The iron chaperone poly(rC)-binding protein 1 regulates iron efflux through intestinal ferroportin in mice.

Iron is an essential nutrient required by all cells but used primarily for red blood cell production. Because humans have no effective mechanism for ridding the body of excess iron, the absorption of dietary iron must be precisely regulated. The critical site of regulation is the transfer of iron from the absorptive enterocyte to the portal circulation via the sole iron efflux transporter, ferroportin. Here, we report that poly(rC)-binding protein 1 (PCBP1), the major cytosolic iron chaperone, is necessary for the regulation of iron flux through ferroportin in the intestine of mice. Mice lacking PCBP1 in the intestinal epithelium exhibit low levels of enterocyte iron, poor retention of dietary iron in enterocyte ferritin, and excess efflux of iron through ferroportin. Excess iron efflux occurred despite lower levels of ferroportin protein in enterocytes and upregulation of the iron regulatory hormone hepcidin. PCBP1 deletion and the resulting unregulated dietary iron absorption led to poor growth, severe anemia on a low-iron diet, and liver oxidative stress with iron loading on a high-iron diet. Ex vivo culture of PCBP1-depleted enteroids demonstrated no defects in hepcidin-mediated ferroportin turnover. However, measurement of kinetically labile iron pools in enteroids competent or blocked for iron efflux indicated that PCBP1 functioned to bind and retain cytosolic iron and limit its availability for ferroportin-mediated efflux. Thus, PCBP1 coordinates enterocyte iron and reduces the concentration of unchaperoned "free" iron to a low level that is necessary for hepcidin-mediated regulation of ferroportin activity.

Vitamin E Induces Liver Iron Depletion and Alters Iron Regulation in Mice.

BACKGROUND: Vitamin E (vit E) is an essential nutrient that functions as a lipophilic antioxidant and is used clinically to treat nonalcoholic fatty liver disease, where it suppresses oxidative damage and impedes the progression of steatosis and fibrosis. Mice lacking a critical liver iron-trafficking protein also manifest steatosis because of iron-mediated oxidative damage and are protected from liver disease by oral vit E supplements. OBJECTIVES: We aimed to examine the role of dietary vit E supplementation in modulating iron-sensing regulatory systems and nonheme iron levels in mouse liver. METHODS: C57Bl/6 male mice, aged 6 wk, were fed purified diets containing normal amounts of iron and either control (45 mg/kg) or elevated (450 mg/kg) levels of 2R-α-tocopherol (vit E) for 18 d. Mouse plasma and liver were analyzed for nonheme iron, levels and activity of iron homeostatic proteins, and markers of oxidative stress. We compared means ± SD for iron and oxidative stress parameters between mice fed the control diet and those fed the vit E diet. RESULTS: The Vit E-fed mice exhibited lower levels of liver nonheme iron (38% reduction, P < 0.0001) and ferritin (74% reduction, P < 0.01) than control-fed mice. The levels of liver mRNA for transferrin receptor 1 and divalent metal transporter 1 were reduced to 42% and 57% of the control, respectively. The mRNA levels for targets of nuclear factor erythroid 2-related factor (Nrf2), a major regulator of the oxidative stress response and iron-responsive genes, were also suppressed in vit E livers. Hepcidin, an iron regulatory hormone, levels were lower in the plasma (P < 0.05), and ferroportin (FPN), the iron exporter regulated by hepcidin, was expressed at higher levels in the liver (P < 0.05). CONCLUSIONS: Oral vit E supplementation in mice can lead to depletion of liver iron stores by suppressing the iron- and redox-sensing transcription factor Nrf2, leading to enhanced iron efflux through liver FPN. Iron depletion may indirectly enhance the antioxidative effects of vit E.

Iron-tracking strategies: Chaperones capture iron in the cytosolic labile iron pool.

