Identification of human genes of iron homeostasis

我们设计了一个基因筛选，以确定人类基因，改变铁稳态时，在酵母中表达。铁储存蛋白铁蛋白的人类基因已在酵母中表达，尽管蛋白质稳定并组装成适当的24亚基寡聚体，但只有少量的铁沉积在铁蛋白核心内。我们推测，胞质铁伴侣，促进铁加载到铁蛋白可能在人类细胞中表达，但不是在酵母，不合成铁蛋白。我们已经分离出多个人cDNA，当在含有人铁蛋白的酵母细胞中表达时，其增加胞质铁向铁蛋白的转移。在培养的人细胞系中使用siRNA，我们还确定了聚C结合蛋白1（PCBP1）的缺失导致铁蛋白铁负载的缺陷。免疫共沉淀实验表明，PCBP1结合铁蛋白在体内，但只有在存在还原铁。PCBP1在体外以微摩尔亲和力结合亚铁，并且可以在体外向铁蛋白提供铁。我们提出PCBP1可以在酵母和人类细胞中作为铁伴侣发挥作用，并增强铁到铁蛋白中的负载。PCBP1是铁蛋白的铁伴侣，我们研究了其他PCBP家族成员是否也作为铁蛋白的铁伴侣。PCBP1是四种同源RNA结合蛋白家族的成员42，PCBP1和2在大多数细胞类型中都以高水平表达。初步数据表明，PCBP2还结合铁，并可以将铁捐赠给酵母中的铁蛋白。目前的努力表明，额外的铁依赖性酶需要多氯联苯在体内的铁输送和激活。

We have designed a genetic screen to identify human genes that alter iron homeostasis when expressed in yeast. Human genes for the iron-storage protein ferritin have been expressed in yeast, and although the proteins are stable and assemble into the proper 24-subunit oligomer, only a small amount of iron is deposited within the ferritin core. We hypothesize that cytosolic iron chaperones that facilitate iron loading into ferritin may be expressed in human cells, but not in yeast, which do not synthesize ferritin. We have isolated multiple human cDNAs which, when expressed in a yeast cell that contains human ferritin, increase the transfer of cytosolic iron into ferritin. Using siRNA in cultured human cell lines, we have also determined that depletion of poly-C binding protein 1 (PCBP1) results in a defect in ferritin iron loading. Co-immunoprecipitation experiments indicate that PCBP1 binds to ferritin in vivo, but only in the presence of reduced iron. PCBP1 binds ferrous iron in vitro with micromolar affinity and can donate iron to ferritin in vitro. We propose that PCBP1 can function in yeast and human cells as an iron chaperone, and enhance the loading of iron into ferritin. PCBP1 is an iron chaperone for ferritin, and we investigated whether other PCBP family members also act as ferritin iron chaperones. PCBP1 is a member of a family of four homologous RNA binding proteins42, and both PCBP1 and 2 are expressed at high levels in most cell types. Preliminary data indicate that PCBP2 also binds iron and can donate iron to ferritin in yeast. Current efforts indicate that additional iron-dependent enzymes require PCBPs for iron delivery and activation in vivo.