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.

An Oxygen-Dependent Interaction between FBXL5 and the CIA-Targeting Complex Regulates Iron Homeostasis.

The iron-sensing protein FBXL5 is the substrate adaptor for a SKP1-CUL1-RBX1 E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins (IRPs). Here, we describe a mechanism of FBXL5 regulation involving its interaction with the cytosolic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, FAM96B, and CIAO1. We demonstrate that the CIA-targeting complex promotes the ability of FBXL5 to degrade IRPs. In addition, the FBXL5-CIA-targeting complex interaction is regulated by oxygen (O2) tension displaying a robust association in 21% O2 that is severely diminished in 1% O2 and contributes to O2-dependent regulation of IRP degradation. Together, these data identify a novel oxygen-dependent signaling axis that links IRP-dependent iron homeostasis with the Fe-S cluster assembly machinery.

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.

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.

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.

The opportunistic pathogen Stenotrophomonas maltophilia utilizes a type IV secretion system for interbacterial killing.

Bacterial type IV secretion systems (T4SS) are a highly diversified but evolutionarily related family of macromolecule transporters that can secrete proteins and DNA into the extracellular medium or into target cells. It was recently shown that a subtype of T4SS harboured by the plant pathogen Xanthomonas citri transfers toxins into target cells. Here, we show that a similar T4SS from the multi-drug-resistant opportunistic pathogen Stenotrophomonas maltophilia is proficient in killing competitor bacterial species. T4SS-dependent duelling between S. maltophilia and X. citri was observed by time-lapse fluorescence microscopy. A bioinformatic search of the S. maltophilia K279a genome for proteins containing a C-terminal domain conserved in X. citri T4SS effectors (XVIPCD) identified twelve putative effectors and their cognate immunity proteins. We selected a putative S. maltophilia effector with unknown function (Smlt3024) for further characterization and confirmed that it is indeed secreted in a T4SS-dependent manner. Expression of Smlt3024 in the periplasm of E. coli or its contact-dependent delivery via T4SS into E. coli by X. citri resulted in reduced growth rates, which could be counteracted by expression of its cognate inhibitor Smlt3025 in the target cell. Furthermore, expression of the VirD4 coupling protein of X. citri can restore the function of S. maltophilia ΔvirD4, demonstrating that effectors from one species can be recognized for transfer by T4SSs from another species. Interestingly, Smlt3024 is homologous to the N-terminal domain of large Ca2+-binding RTX proteins and the crystal structure of Smlt3025 revealed a topology similar to the iron-regulated protein FrpD from Neisseria meningitidis which has been shown to interact with the RTX protein FrpC. This work expands our current knowledge about the function of bacteria-killing T4SSs and increases the panel of effectors known to be involved in T4SS-mediated interbacterial competition, which possibly contribute to the establishment of S. maltophilia in clinical and environmental settings.

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.

SMAD family member 3 (SMAD3) and SMAD4 repress HIF2α-dependent iron-regulatory genes.

Hypoxia-inducible factor 2α (HIF2α) directly regulates a battery of genes essential for intestinal iron absorption. Interestingly, iron deficiency and overload disorders do not result in increased intestinal expression of glycolytic or angiogenic HIF2α target genes. Similarly, inflammatory and tumor foci can induce a distinct subset of HIF2α target genes in vivo These observations indicate that different stimuli activate distinct subsets of HIF2α target genes via mechanisms that remain unclear. Here, we conducted a high-throughput siRNA-based screen to identify genes that regulate HIF2α's transcriptional activity on the promoter of the iron transporter gene divalent metal transporter-1 (DMT1). SMAD family member 3 (SMAD3) and SMAD4 were identified as potential transcriptional repressors. Further analysis revealed that SMAD4 signaling selectively represses iron-absorptive gene promoters but not the inflammatory or glycolytic HIF2α or HIF1α target genes. Moreover, the highly homologous SMAD2 did not alter HIF2α transcriptional activity. During iron deficiency, SMAD3 and SMAD4 expression was significantly decreased via proteasomal degradation, allowing for derepression of iron target genes. Several iron-regulatory genes contain a SMAD-binding element (SBE) in their proximal promoters; however, mutation of the putative SBE on the DMT1 promoter did not alter the repressive function of SMAD3 or SMAD4. Importantly, the transcription factor forkhead box protein A1 (FOXA1) was critical in SMAD4-induced DMT1 repression, and DNA binding of SMAD4 was essential for the repression of HIF2α activity, suggesting an indirect repressive mechanism through DNA binding. These results provide mechanistic clues to how HIF signaling can be regulated by different cellular cues.

