Proximity Ligation Assay for the Analysis of Iron-Mediated Protein-Protein Interactions in the Nucleus.

Iron forms essential cofactors used by many nuclear enzymes involved in genome maintenance. However, unchaperoned nuclear iron may represent a threat to the surrounding genetic material as it promotes redox toxicity that may affect DNA integrity. Safely handling intracellular iron implies metal transfer and cofactor assembly processes based on protein-protein interactions. Identifying those interactions commonly occurs via high-throughput approaches using affinity purification or proximity labeling coupled with mass spectrometry analysis. However, these methods do not identify the subcellular location of the interactions. The one-on-one confirmation of proposed nuclear interactions is also challenging. Many approaches used to look at protein interactions are not tailored for looking at the nucleus because the methods used to solubilize nuclear content are harsh enough to disrupt those transient interactions. Here, we describe step-by-step the use of Proximity Ligation Assay (PLA) to analyze iron-mediated protein-protein interactions in the nucleus of cultured human cells. PLA allows the subcellular visualization of the interactions via the in situ detection of the two interacting proteins using fluorescence confocal microscopy. Briefly, cells are fixed, blocked, permeabilized, and incubated with primary antibodies directed to target proteins. Primary antibodies are recognized using PLA probes consisting of one PLUS and one MINUS oligonucleotide-labeled secondary antibody. If the two proteins are close enough (<40 nm), the PLA probes are ligated and used as the template for rolling circle amplification (RCA) with fluorescently labeled oligonucleotides that yield a signal detectable using fluorescence confocal microscopy. A fluorescently labeled membrane-specific stain (WGA) and the DNA-specific probe DAPI are used to identify cellular and nuclear boundaries, respectively. Confocal images are then analyzed using the CellProfiler software to confirm the abundance and localization of the studied protein-protein interactions.

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.

PCBP1 regulates alternative splicing of AARS2 in congenital cardiomyopathy.

Alanyl-transfer RNA synthetase 2 (AARS2) is a nuclear encoded mitochondrial tRNA synthetase that is responsible for charging of tRNA-Ala with alanine during mitochondrial translation. Homozygous or compound heterozygous mutations in the Aars2 gene, including those affecting its splicing, are linked to infantile cardiomyopathy in humans. However, how Aars2 regulates heart development, and the underlying molecular mechanism of heart disease remains unknown. Here, we found that poly(rC) binding protein 1 (PCBP1) interacts with the Aars2 transcript to mediate its alternative splicing and is critical for the expression and function of Aars2. Cardiomyocyte-specific deletion of Pcbp1 in mice resulted in defects in heart development that are reminiscent of human congenital cardiac defects, including noncompaction cardiomyopathy and a disruption of the cardiomyocyte maturation trajectory. Loss of Pcbp1 led to an aberrant alternative splicing and a premature termination of Aars2 in cardiomyocytes. Additionally, Aars2 mutant mice with exon-16 skipping recapitulated heart developmental defects observed in Pcbp1 mutant mice. Mechanistically, we found dysregulated gene and protein expression of the oxidative phosphorylation pathway in both Pcbp1 and Aars2 mutant hearts; these date provide further evidence that the infantile hypertrophic cardiomyopathy associated with the disorder oxidative phosphorylation defect type 8 (COXPD8) is mediated by Aars2. Our study therefore identifies Pcbp1 and Aars2 as critical regulators of heart development and provides important molecular insights into the role of disruptions in metabolism on congenital heart defects.

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.

Achieving Life through Death: Redox Biology of Lipid Peroxidation in Ferroptosis.

