays dominant role has not been reported. In our study with IRP2-/- mice, we found that knockout of IRP2 increased FPN1 expression in the cerebral cortex of IRP2-/- mice, whereas the upregulation of FPN1 was more significant in IRP1/IRP2 dual knockdown fibroblasts. Interestingly, we found that the knockout of IRP2 severely affected the regulation effect of hepcidin on FPN1 in mouse brain. FPN1 level decreased dramatically in the brain of wild-type mice injected with hepcidin, but it did not decrease much in IRP2 knockout mice. Further investigation disclosed that the compromised hepcidin-FPN1 regulation in IRP2-/- cells was directly dependent on the existence of iron-responsive element (IRE) in FPN1 messenger RNA. These results indicate that IRPs and hepcidin coordinately regulate the FPN1 level in mice. This study will provide a more comprehensive understanding of the regulatory mechanisms of FPN1 expression.

Entamoeba histolytica is a human parasite that causes amoebiasis, a disease that affects the colon and liver and is prevalent worldwide. This protozoan requires a high concentration of iron to survive and reproduce. Iron modulates the expression of parasite virulence factors, including hemoglobinases, hemoglobin-binding proteins and cysteine proteases, as well as proteins related to the amoebic cytoskeleton. This review summarizes the virulence factors that are affected by iron, resulting in upregulation or downregulation of E. histolytica genes. This review also discusses the functionality of iron in the mechanisms of pathogenesis.

Candida albicans is a commensal fungal species that commonly colonizes a heterogeneous mixture of human body where it intimately interacts with other microbes in the host environment such as the gastrointestinal (GI) tract. Most studies in fungal-bacterial interactions are about synergistic or antagonistic effects of bacterial functions on fungal physiological activities including pathogenicity. Very few studies have been demonstrated about the role of fungi on bacteria. In this study, we investigated the interactions between C. albicans and the bacterium Escherichia coli and unexpectedly observed that C. albicans enhances growth and proliferation of Escherichia coli strain K12 by facilitating its cell division. Importantly, we found, based on our genetic screens, that both fungus- and bacterium-derived factors, including the iron-responsive transcription factors Sef1 and Sfu1 in C. albicans and the siderophere enterobactin transporters FepD and FepG in E. coli, actively contribute to this transkingdom interaction. Deletion of SFU1 or SEF1 caused a dramatic reduction in growth enhancement of E. coli. Compared to the wild type E. coli, the enhanced growth of both fepD and fepG null mutants were largely dampened. However, the E. coli mutant lacking entB, a key enzyme catalyzing the biosynthesis of siderophore enterobactin, showed similar growth enhancement as the wild type when co-inoculated with C. albicans. C. albicans promotes growth and proliferation of the commensal bacterium E. coli and an iron-responsive signaling pathway appears to be required. C. albicans may act to supply a siderophere-like molecule that captures the environmental iron to promote the growth of E. coli. Our studies gave insight into a novel interacting mechanism operative in interspecies communication that occurs when bacteria and fungi co-exist.

Intracellular iron is tightly regulated by coordinated expression of iron transport and storage genes, such as transferrin receptor-1 (TfR1) and ferritin. They are primarily regulated by iron through iron-induced dissociation of iron-regulatory proteins (IRPs) from iron-responsive elements (IREs) in the 3′-UTR (untranslated region) of TfR1 or 5′-UTR of ferritin mRNA, resulting in destabilization of TfR1 mRNA and release of ferritin translation block. Thus high iron decreases iron transport via TfR1 mRNA degradation and increases iron storage via ferritin translational up-regulation. However, the molecular mechanism of TfR1 mRNA destabilization in response to iron remains elusive. Here, we demonstrate that miR-7-5p and miR-141-3p target 3′-TfR1 IREs and down-regulate TfR1 mRNA and protein expression. Conversely, miR-7-5p and miR-141-3p antagomiRs partially but significantly blocked iron- or IRP knockdown-induced down-regulation of TfR1 mRNA, suggesting the interplay between these microRNAs and IRPs along with involvement of another uncharacterized mechanism in TfR1 mRNA degradation. Luciferase reporter assays using 3′-UTR TfR1 IRE mutants suggested that the IREs C and E are targets of miR-7-5p and miR-141-3p, respectively. Furthermore, miR-7 expression was inversely correlated with TfR1 mRNA in human pancreatic adenocarcinoma patient samples. These results suggest a role of microRNAs in the TfR1 regulation in the IRP–IRE system.

The essentiality of iron for cell growth and proliferation, coupled with its capacity to promote damaging free radical production, has made it a desirable target for cancer treatment and prevention. One such approach may be through the activation of ferroptosis, a form of iron-mediated programmed cell death. However, we must first understand how cancer cells manipulate the homeostatic regulators of iron metabolism to promote malignancy before we can fully harness iron's therapeutic potential. The iron regulatory proteins 1 and 2 (IRP1 and IRP2) are the master regulators of intracellular iron homeostasis because they coordinate the expression of proteins involved in iron storage, uptake, and utilization. Yet, the roles and regulation of IRPs during cellular ferroptosis remain unknown. The primary objective of this work was to examine the reciprocal relationship between ferroptosis activation and IRP mRNA binding activity. Utilizing tetracycline-inducible plasmids, we found that overexpression of IRP1 or IRP2 significantly increases sensitivity to ferroptotic cell death in HEK293T cells. Intriguingly however, ferroptosis induction by erastin treatment differentially influences IRP1 and IRP2 mRNA binding activity in a cell-type dependent manner, with those cell types expressing higher levels IRP2 exhibiting increased sensitivity to ferroptosis activation. Yet, IRP2 knockout cells are still subject to ferroptotic cell death, as are cells that lack IRP1 expression. Collectively, our findings suggest that while increased expression of IRP1 and IRP2 can promote ferroptotic cell death, expression of IRP1 or IRP2 is sufficient to convey sensitivity to ferroptosis activation. To identify mechanism driving increased IRP mRNA binding activity during cellular ferroptosis, we are currently assessing both iron-dependent and -independent regulators of IRP1 and IRP2 function and stability. As iron is an essential, yet potentially toxic nutrient, such findings will be important because they are expected to provide novel insights for exploiting the toxic nature of iron without compromising the essential homeostatic control mechanisms. Citation Format: Evan Hermann, Thais Oliveira, McKale Montgomery. The contributions of iron regulatory proteins 1 and 2 to ferroptosis activation and ferroptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2006.