Regulation of transferrin receptor-1 mRNA by the interplay between IRE-binding proteins and miR-7/miR-141 in the 3'-IRE stem-loops.

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.

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.

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.

Iron regulatory protein is involved in the immune defense of the Chinese mitten crab Eriocheir sinensis.

Iron homeostasis is vital to organismal health; it is maintained by the iron regulatory protein (IRP)-iron-responsive element (IRE) signaling pathway. In the Chinese mitten crab Eriocheir sinensis, EsFer-1 and EsFer-2 reportedly have a putative IRE, but an IRP has not yet been identified. In this study, we successfully amplified the full-length cDNA of EsIRP using gene cloning and rapid amplification of cDNA ends techniques. The length of this cDNA was 4474 bp, and it included a 2682-bp open reading frame encoding 893 amino acids. Using quantitative real-time PCR, mRNA transcripts of EsIRP were detected in various tissues. The highest and lowest expression level was detected in the muscle and gills, respectively. In response to Staphylococcus aureus and Vibrio parahaemolyticus challenge, the transcription level of EsIRP was downregulated and that of EsFer-1 and EsFer-2 was upregulated in hemocytes. EsIRP knockdown resulted in increased expression of both EsFer-1 and EsFer-2. After EsFer-1 and EsFer-2 knockdown, the bacterial clearance ability of E. sinensis against S. aureus and V. parahaemolyticus was impaired. In conclusion, our results suggest that the IRP-IRE signaling pathway plays an important role in the innate immune system response in E. sinensis.

Competing for Iron: Duplication and Amplification of the isd Locus in Staphylococcus lugdunensis HKU09-01 Provides a Competitive Advantage to Overcome Nutritional Limitation.

Staphylococcus lugdunensis is a coagulase negative bacterial pathogen that is particularly associated with severe cases of infectious endocarditis. Unique amongst the coagulase-negative staphylococci, S. lugdunensis harbors an iron regulated surface determinant locus (isd). This locus facilitates the acquisition of heme as a source of nutrient iron during infection and allows iron limitation caused by "nutritional immunity" to be overcome. The isd locus is duplicated in S. lugdunensis HKU09-01 and we show here that the duplication is intrinsically unstable and undergoes accordion-like amplification and segregation leading to extensive isd copy number variation. Amplification of the locus increased the level of expression of Isd proteins and improved binding of hemoglobin to the cell surface of S. lugdunensis. Furthermore, Isd overexpression provided an advantage when strains were competing for a limited amount of hemoglobin as the sole source of iron. Gene duplications and amplifications (GDA) are events of fundamental importance for bacterial evolution and are frequently associated with antibiotic resistance in many species. As such, GDAs are regarded as evolutionary adaptions to novel selective pressures in hostile environments pointing towards a special importance of isd for S. lugdunensis. For the first time we show an example of a GDA that involves a virulence factor of a Gram-positive pathogen and link the GDA directly to a competitive advantage when the bacteria were struggling with selective pressures mimicking "nutritional immunity".

Targeting the Iron-Response Elements of the mRNAs for the Alzheimer's Amyloid Precursor Protein and Ferritin to Treat Acute Lead and Manganese Neurotoxicity.

The therapeutic value of inhibiting translation of the amyloid precursor protein (APP) offers the possibility to reduce neurotoxic amyloid formation, particularly in cases of familial Alzheimer's disease (AD) caused by APP gene duplications (Dup⁻APP) and in aging Down syndrome individuals. APP mRNA translation inhibitors such as the anticholinesterase phenserine, and high throughput screened molecules, selectively inhibited the uniquely folded iron-response element (IRE) sequences in the 5'untranslated region (5'UTR) of APP mRNA and this class of drug continues to be tested in a clinical trial as an anti-amyloid treatment for AD. By contrast, in younger age groups, APP expression is not associated with amyloidosis, instead it acts solely as a neuroprotectant while facilitating cellular ferroportin-dependent iron efflux. We have reported that the environmental metallotoxins Lead (Pb) and manganese (Mn) cause neuronal death by interfering with IRE dependent translation of APP and ferritin. The loss of these iron homeostatic neuroprotectants thereby caused an embargo of iron (Fe) export from neurons as associated with excess unstored intracellular iron and the formation of toxic reactive oxidative species (ROS). We propose that APP 5'UTR directed translation activators can be employed therapeutically to protect neurons exposed to high acute Pb and/or Mn exposure. Certainly, high potency APP translation activators, exemplified by the Food and Drug Administration (FDA) pre-approved M1 muscarinic agonist AF102B and high throughput-screened APP 5'UTR translation activators, are available for drug development to treat acute toxicity caused by Pb/Mn exposure to neurons. We conclude that APP translation activators can be predicted to prevent acute metal toxicity to neurons by a mechanism related to the 5'UTR specific yohimbine which binds and targets the canonical IRE RNA stem loop as an H-ferritin translation activator.