Redox balance is essential for normal brain, hence dis-coordinated oxidative reactions leading to neuronal death, including programs of regulated death, are commonly viewed as an inevitable pathogenic penalty for acute neuro-injury and neurodegenerative diseases. Ferroptosis is one of these programs triggered by dyshomeostasis of three metabolic pillars: iron, thiols, and polyunsaturated phospholipids. This review focuses on: (1) lipid peroxidation (LPO) as the major instrument of cell demise, (2) iron as its catalytic mechanism, and (3) thiols as regulators of pro-ferroptotic signals, hydroperoxy lipids. Given the central role of LPO, we discuss the engagement of selective and specific enzymatic pathways versus random free radical chemical reactions in the context of the phospholipid substrates, their biosynthesis, intracellular location, and related oxygenating machinery as participants in ferroptotic cascades. These concepts are discussed in the light of emerging neuro-therapeutic approaches controlling intracellular production of pro-ferroptotic phospholipid signals and their non-cell-autonomous spreading, leading to ferroptosis-associated necroinflammation.

PCBP2 post-transcriptionally regulates sortilin expression by binding to a C-rich element in its 3' UTR.

Post-transcriptional regulation of cytokine production is crucial to ensure appropriate immune responses. We previously demonstrated that poly-rC-binding protein-1 (PCBP1) can act as a trans-acting factor to stabilize transcripts encoding sortilin, which mediates cytokine trafficking. Here, we report that PCBP2, which strongly resembles PCBP1, can stabilize sortilin transcripts in macrophages using the same mechanism employed by PCBP1. PCBP2 recognized the C-rich element in the 3' UTR of sortilin mRNA, and PCBP2 knockdown decreased sortilin transcripts, indicating that PCBP2 stabilizes sortilin mRNA by binding to its 3' UTR. Zn2+ reversibly inhibited the nucleotide binding ability of PCBP2 in vitro. These findings suggest that both PCBP2 and PCBP1 may control the stability of sortilin transcripts by sensing intracellular Zn2+ levels in immune cells.

The Human-Specific BOLA2 Duplication Modifies Iron Homeostasis and Anemia Predisposition in Chromosome 16p11.2 Autism Individuals.

Human-specific duplications at chromosome 16p11.2 mediate recurrent pathogenic 600 kbp BP4-BP5 copy-number variations, which are among the most common genetic causes of autism. These copy-number polymorphic duplications are under positive selection and include three to eight copies of BOLA2, a gene involved in the maturation of cytosolic iron-sulfur proteins. To investigate the potential advantage provided by the rapid expansion of BOLA2, we assessed hematological traits and anemia prevalence in 379,385 controls and individuals who have lost or gained copies of BOLA2: 89 chromosome 16p11.2 BP4-BP5 deletion carriers and 56 reciprocal duplication carriers in the UK Biobank. We found that the 16p11.2 deletion is associated with anemia (18/89 carriers, 20%, p = 4e-7, OR = 5), particularly iron-deficiency anemia. We observed similar enrichments in two clinical 16p11.2 deletion cohorts, which included 6/63 (10%) and 7/20 (35%) unrelated individuals with anemia, microcytosis, low serum iron, or low blood hemoglobin. Upon stratification by BOLA2 copy number, our data showed an association between low BOLA2 dosage and the above phenotypes (8/15 individuals with three copies, 53%, p = 1e-4). In parallel, we analyzed hematological traits in mice carrying the 16p11.2 orthologous deletion or duplication, as well as Bola2+/- and Bola2-/- animals. The Bola2-deficient mice and the mice carrying the deletion showed early evidence of iron deficiency, including a mild decrease in hemoglobin, lower plasma iron, microcytosis, and an increased red blood cell zinc-protoporphyrin-to-heme ratio. Our results indicate that BOLA2 participates in iron homeostasis in vivo, and its expansion has a potential adaptive role in protecting against iron deficiency.

A PCBP1-BolA2 chaperone complex delivers iron for cytosolic [2Fe-2S] cluster assembly.

