Using Biotinylated Iron-Responsive Element to Analyze the Activity of Iron Regulatory Proteins.

Iron regulatory proteins (IRP1 and IRP2) are the master regulators of mammalian iron homeostasis. They bind to the iron-responsive elements (IREs) of the transcripts of iron-related genes to regulate their expression, thereby maintaining cellular iron availability. The primary method to measure the IRE-binding activity of IRPs is the electrophoresis mobility shift assay (EMSA). This method is particularly useful for evaluating IRP1 activity, since IRP1 is a bifunctional enzyme and its protein levels remain similar during conversion between the IRE-binding protein and cytosolic aconitase forms. Here, we exploited a method of using a biotinylated-IRE probe to separate IRE-binding IRPs followed by immunoblotting to analyze the IRE-binding activity. This method allows for the successful measurement of IRP activity in cultured cells and mouse tissues under various iron conditions. By separating IRE-binding IRPs from the rest of the lysates, this method increases the specificity of IRP antibodies and verifies whether a band represents an IRP, thereby revealing some previously unrecognized information about IRPs. With this method, we showed that the S711-phosphorylated IRP1 was found only in the IRE-binding form in PMA-treated Hep3B cells. Second, we found a truncated IRE-binding IRP2 isoform that is generated by proteolytic cleavage on sites in the 73aa insert region of the IRP2 protein. Third, we found that higher levels of SDS, compared to 1-2% SDS in regular loading buffer, could dramatically increase the band intensity of IRPs in immunoblots, especially in HL-60 cells. Fourth, we found that the addition of SDS or LDS to cell lysates activated protein degradation at 37 °C or room temperature, especially in HL-60 cell lysates. As this method is more practical, sensitive, and cost-effective, we believe that its application will enhance future research on iron regulation and metabolism.

The interplay between electron transport chain function and iron regulatory factors influences melanin formation in Cryptococcus neoformans.

Mitochondrial functions are critical for the ability of the fungal pathogen Cryptococcus neoformans to cause disease. However, mechanistic connections between key functions such as the mitochondrial electron transport chain (ETC) and virulence factor elaboration have yet to be thoroughly characterized. Here, we observed that inhibition of ETC complex III suppressed melanin formation, a major virulence factor. This inhibition was partially overcome by defects in Cir1 or HapX, two transcription factors that regulate iron acquisition and use. In this regard, loss of Cir1 derepresses the expression of laccase genes as a potential mechanism to restore melanin, while HapX may condition melanin formation by controlling oxidative stress. We hypothesize that ETC dysfunction alters redox homeostasis to influence melanin formation. Consistent with this idea, inhibition of growth by hydrogen peroxide was exacerbated in the presence of the melanin substrate L-DOPA. In addition, loss of the mitochondrial chaperone Mrj1, which influences the activity of ETC complex III and reduces ROS accumulation, also partially overcame antimycin A inhibition of melanin. The phenotypic impact of mitochondrial dysfunction was consistent with RNA-Seq analyses of WT cells treated with antimycin A or L-DOPA, or cells lacking Cir1 that revealed influences on transcripts encoding mitochondrial functions (e.g., ETC components and proteins for Fe-S cluster assembly). Overall, these findings reveal mitochondria-nuclear communication via ROS and iron regulators to control virulence factor production in C. neoformans.IMPORTANCEThere is a growing appreciation of the importance of mitochondrial functions and iron homeostasis in the ability of fungal pathogens to sense the vertebrate host environment and cause disease. Many mitochondrial functions such as heme and iron-sulfur cluster biosynthesis, and the electron transport chain (ETC), are dependent on iron. Connections between factors that regulate iron homeostasis and mitochondrial activities are known in model yeasts and are emerging for fungal pathogens. In this study, we identified connections between iron regulatory transcription factors (e.g., Cir1 and HapX) and the activity of complex III of the ETC that influence the formation of melanin, a key virulence factor in the pathogenic fungus Cryptococcus neoformans. This fungus causes meningoencephalitis in immunocompromised people and is a major threat to the HIV/AIDS population. Thus, understanding how mitochondrial functions influence virulence may support new therapeutic approaches to combat diseases caused by C. neoformans and other fungi.

