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。多聚腺苷酸结合蛋白1（PCBP1）是铁蛋白的铁分子伴侣; PCBP1和它的伴侣PCBP2都是将铁传递到调节HIF 1的脯氨酰羟化酶所必需的。在这里，我们表明PCBP2也是铁蛋白的铁伴侣。PCBP2和人铁蛋白在酵母中的共表达激活了缺铁反应，并增加了铁沉积到铁蛋白中。Huh 7细胞中PCBP 2的耗尽减少了铁掺入铁蛋白中。PCBP1和PCBP2均与HEK293细胞中的铁蛋白共免疫沉淀，并且两种PCBP的表达是细胞中铁蛋白复合物形成所需的。PCBP 1和2在体外表现出与铁蛋白的高亲和力结合。哺乳动物基因组编码4种PCBP，包括最低表达的PCBP 3和4。PCBP3和4在酵母中的表达激活了缺铁反应，但只有PCBP3表现出与铁蛋白的强相互作用。PCBP1和铁蛋白在铁敏感的ccc1酵母菌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形成一个复杂的铁传递到铁蛋白和所有的PCBPs可以共享铁伴侣活性。 PCBP1和PCBP2还将铁递送至脱氧羟腐胺赖氨酸羟化酶（DOHH），所述脱氧羟腐胺赖氨酸羟化酶是真核翻译因子eIF5A的羟腐胺赖氨酸修饰所需的双核铁酶。PCBP1或PCBP2耗竭的细胞表现出DOHH活性的丧失和细胞中酶的完整形式的丧失，特别是当细胞轻度缺铁时。含有PCBP1和PCBP2的裂解物在体外将apo-DOHH转化为holo-DOHH的效率高于缺乏PCBP1或PCBP2的裂解物。PCBP1在铁处理的细胞中与DOHH结合，但在对照或铁缺乏的细胞中不结合。PCBP1或PCBP2的耗竭对胞质Fe-S簇酶黄嘌呤氧化酶没有影响，但导致胞质乌头酸酶活性丧失。乌头酸酶活性的损失并不伴随着RNA结合活性的获得，这表明4Fe-4S簇的不完全解体的模式。PCBP耗竭对总细胞铁，线粒体铁水平和血红素合成的影响最小。因此，PCBP1和PCBP2可以作为铁分子伴侣，以多种类型的细胞质非血红素铁酶，并可能有一个特殊的作用，在恢复金属辅因子，自发失去缺铁细胞。