Identification of human genes of iron homeostasis

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

1) 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. 2) Significance: Poly rC binding protein 1 (PCBP1) is an important regulator of multiple cellular processes; many studies have focused on its role in tumor formation and metastasis through its capacity to bind nucleic acids. PCBP1 binds to DNA and RNA and mediates interactions with other proteins that affect transcription and mRNA processing. PCBP1 is also an iron chaperone. It binds cytosolic iron as an Fe-glutathione complex and controls the chemical reactivity, sensing, and trafficking of iron in the cell. How are these distinct activities coordinated in a single protein? We identify the iron-binding sites on PCBP1 and show that iron- and RNA-binding occur independently at separate sites. Iron-binding and RNA-binding activity separately function to prevent DNA damage and prevent cell death. 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 co-evolution of DNA/RNA binding and iron chaperone activities on a single protein may prove advantageous for nucleic acid processing that depends on enzymes with iron cofactors. 3)Acute blood loss, whether through hemorrhage, hemolysis, or phlebotomy, triggers erythropoiesis. Expansion of developing erythroid precursors necessitates mobilization of stored iron to accommodate increased synthesis of heme in the erythron. Where does all this iron come from? The short answer is, ferritin. Dietary iron absorption is increased during stress erythropoiesis, the most rapidly available source of iron is in this ubiquitous iron storage protein. Iron deposition into ferritin and mobilization out of ferritin are tightly controlled in mammals and multiple modes of regulation affect both the ins and the outs. In 2014, Nuclear coactivator 4 (NCOA4) was identified as an autophagic receptor for ferritin, with a capacity to direct ferritin into autophagosomes destined for destruction in the lysosome. In this review, we describe the recent work describing the role of NCOA4 in iron homeostasis. Most recently, Li and others show that NCOA4 is required to mobilize the iron stored in hepatocyte ferritin so that it may be used for stress erythropoiesis. Their work also suggests a new mode of iron-dependent transcriptional regulation for NCOA4 that involves the hypoxia-inducible factors 1 and 2.