The N-Terminus of Iron-Sulfur Cluster Assembly Factor ISD11 Is Crucial for Subcellular Targeting and Interaction with l-Cysteine Desulfurase NFS1.

Assembly of iron-sulfur (FeS) clusters is an important process in living cells. The initial sulfur mobilization step for FeS cluster biosynthesis is catalyzed by l-cysteine desulfurase NFS1, a reaction that is localized in mitochondria in humans. In humans, the function of NFS1 depends on the ISD11 protein, which is required to stabilize its structure. The NFS1/ISD11 complex further interacts with scaffold protein ISCU and regulator protein frataxin, thereby forming a quaternary complex for FeS cluster formation. It has been suggested that the role of ISD11 is not restricted to its role in stabilizing the structure of NFS1, because studies of single-amino acid variants of ISD11 additionally demonstrated its importance for the correct assembly of the quaternary complex. In this study, we are focusing on the N-terminal region of ISD11 to determine the role of N-terminal amino acids in the formation of the complex with NFS1 and to reveal the mitochondrial targeting sequence for subcellular localization. Our in vitro studies with the purified proteins and in vivo studies in a cellular system show that the first 10 N-terminal amino acids of ISD11 are indispensable for the activity of NFS1 and especially the conserved "LYR" motif is essential for the role of ISD11 in forming a stable and active complex with NFS1.

Human Mitochondrial Ferredoxin 1 (FDX1) and Ferredoxin 2 (FDX2) Both Bind Cysteine Desulfurase and Donate Electrons for Iron-Sulfur Cluster Biosynthesis.

Ferredoxins play an important role as an electron donor in iron-sulfur (Fe-S) cluster biosynthesis. Two ferredoxins, human mitochondrial ferredoxin 1 (FDX1) and human mitochondrial ferredoxin 2 (FDX2), are present in the matrix of human mitochondria. Conflicting results have been reported regarding their respective function in mitochondrial iron-sulfur cluster biogenesis. We report here biophysical studies of the interaction of these two ferredoxins with other proteins involved in mitochondrial iron-sulfur cluster assembly. Results from nuclear magnetic resonance spectroscopy show that both FDX1 and FDX2 (in both their reduced and oxidized states) interact with the protein complex responsible for cluster assembly, which contains cysteine desulfurase (NFS1), ISD11 (also known as LYRM4), and acyl carrier protein (Acp). In all cases, ferredoxin residues close to the Fe-S cluster are involved in the interaction with this complex. Isothermal titration calorimetry results showed that FDX2 binds more tightly to the cysteine desulfurase complex than FDX1 does. The reduced form of each ferredoxin became oxidized in the presence of the cysteine desulfurase complex when l-cysteine was added, leading to its conversion to l-alanine and the generation of sulfide. In an in vitro reaction, the reduced form of each ferredoxin was found to support Fe-S cluster assembly on ISCU; the rate of cluster assembly was faster with FDX2 than with FDX1. Taken together, these results show that both FDX1 and FDX2 can function in Fe-S cluster assembly in vitro.

Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery.

Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human machinery remains undefined, we co-expressed in Escherichia coli the following four proteins involved in the initial step of Fe-S cluster synthesis: FXN42-210 (iron donor); [NFS1]·[ISD11] (sulfur donor); and ISCU (scaffold upon which new clusters are assembled). We purified a stable, active complex consisting of all four proteins with 1:1:1:1 stoichiometry. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional model of the complex with ∼14 Å resolution. Molecular dynamics flexible fitting of protein structures docked into the EM map of the model revealed a [FXN42-210]24·[NFS1]24·[ISD11]24·[ISCU]24 complex, consistent with the measured 1:1:1:1 stoichiometry of its four components. The complex structure fulfills distance constraints obtained from chemical cross-linking of the complex at multiple recurring interfaces, involving hydrogen bonds, salt bridges, or hydrophobic interactions between conserved residues. The complex consists of a central roughly cubic [FXN42-210]24·[ISCU]24 sub-complex with one symmetric ISCU trimer bound on top of one symmetric FXN42-210 trimer at each of its eight vertices. Binding of 12 [NFS1]2·[ISD11]2 sub-complexes to the surface results in a globular macromolecule with a diameter of ∼15 nm and creates 24 Fe-S cluster assembly centers. The organization of each center recapitulates a previously proposed conserved mechanism for sulfur donation from NFS1 to ISCU and reveals, for the first time, a path for iron donation from FXN42-210 to ISCU.