Cells express hundreds of iron-dependent enzymes that rely on the iron cofactors heme, iron-sulfur clusters, and mono-or di-nuclear iron centers for activity. Cells require systems for both the assembly and the distribution of iron cofactors to their cognate enzymes. Proteins involved in the binding and trafficking of iron ions in the cytosol, called cytosolic iron chaperones, have been identified and characterized in mammalian cells. The first identified iron chaperone, poly C-binding protein 1 (PCBP1), has also been studied in mice using genetic models of conditional deletion in tissues specialized for iron handling. Studies of iron trafficking in mouse tissues have necessitated the development of new approaches, which have revealed new roles for PCBP1 in the management of cytosolic iron. These approaches can be applied to investigate use of other nutrient metals in mammals.

Mitochondrial dysfunction in mouse livers depleted of iron chaperone PCBP1.

Iron is an essential nutrient that forms cofactors required for the activity of hundreds of cellular proteins. However, iron can be toxic and must be precisely managed. Poly r(C) binding protein 1 (PCBP1) is an essential, multifunctional protein that binds both iron and nucleic acids, regulating the fate of both. As an iron chaperone, PCBP1 binds cytosolic iron and delivers it to iron enzymes for activation and to ferritin for storage. Mice deleted for PCBP1 in the liver exhibit dysregulated iron balance, with lower levels of liver iron stores and iron enzymes, but higher levels of chemically-reactive iron. Unchaperoned iron triggers the formation of reactive oxygen species, leading to lipid peroxidation and ferroptotic cell death. Hepatic PCBP1 deletion produces chronic liver disease in mice, with steatosis, triglyceride accumulation, and elevated plasma ALT levels. Human and mouse models of fatty liver disease are associated with mitochondrial dysfunction. Here we show that, although deletion of PCBP1 does not affect mitochondrial iron balance, it does affect mitochondrial function. PCBP1 deletion affected mitochondrial morphology and reduced levels of respiratory complexes II and IV, oxygen consumption, and ATP production. Depletion of mitochondrial lipids cardiolipin and coenzyme Q, along with reduction of mitochondrial oxygen consumption, were the first manifestations of mitochondrial dysfunction. Although dietary supplementation with vitamin E ameliorated the liver disease in mice with hepatic PCBP1 deletion, supplementation with coenzyme Q was required to fully restore mitochondrial lipids and function. In conclusion, our studies indicate that mitochondrial function can be restored in livers subjected to ongoing oxidative damage from unchaperoned iron by supplementation with coenzyme Q, a mitochondrial lipid essential for respiration that also functions as a lipophilic radical-trapping agent.

The iron chaperone and nucleic acid-binding activities of poly(rC)-binding protein 1 are separable and independently essential.

Poly(rC)-binding protein (PCBP1) is a multifunctional adaptor protein that can coordinate single-stranded nucleic acids and iron-glutathione complexes, altering the processing and transfer of these ligands through interactions with other proteins. Multiple phenotypes are ascribed to cells lacking PCBP1, but the relative contribution of RNA, DNA, or iron chaperone activity is not consistently clear. Here, we report the identification of amino acid residues required for iron coordination on each structural domain of PCBP1 and confirm the requirement of iron coordination for binding target proteins BolA2 and ferritin. We further construct PCBP1 variants that lack either nucleic acid- or iron-binding activity and examine their functions in human cells and mouse tissues depleted of endogenous PCBP1. We find that these activities are separable and independently confer essential functions. While iron chaperone activity controls cell cycle progression and suppression of DNA damage, RNA/DNA-binding activity maintains cell viability in both cultured cell and mouse models. The coevolution of RNA/DNA binding and iron chaperone activities on a single protein may prove advantageous for nucleic acid processing that depends on enzymes with iron cofactors.

Iron Chaperone Poly rC Binding Protein 1 Protects Mouse Liver From Lipid Peroxidation and Steatosis.