Iron regulatory protein from the hard tick Haemaphysalis longicornis: characterization, function and assessment as a protective antigen.

BACKGROUND: Ticks are blood-feeding ectoparasites with different host specificities and are capable of pathogen transmission. Iron regulatory proteins (IRPs) play crucial roles in iron homeostasis in vertebrates. However, their functions in ticks remain poorly understood. The aim of the present study was to investigate the characteristics, functions, molecular mechanisms, and the vaccine efficacy of IRP in the hard tick Haemaphysalis longicornis. RESULTS: The full-length complementary DNA of IRP from Haemaphysalis longicornis (HlIRP) was 2973 bp, including a 2772 bp open reading frame. It is expressed throughout three developmental stages (larvae, nymphs, and adult females) and in various tissues (salivary glands, ovaries, midgut, and Malpighian tubules). Recombinant Haemaphysalis longicornis IRP (rHlIRP) was obtained via a prokaryotic expression system and exhibited aconitase, iron chelation, radical-scavenging, and hemolytic activities in vitro. RNA interference-mediated IRP knockdown reduced tick engorgement weight, ovary weight, egg mass weight, egg hatching rate, and ovary vitellin content, as well as prolonging the egg incubation period. Proteomics revealed that IRP may affect tick reproduction and development through proteasome pathway-associated, ribosomal, reproduction-related, and iron metabolism-related proteins. A trial on rabbits against adult Haemaphysalis longicornis infestation demonstrated that rHlIRP vaccine could significantly decrease engorged weight (by 10%), egg mass weight (by 16%) and eggs hatching rate (by 22%) of ticks. The overall immunization efficacy using rHlIRP against adult females was 41%. CONCLUSION: IRP could limit reproduction and development in Haemaphysalis longicornis, and HlIRP was confirmed as a candidate vaccine antigen to impair tick iron metabolism and protect the host against tick infestation. © 2024 Society of Chemical Industry.

Mechanisms controlling cellular and systemic iron homeostasis.

In mammals, hundreds of proteins use iron in a multitude of cellular functions, including vital processes such as mitochondrial respiration, gene regulation and DNA synthesis or repair. Highly orchestrated regulatory systems control cellular and systemic iron fluxes ensuring sufficient iron delivery to target proteins is maintained, while limiting its potentially deleterious effects in iron-mediated oxidative cell damage and ferroptosis. In this Review, we discuss how cells acquire, traffick and export iron and how stored iron is mobilized for iron-sulfur cluster and haem biogenesis. Furthermore, we describe how these cellular processes are fine-tuned by the combination of various sensory and regulatory systems, such as the iron-regulatory protein (IRP)-iron-responsive element (IRE) network, the nuclear receptor co-activator 4 (NCOA4)-mediated ferritinophagy pathway, the prolyl hydroxylase domain (PHD)-hypoxia-inducible factor (HIF) axis or the nuclear factor erythroid 2-related factor 2 (NRF2) regulatory hub. We further describe how these pathways interact with systemic iron homeostasis control through the hepcidin-ferroportin axis to ensure appropriate iron fluxes. This knowledge is key for the identification of novel therapeutic opportunities to prevent diseases of cellular and/or systemic iron mismanagement.

Iron response elements (IREs)-mRNA of Alzheimer's amyloid precursor protein binding to iron regulatory protein (IRP1): a combined molecular docking and spectroscopic approach.