The Irr and RirA Proteins Participate in a Complex Regulatory Circuit and Act in Concert To Modulate Bacterioferritin Expression in Ensifer meliloti 1021.

In this work we found that the bfr gene of the rhizobial species Ensifer meliloti, encoding a bacterioferritin iron storage protein, is involved in iron homeostasis and the oxidative stress response. This gene is located downstream of and overlapping the smc03787 open reading frame (ORF). No well-predicted RirA or Irr boxes were found in the region immediately upstream of the bfr gene although two presumptive RirA boxes and one presumptive Irr box were present in the putative promoter of smc03787 We demonstrate that bfr gene expression is enhanced under iron-sufficient conditions and that Irr and RirA modulate this expression. The pattern of bfr gene expression as well as the response to Irr and RirA is inversely correlated to that of smc03787 Moreover, our results suggest that the small RNA SmelC759 participates in RirA- and Irr-mediated regulation of bfr expression and that additional unknown factors are involved in iron-dependent regulation.IMPORTANCEE. meliloti belongs to the Alphaproteobacteria, a group of bacteria that includes several species able to associate with eukaryotic hosts, from mammals to plants, in a symbiotic or pathogenic manner. Regulation of iron homeostasis in this group of bacteria differs from that found in the well-studied Gammaproteobacteria In this work we analyzed the effect of rirA and irr mutations on bfr gene expression. We demonstrate the effect of an irr mutation on iron homeostasis in this bacterial genus. Moreover, results obtained indicate a complex regulatory circuit where multiple regulators, including RirA, Irr, the small RNA SmelC759, and still unknown factors, act in concert to balance bfr gene expression.

Upregulation of transferrin receptor-1 induces cholangiocarcinoma progression via induction of labile iron pool.

Labile iron pool is a cellular source of ions available for Fenton reactions resulting in oxidative stress. Living organisms avoid an excess of free irons by a tight control of iron homeostasis. We investigated the altered expression of iron regulatory proteins and iron discrimination in the development of liver fluke-associated cholangiocarcinoma. Additionally, the levels of labile iron pool and the functions of transferrin receptor-1 on cholangiocarcinoma development were also identified. Iron deposition was determined using the Prussian blue staining method in human cholangiocarcinoma tissues. We investigated the alteration of iron regulatory proteins including transferrin, transferrin receptor-1, ferritin, ferroportin, hepcidin, and divalent metal transporter-1 in cholangiocarcinoma tissues using immunohistochemistry. The clinicopathological data of cholangiocarcinoma patients and the expressions of proteins were analyzed. Moreover, the level of intracellular labile iron pool in cholangiocarcinoma cell lines was identified by the RhoNox-1 staining method. We further demonstrated transferrin receptor-1 functions on cell proliferation and migration upon small interfering RNA for human transferrin receptor 1 transfection. Results show that Iron was strongly stained in tumor tissues, whereas negative staining was observed in normal bile ducts of healthy donors. Interestingly, high iron accumulation was significantly correlated with poor prognosis of cholangiocarcinoma patients. The expressions of iron regulatory proteins in human cholangiocarcinoma tissues and normal liver from cadaveric donors revealed that transferrin receptor-1 expression was increased in the cancer cells of cholangiocarcinoma tissues when compared with the adjacent normal bile ducts and was significantly correlated with cholangiocarcinoma metastasis. Labile iron pool level and transferrin receptor-1 expression were significantly increased in KKU-214 and KKU-213 when compared with cholangiocyte cells (MMNK1). Additionally, the suppression of transferrin receptor-1 expression significantly decreased intracellular labile iron pool, cholangiocarcinoma migration, and cell proliferation when compared with control media and control small interfering RNA. In Conclusion, high expression of transferrin receptor-1 resulting in iron uptake contributes to increase in the labile iron pool which plays roles in cholangiocarcinoma progression with aggressive clinical outcomes.