BACKGROUND AND AIMS: Iron is essential yet also highly chemically reactive and potentially toxic. The mechanisms that allow cells to use iron safely are not clear; defects in iron management are a causative factor in the cell-death pathway known as ferroptosis. Poly rC binding protein 1 (PCBP1) is a multifunctional protein that serves as a cytosolic iron chaperone, binding and transferring iron to recipient proteins in mammalian cells. Although PCBP1 distributes iron in cells, its role in managing iron in mammalian tissues remains open for study. The liver is highly specialized for iron uptake, utilization, storage, and secretion. APPROACH AND RESULTS: Mice lacking PCBP1 in hepatocytes exhibited defects in liver iron homeostasis with low levels of liver iron, reduced activity of iron enzymes, and misregulation of the cell-autonomous iron regulatory system. These mice spontaneously developed liver disease with hepatic steatosis, inflammation, and degeneration. Transcriptome analysis indicated activation of lipid biosynthetic and oxidative-stress response pathways, including the antiferroptotic mediator, glutathione peroxidase type 4. Although PCBP1-deleted livers were iron deficient, dietary iron supplementation did not prevent steatosis; instead, dietary iron restriction and antioxidant therapy with vitamin E prevented liver disease. PCBP1-deleted hepatocytes exhibited increased labile iron and production of reactive oxygen species (ROS), were hypersensitive to iron and pro-oxidants, and accumulated oxidatively damaged lipids because of the reactivity of unchaperoned iron. CONCLUSIONS: Unchaperoned iron in PCBP1-deleted mouse hepatocytes leads to production of ROS, resulting in lipid peroxidation (LPO) and steatosis in the absence of iron overload. The iron chaperone activity of PCBP1 is therefore critical for limiting the toxicity of cytosolic iron and may be a key factor in preventing the LPO that triggers the ferroptotic cell-death pathway.

Management versus miscues in the cytosolic labile iron pool: The varied functions of iron chaperones.

Iron-containing proteins rely on the incorporation of a set of iron cofactors for activity. The cofactors must be synthesized or assembled from raw materials located within the cell. The chemical nature of this pool of raw material - referred to as the labile iron pool - has become clearer with the identification of micro- and macro-molecules that coordinate iron within the cell. These molecules function as a buffer system for the management of intracellular iron and are the focus of this review, with emphasis on the major iron chaperone protein coordinating the labile iron pool: poly C-binding protein 1.

A lower affinity to cytosolic proteins reveals VDAC3 isoform-specific role in mitochondrial biology.

Voltage-dependent anion channel (VDAC) is the major pathway for the transport of ions and metabolites across the mitochondrial outer membrane. Among the three known mammalian VDAC isoforms, VDAC3 is the least characterized, but unique functional roles have been proposed in cellular and animal models. Yet, a high-sequence similarity between VDAC1 and VDAC3 is indicative of a similar pore-forming structure. Here, we conclusively show that VDAC3 forms stable, highly conductive voltage-gated channels that, much like VDAC1, are weakly anion selective and facilitate metabolite exchange, but exhibit unique properties when interacting with the cytosolic proteins α-synuclein and tubulin. These two proteins are known to be potent regulators of VDAC1 and induce similar characteristic blockages (on the millisecond time scale) of VDAC3, but with 10- to 100-fold reduced on-rates and altered α-synuclein blocking times, indicative of an isoform-specific function. Through cysteine scanning mutagenesis, we found that VDAC3's cysteine residues regulate its interaction with α-synuclein, demonstrating VDAC3-unique functional properties and further highlighting a general molecular mechanism for VDAC isoform-specific regulation of mitochondrial bioenergetics.

PCBP1 and NCOA4 regulate erythroid iron storage and heme biosynthesis.

Developing erythrocytes take up exceptionally large amounts of iron, which must be transferred to mitochondria for incorporation into heme. This massive iron flux must be precisely controlled to permit the coordinated synthesis of heme and hemoglobin while avoiding the toxic effects of chemically reactive iron. In cultured animal cells, iron chaperones poly rC-binding protein 1 (PCBP1) and PCBP2 deliver iron to ferritin, the sole cytosolic iron storage protein, and nuclear receptor coactivator 4 (NCOA4) mediates the autophagic turnover of ferritin. The roles of PCBP, ferritin, and NCOA4 in erythroid development remain unclear. Here, we show that PCBP1, NCOA4, and ferritin are critical for murine red cell development. Using a cultured cell model of erythroid differentiation, depletion of PCBP1 or NCOA4 impaired iron trafficking through ferritin, which resulted in reduced heme synthesis, reduced hemoglobin formation, and perturbation of erythroid regulatory systems. Mice lacking Pcbp1 exhibited microcytic anemia and activation of compensatory erythropoiesis via the regulators erythropoietin and erythroferrone. Ex vivo differentiation of erythroid precursors from Pcbp1-deficient mice confirmed defects in ferritin iron flux and heme synthesis. These studies demonstrate the importance of ferritin for the vectorial transfer of imported iron to mitochondria in developing red cells and of PCBP1 and NCOA4 in mediating iron flux through ferritin.

