Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin
Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin
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DOI：
10.1182/blood-2017-05-786590
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发表时间：
2018-02-22
期刊：
影响因子：
20.3
通讯作者：
Nemeth, Elizabeta
中科院分区：
文献类型：
--
作者：
Aschemeyer, Sharraya;Qiao, Bo;Nemeth, Elizabeta
非经典型的铁转运蛋白病(FD)是一种遗传性血色素沉着症，由铁转运蛋白(FPN)基因突变引起，导致实质铁超载。FPN受荷尔蒙海普西丁的调节，它诱导FPN内吞和细胞铁滞留。我们使用稳定的、可诱导的等基因细胞系鉴定了11个临床相关的和5个非临床的FPN突变。所有临床突变体都对海普西丁产生了功能性耐药，这是由于海普西丁结合受损或海普西丁依赖泛素化受损所致，尽管海普西丁结合完整。将这些残基映射到人类FPN结构的两个计算模型上表明：(1)导致泛素化耐药的突变被定位在螺旋-螺旋界面上，很可能阻止了海普西丁引起的构象变化；(2)海普西丁与FPN的中央空腔内发生了结合；(3)海普西丁与多达4个螺旋相互作用；以及(4)海普西丁结合应该阻断FPN并干扰铁的输出，而不依赖于内吞作用。我们在实验中证实，在没有内吞作用的情况下，海普西丁介导的FPN在多种细胞系统中被阻断：表达内吞缺陷FPN突变体(K8R)的HEK293细胞，表达野生型或K8R FPN的非洲爪哇卵母细胞，以及成熟的人红细胞。我们得出结论，非经典FD是由FPN突变引起的，这种突变减少了海普西丁的结合或阻碍了FPN泛素化和内吞所需的构象变化。新发现的海普西丁及其激动剂阻断铁转运的能力可能有助于开发广泛有效的遗传性铁超载障碍的治疗方法。
Nonclassical ferroportin disease (FD) is a form of hereditary hemochromatosis caused by mutations in the iron transporter ferroportin (Fpn), resulting in parenchymal iron overload. Fpn is regulated by the hormone hepcidin, which induces Fpn endocytosis and cellular iron retention. We characterized 11 clinically relevant and 5 nonclinical Fpn mutations using stably transfected, inducible isogenic cell lines. All clinical mutants were functionally resistant to hepcidin as a consequence of either impaired hepcidin binding or impaired hepcidin-dependent ubiquitination despite intact hepcidin binding. Mapping the residues onto 2 computational models of the human Fpn structure indicated that (1) mutations that caused ubiquitination-resistance were positioned at helix-helix interfaces, likely preventing the hepcidin-induced conformational change, (2) hepcidin binding occurred within the central cavity of Fpn, (3) hepcidin interacted with up to 4 helices, and (4) hepcidin binding should occlude Fpn and interfere with iron export independently of endocytosis. We experimentally confirmed hepcidin-mediated occlusion of Fpn in the absence of endocytosis in multiple cellular systems: HEK293 cells expressing an endocytosis-defective Fpn mutant (K8R), Xenopus oocytes expressing wild-type or K8R Fpn, and mature human red blood cells. We conclude that nonclassical FD is caused by Fpn mutations that decrease hepcidin binding or hinder conformational changes required for ubiquitination and endocytosis of Fpn. The newly documented ability of hepcidin and its agonists to occlude iron transport may facilitate the development of broadly effective treatments for hereditary iron overload disorders.