The interaction between the stem-loop structure of the Alzheimer's amyloid precursor protein IRE mRNA and iron regulatory protein was examined by employing molecular docking and multi-spectroscopic techniques. A detailed molecular docking analysis of APP IRE mRNA∙IRP1 reveals that 11 residues are involved in hydrogen bonding as the main driving force for the interaction. Fluorescence binding results revealed a strong interaction between APP IRE mRNA and IRP1 with a binding affinity and an average binding sites of 31.3 × 106 M-1 and 1.0, respectively. Addition of Fe2+(anaerobic) showed a decreased (3.3-fold) binding affinity of APP mRNA∙IRP1. Further, thermodynamic parameters of APP mRNA∙IRP1 interactions were an enthalpy-driven and entropy-favored event, with a large negative ΔH (-25.7 ± 2.5 kJ/mol) and a positive ΔS (65.0 ± 3.7 J/mol·K). A negative ΔH value for the complex formation suggested the contribution of hydrogen bonds and van der Waals forces. The addition of iron increased the enthalpic contribution by 38% and decreased the entropic influence by 97%. Furthermore, the stopped-flow kinetics of APP IRE mRNA∙IRP1 also confirmed the complex formation, having the rate of association (kon) and the rate of dissociation (koff) as 341 µM-1 s-1, and 11 s-1, respectively. The addition of Fe2+ has decreased the rate of association (kon) by ~ three-fold, whereas the rate of dissociation (koff) has increased by ~ two-fold. The activation energy for APP mRNA∙IRP1 complex was 52.5 ± 2.1 kJ/mol. The addition of Fe2+ changed appreciably the activation energy for the binding of APP mRNA with IRP1. Moreover, circular dichroism spectroscopy has confirmed further the APP mRNA∙IRP1 complex formation and IRP1 secondary structure change with the addition of APP mRNA. In the interaction between APP mRNA and IRP1, iron promotes structural changes in the APP IRE mRNA∙IRP1 complexes by changing the number of hydrogen bonds and promoting a conformational change in the IRP1 structure when it is bound to the APP IRE mRNA. It further illustrates how IRE stem-loop structure influences selectively the thermodynamics and kinetics of these protein-RNA interactions.

Iron-dependent post transcriptional control of mitochondrial aconitase expression.

Iron regulatory proteins (IRPs) control the translation of animal cell mRNAs encoding proteins with diverse roles. This includes the iron storage protein ferritin and the tricarboxylic cycle (TCA) enzyme mitochondrial aconitase (ACO2) through iron-dependent binding of IRP to the iron responsive element (IRE) in the 5' untranslated region (UTR). To further elucidate the mechanisms allowing IRPs to control translation of 5' IRE-containing mRNA differentially, we focused on Aco2 mRNA, which is weakly controlled versus the ferritins. Rat liver contains two classes of Aco2 mRNAs, with and without an IRE, due to alterations in the transcription start site. Structural analysis showed that the Aco2 IRE adopts the canonical IRE structure but lacks the dynamic internal loop/bulge five base pairs 5' of the CAGUG(U/C) terminal loop in the ferritin IREs. Unlike ferritin mRNAs, the Aco2 IRE lacks an extensive base-paired flanking region. Using a full-length Aco2 mRNA expression construct, iron controlled ACO2 expression in an IRE-dependent and IRE-independent manner, the latter of which was eliminated with the ACO23C3S mutant that cannot bind the FeS cluster. Iron regulation of ACO23C3S encoded by the full-length mRNA was completely IRE-dependent. Replacement of the Aco23C3S 5' UTR with the Fth1 IRE with base-paired flanking sequences substantially improved iron responsiveness, as did fusing of the Fth1 base-paired flanking sequences to the native IRE in the Aco3C3S construct. Our studies further define the mechanisms underlying the IRP-dependent translational regulatory hierarchy and reveal that Aco2 mRNA species lacking the IRE contribute to the expression of this TCA cycle enzyme.

Ferritin Iron Responsive Elements (IREs) mRNA Interacts with eIF4G and Activates In Vitro Translation.

BACKGROUND: 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. METHODS: In vitro translation assay was performed in depleted wheat germ lysate with supplementation of initiation factors. Fluorescence spectroscopy was used to characterize eIF4F/IRE binding. RESULTS: 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 (Kd = 63 nM), but not eIF4E. Further equilibrium studies showed that iron enhanced (~4-fold) the ferritin IRE binding to eIF4G. The equilibrium binding effects of iron on ferritin IRE RNA/eIFs interaction and the temperature dependence of this reaction were measured and compared. The Kd values for the IRE binding to eIF4G ranging from 18.2 nM to 63.0 nM as temperature elevated from 5 °C to 25 °C, while the presence of iron showed much stronger affinity over the same range of temperatures. Thermodynamic parameter revealed that IRE RNA binds to eIF4G with ΔH = -42.6 ± 3.3 kJ. mole-1, ΔS = -11.5 ± 0.4 J. mole-1K-1, and ΔG = -39.2 ± 2.7 kJ. mole-1, respectively. Furthermore, addition of iron significantly changed the values of thermodynamic parameters, favoring stable complex formation, thus favoring efficient protein synthesis. This study first time demonstrate the participation of eIF4G in ferritin IRE mRNA translation. CONCLUSIONS: eIF4G specifically interacts with ferritin IRE RNA and promotes eIF4G-dependent translation.