Hundreds of cellular proteins require iron cofactors for activity, and cells express systems for their assembly and distribution. Molecular details of the cytosolic iron pool used for iron cofactors are lacking, but iron chaperones of the poly(rC)-binding protein (PCBP) family play a key role in ferrous ion distribution. Here we show that, in cells and in vitro, PCBP1 coordinates iron via conserved cysteine and glutamate residues and a molecule of noncovalently bound glutathione (GSH). Proteomics analysis of PCBP1-interacting proteins identified BolA2, which functions, in complex with Glrx3, as a cytosolic [2Fe-2S] cluster chaperone. The Fe-GSH-bound form of PCBP1 complexes with cytosolic BolA2 via a bridging Fe ligand. Biochemical analysis of PCBP1 and BolA2, in cells and in vitro, indicates that PCBP1-Fe-GSH-BolA2 serves as an intermediate complex required for the assembly of [2Fe-2S] clusters on BolA2-Glrx3, thereby linking the ferrous iron and Fe-S distribution systems in cells.

The ins and outs of iron: Escorting iron through the mammalian cytosol.

Mammalian cells contain thousands of metalloproteins and have evolved sophisticated systems for ensuring that metal cofactors are correctly assembled and delivered to their proper destinations. Equally critical in this process are the strategies to avoid the insertion of the wrong metal cofactor into apo-proteins and to avoid the damage that redox-active metals can catalyze in the cellular milieu. Iron and zinc are the most abundant metal cofactors in cells and iron cofactors include heme, iron-sulfur clusters, and mono- and dinuclear iron centers. Systems for the intracellular trafficking of iron cofactors are being characterized. This review focuses on the trafficking of ferrous iron cofactors in the cytosol of mammalian cells, a process that involves specialized iron-binding proteins, termed iron chaperones, of the poly rC-binding protein family.

The flux of iron through ferritin in erythrocyte development.

PURPOSE OF REVIEW: Terminal differentiation of erythropoietic progenitors requires the rapid accumulation of large amounts of iron, which is transported to the mitochondria, where it is incorporated into heme. Ferritin is the sole site of iron storage present in the cytosol. Yet the role of iron accumulation into ferritin in the context of red cell development had not been clearly defined. Early studies indicated that at the onset of terminal differentiation, iron initially accumulates in ferritin and precedes heme synthesis. Whether this accumulation is physiologically important for red cell development was unclear until recent studies defined an obligatory pathway of iron flux through ferritin. RECENT FINDINGS: The iron chaperone functions of poly rC-binding protein 1 (PCBP1) and the autophagic cargo receptor for ferritin, nuclear co-activator 4 (NCOA4) are required for the flux of iron through ferritin in developing red cells. In the absence of these functions, iron delivery to mitochondria for heme synthesis is impaired. SUMMARY: The regulated trafficking of iron through ferritin is important for maintaining a consistent flow of iron to mitochondria without releasing potentially damaging redox-active species in the cell. Other components of the iron trafficking machinery are likely to be important in red cell development.

Ferritin iron regulators, PCBP1 and NCOA4, respond to cellular iron status in developing red cells.

Developing red blood cells exhibit multiple, redundant systems for regulating and coordinating the uptake of iron, the synthesis of heme, and the formation of hemoglobin during terminal differentiation. We recently described the roles of poly rC-binding protein (PCBP1) and nuclear coactivator 4 (NCOA4) in mediating the flux of iron through ferritin in developing erythroid cells, with PCBP1, an iron chaperone, delivering iron to ferritin and NCOA4, an autophagic cargo receptor, directing ferritin to the lysosome for degradation and iron release. Ferritin iron flux is critical, as mice lacking these factors develop microcytic anemia. Here we report that these processes are regulated by cellular iron levels in a murine model of ex vivo terminal differentiation. PCBP1 delivers iron to ferritin via a direct protein-protein interaction. This interaction is developmentally regulated, enhanced by iron deprivation, and inhibited by iron excess, both in developing cells and in vitro. NCOA4 activity also exhibited developmental regulation and regulation by cellular iron levels. Excess iron uptake during differentiation triggered lysosomal degradation of NCOA4, which was dependent on the E3 ubiquitin ligase HERC2. Thus, developing red blood cells express a series of proteins that both mediate and regulate the flux of iron to the mitochondria.

Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells.

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.