Identification of human genes of iron homeostasis

人类铁稳态基因的鉴定

• 批准号: 8939621
• 负责人: Caroline Philpott
• 金额: $ 147.45万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939621
• 关键词: Aconitate Hydratase; Affinity; Binding; Binding Proteins; Biochemical; Biological; Cells; Complex; Deposition; Enzymes; Exhibits; Ferritin; Friedreich Ataxia; Genes; Genetic; Heme; Hereditary Disease; Hereditary hemochromatosis; Homeostasis; Human; Human Identifications; In Vitro; Intracellular Transport; Iron; Iron Overload; Metals; Mitochondria; Modification; Molecular Chaperones; Nutrient; Organism; Pattern; Process; Procollagen-Proline Dioxygenase; RNA Binding; Regulation; Role; Toxic effect; Toxin; Xanthine Oxidase; Yeasts; cofactor; deoxyhypusine monooxygenase; human disease; hypoxia inducible factor 1; hypusine; iron deficiency; iron metabolism; mammalian genome; paralogous gene; response; translation factor; uptake

The mechanisms through which iron-dependent enzymes receive their metal cofactors are largely unknown. Poly r(C) Binding Protein 1 (PCBP1) is an iron chaperone for ferritin; both PCBP1 and its paralog PCBP2 are required for iron delivery to the prolyl hydroxylase that regulates HIF1. Here we show that PCBP2 is also an iron chaperone for ferritin. Co-expression of PCBP2 and human ferritins in yeast activated the iron deficiency response and increased iron deposition into ferritin. Depletion of PCBP2 in Huh7 cells diminished iron incorporation into ferritin. Both PCBP1 and PCBP2 were co-immunoprecipitated with ferritin in HEK293 cells and expression of both PCBPs was required for ferritin complex formation in cells. PCBP1 and 2 exhibited high-affinity binding to ferritin in vitro. Mammalian genomes encode 4 PCBPs, including the minimally expressed PCBPs 3 and 4. Expression of PCBP3 and 4 in yeast activated the iron deficiency response, but only PCBP3 exhibited strong interactions with ferritin. Expression of PCBP1 and ferritin in an iron sensitive, ccc1 yeast strain intensified the toxic effects of iron, while expression of PCBP4 protected the cells from iron toxicity. Thus, PCBP1 and 2 form a complex for iron delivery to ferritin and all PCBPs may share iron chaperone activity. PCBP1 and PCBP2 also deliver iron to deoxyhypusine hydroxylase (DOHH), the dinuclear iron enzyme required for hypusine modification of the eukaryotic translation factor eIF5A. Cells depleted of PCBP1 or PCBP2 exhibited loss of DOHH activity and loss of the holo-form of the enzyme in cells, particularly when cells were made mildly iron deficient. Lysates containing PCBP1 and PCBP2 converted apo-DOHH to holo-DOHH in vitro with greater efficiency than lysates lacking PCBP1 or PCBP2. PCBP1 bound to DOHH in iron-treated cells, but not in control or iron-deficient cells. Depletion of PCBP1 or PCBP2 had no effect on the cytosolic Fe-S cluster enzyme xanthine oxidase but led to loss of cytosolic aconitase activity. Loss of aconitase activity was not accompanied by gain of RNA binding activity, a pattern suggesting the incomplete disassembly of the 4Fe-4S cluster. PCBP depletions had minimal effects on total cellular iron, mitochondrial iron levels, and heme synthesis. Thus, PCBP1 and PCBP2 may serve as iron chaperones to multiple classes of cytosolic non-heme iron enzymes and may have a particular role in restoring metal cofactors that are spontaneously lost in iron deficient cells.

铁依赖性酶接受其金属辅因子的机制在很大程度上是未知的。聚r(C)结合蛋白1 （PCBP1）是铁蛋白的铁伴侣蛋白；PCBP1及其相似的PCBP2都是铁传递到调节HIF1的脯氨酰羟化酶所必需的。我们发现PCBP2也是铁蛋白的铁伴侣。酵母中PCBP2和人铁蛋白的共表达激活了缺铁反应，增加了铁沉积到铁蛋白中。Huh7细胞中PCBP2的缺失减少了铁蛋白中的铁掺入。在HEK293细胞中，PCBP1和PCBP2与铁蛋白共免疫沉淀，细胞中铁蛋白复合物的形成需要这两种PCBPs的表达。PCBP1和2在体外与铁蛋白表现出高亲和力结合。哺乳动物基因组编码4个PCBPs，包括最低表达的PCBPs 3和4。酵母中PCBP3和4的表达激活了铁缺乏反应，但只有PCBP3与铁蛋白表现出强烈的相互作用。PCBP1和铁蛋白在铁敏感酵母ccc1株中的表达增强了铁的毒性作用，而PCBP4的表达保护细胞免受铁毒性。因此，PCBP1和2形成一个复合体，将铁传递给铁蛋白，所有PCBPs可能具有铁伴侣活性。