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.

铁依赖的酶获得金属辅因子的机制在很大程度上是未知的。多聚r(C)结合蛋白1(PCBP1)是铁蛋白的铁伴侣；PCBP1及其类似物PCBP2都是铁转运到调节HIF1的脯氨酸羟基酶所必需的。这里我们证明了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铁缺乏反应，并增加了铁沉积到铁蛋白中。Huh7细胞中PCBP2的缺失减少了铁与铁蛋白的结合。在HEK293细胞中，PCBP1和PCBP2都与铁蛋白共沉淀，这两种PCBPs的表达是细胞形成铁蛋白复合体所必需的。PCBP1和PCB2在体外表现出与铁蛋白高亲和力结合。哺乳动物基因组编码4个PCBPs，包括最低表达的PCBP3和4。PCBP3和4在酵母中的表达激活了缺铁反应，但只有PCBP3与铁蛋白有很强的相互作用。PCBP1和铁蛋白在铁敏感的CCc1酵母菌株中的表达加强了铁的毒性作用，而PCBP4的表达则保护细胞免受铁的毒性。因此，PCBP1和2形成了一个向铁蛋白运送铁的复合体，所有的PCBP都可能分享铁伴侣的活性。 PCBP1和PCBP2还将铁输送到脱氧亚精氨酸羟基酶(DOHH)，这是真核细胞翻译因子eIF5A亚硫氨酸修饰所需的双核铁酶。缺乏PCBP1或PCBP2的细胞表现出DOHH活性的丧失和细胞内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的效率比不含PCBP1或PCBP2的裂解物更高。在铁处理的细胞中，PCBP1与DOHH结合，而在对照或缺铁的细胞中不结合。PCBP_1或PCBP_2的耗竭对胞质Fe-S簇酶黄嘌呤氧化酶无影响，但导致胞质乌头酸酶活性丧失。乌头酸酶活性的丧失并没有伴随着RNA结合活性的增加，这一模式表明4Fe-4S簇是不完全分解的。PCBP的消耗对细胞总铁、线粒体铁水平和血红素合成的影响很小。因此，PCBP1和PCBP2可能是多种胞质非血红素铁酶的铁伴侣，在修复缺铁细胞中自发丢失的金属辅因子方面可能具有特殊的作用。