Heme uptake by Leishmania amazonensis is mediated by the transmembrane protein LHR1.

Trypanosomatid protozoan parasites lack a functional heme biosynthetic pathway, so must acquire heme from the environment to survive. However, the molecular pathway responsible for heme acquisition by these organisms is unknown. Here we show that L. amazonensis LHR1, a homolog of the C. elegans plasma membrane heme transporter HRG-4, functions in heme transport. Tagged LHR1 localized to the plasma membrane and to endocytic compartments, in both L. amazonensis and mammalian cells. Heme deprivation in L. amazonensis increased LHR1 transcript levels, promoted uptake of the fluorescent heme analog ZnMP, and increased the total intracellular heme content of promastigotes. Conversely, deletion of one LHR1 allele reduced ZnMP uptake and the intracellular heme pool by approximately 50%, indicating that LHR1 is a major heme importer in L. amazonensis. Viable parasites with correct replacement of both LHR1 alleles could not be obtained despite extensive attempts, suggesting that this gene is essential for the survival of promastigotes. Notably, LHR1 expression allowed Saccharomyces cerevisiae to import heme from the environment, and rescued growth of a strain deficient in heme biosynthesis. Syntenic genes with high sequence identity to LHR1 are present in the genomes of several species of Leishmania and also Trypanosoma cruzi and Trypanosoma brucei, indicating that therapeutic agents targeting this transporter could be effective against a broad group of trypanosomatid parasites that cause serious human disease.

Metabolic response to iron deficiency in Saccharomyces cerevisiae.

Iron is an essential cofactor for enzymes involved in numerous cellular processes, yet little is known about the impact of iron deficiency on cellular metabolism or iron proteins. Previous studies have focused on changes in transcript and proteins levels in iron-deficient cells, yet these changes may not reflect changes in transport activity or flux through a metabolic pathway. We analyzed the metabolomes and transcriptomes of yeast grown in iron-rich and iron-poor media to determine which biosynthetic processes are altered when iron availability falls. Iron deficiency led to changes in glucose metabolism, amino acid biosynthesis, and lipid biosynthesis that were due to deficiencies in specific iron-dependent enzymes. Iron-sulfur proteins exhibited loss of iron cofactors, yet amino acid synthesis was maintained. Ergosterol and sphingolipid biosynthetic pathways had blocks at points where heme and diiron enzymes function, whereas Ole1, the essential fatty acid desaturase, was resistant to iron depletion. Iron-deficient cells exhibited depletion of most iron enzyme activities, but loss of activity during iron deficiency did not consistently disrupt metabolism. Amino acid homeostasis was robust, but iron deficiency impaired lipid synthesis, altering the properties and functions of cellular membranes.

Mutations and environmental factors affecting regulation of riboflavin synthesis and iron assimilation also cause oxidative stress in the yeast Pichia guilliermondii.

Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. However, the mechanisms of such regulation are not known. We found that mutations causing riboflavin overproduction and iron hyperaccumulation (rib80, rib81 and hit1), as well as cobalt excess or iron deficiency all provoke oxidative stress in the Pichia guilliermondii yeast. Iron content in the cells, production both of riboflavin and malondialdehyde by P. guilliermondii wild type and hit1 mutant strains depend on a type of carbon source used in cultivation media. The data suggest that the regulation of riboflavin biosynthesis and iron assimilation in P. guilliermondii are linked with cellular oxidative state.