Prospective role and immunotherapeutic targets of sideroflexin protein family in lung adenocarcinoma: evidence from bioinformatics validation.

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.

Multimodal approaches for the improvement of the cellular folding of a recombinant iron regulatory protein in E. coli.

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.

Root-to-shoot iron partitioning in Arabidopsis requires IRON-REGULATED TRANSPORTER1 (IRT1) protein but not its iron(II) transport function.

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 55 Fe 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.

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.

Hereditary Hyperferritinemia Cataract Syndrome: Ferritin L Gene and Physiopathology behind the Disease-Report of New Cases.

Hereditary hyperferritinemia-cataract syndrome (HHCS) is a rare disease characterized by high serum ferritin levels, congenital bilateral cataracts, and the absence of tissue iron overload. This disorder is produced by mutations in the iron responsive element (IRE) located in the 5' untranslated regions (UTR) of the light ferritin (FTL) gene. A canonical IRE is a mRNA structure that interacts with the iron regulatory proteins (IRP1 and IRP2) to post-transcriptionally regulate the expression of proteins related to iron metabolism. Ferritin L and H are the proteins responsible for iron storage and intracellular distribution. Mutations in the FTL IRE abrogate the interaction of FTL mRNA with the IRPs, and de-repress the expression of FTL protein. Subsequently, there is an overproduction of ferritin that accumulates in serum (hyperferritinemia) and excess ferritin precipitates in the lens, producing cataracts. To illustrate this disease, we report two new families affected with hereditary hyperferritinemia-cataract syndrome with previous known mutations. In the diagnosis of congenital bilateral cataracts, HHCS should be taken into consideration and, therefore, it is important to test serum ferritin levels in patients with cataracts.

Iron enhances the binding rates and translational efficiency of iron responsive elements (IREs) mRNA with initiation factor eIF4F.

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.

The Iron-Responsive Genome of the Chiton Acanthopleura granulata.

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.

Differential translational control of 5' IRE-containing mRNA in response to dietary iron deficiency and acute iron overload.

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.

Distinct TP53 Mutation Types Exhibit Increased Sensitivity to Ferroptosis Independently of Changes in Iron Regulatory Protein Activity.

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 "hotspot" 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.

Cobalt accumulation and iron-regulatory protein profile expression in immature mouse brain after perinatal exposure to cobalt chloride.

Developing brain is very sensitive to the influence of environmental factors during gestation and the neonatal period. The aim of the study is to assess cobalt and iron accumulation in the brain as well as changes in the expression of iron-regulatory proteins transferrin receptor 1, hepcidin, and ferroportin in suckling mice. Perinatal exposure to cobalt chloride increased significantly cobalt content in brain tissue homogenates of 18-day-old (d18) and 25-day-old (d25) mice inducing alterations in brain iron homeostasis. Higher degree of transferrin receptor 1 expression was demonstrated in cobalt chloride-exposed mice with no substantial changes between d18 and d25 mice. A weak ferroportin expression was found in 18-day-old control and cobalt-treated mouse brain. Cobalt exposure of d25 mice resulted in increased ferroportin expression in brain compared to the untreated age-matched control group. Hepcidin level in cobalt-exposed groups was decreased in d18 mice and slightly increased in d25 mice. The obtained data contribute for the better understanding of metal toxicity impact on iron homeostasis in the developing brain with further possible implications in neurodegeneration.

Altered iron metabolism in cystic fibrosis macrophages: the impact of CFTR modulators and implications for Pseudomonas aeruginosa survival.

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.

Sequential recruitment of the mRNA decay machinery to the iron-regulated protein Cth2 in Saccharomyces cerevisiae.

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.

Expression of iron regulatory proteins in full-term swine placenta.

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-1ß (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.