As predicted by the model, we also experimentally demonstrated that hepcidin can function by occluding Fpn and preventing iron export even in the absence of endocytosis. We therefore used the available structural data from related members of the major facilitator superfamily, including eventually BdFPN, and incorporated the experimental results from the Fpn mutant analysis to develop a new model of hepcidin-Fpn interaction, which segregates the Fpn residues important for hepcidin-induced conformational change from those critical for hepcidin binding. 33 However, hepcidin itself and the hepcidin-binding site within Fpn are found only in vertebrates, 34 so in this context, the BdFPN structure can only serve as a modeling constraint. 28, 30-32 Recently, Taniguchi et al identified the prokaryotic protein, BdFPN, from the bacterium Bdellovibrio bacteriovorus as an ortholog of Fpn and obtained its structure by X-ray crystallography. The lack of information about Fpn structure has also hampered understanding of hepcidin-Fpn interaction. Iron loading in hepatocytes, Kupffer cells, and PTMs, mild steatosis Mixed iron loading, but mainly hepatocytes heterogeneous phenotypeįatigue, fibrosis, iron loading in hepatocytes and Kupffer cells, hepatitis C Iron loading in hepatocytes, mild steatosis, and siderosis Iron loading in Kupffer cells and hepatocytes, fatigue and tremors Iron loading primarily in hepatocytes, but also in Kupffer cells, BDECs, and PTMs, fibrosis, and hepatic steatosis Iron loading in hepatocytes and BDECs, cirrhosis Understanding the mechanism of hepcidin-ferroportin interaction would allow improved targeting of this regulatory axis and development of novel treatments for iron disorders. 7, 8 In these disorders, Fpn is hyperactive, usually as a result of hepcidin deficiency, and leads to excessive iron absorption and toxic iron deposition in hepatocytes and other parenchymal cells, but relative iron depletion of macrophages.
The other extreme of the spectrum of iron disorders is exemplified by hereditary hemochromatosis or β-thalassemia intermedia. The condition is exemplified by iron-refractory iron deficiency anemia (IRIDA) or anemia of inflammation, 5, 6 in which iron accumulates in recycling macrophages and enterocytes, but may become insufficient in other tissues. 2-4 Decreased Fpn activity, usually caused by high hepcidin levels, is manifested as an iron-restriction syndrome.
Hepcidin binding to Fpn causes the ubiquitination, endocytosis, and degradation of the ligand-receptor complex, thereby decreasing iron supply to plasma. Hepcidin is a systemically acting iron-regulatory peptide hormone and the only known natural Fpn ligand. The main tissues expressing Fpn include duodenal enterocytes absorbing dietary iron, hepatic and splenic iron-recycling macrophages, and hepatocytes, which export stored iron when demand is high. Fpn is the only known cellular iron exporter in vertebrates and is the conduit through which iron is delivered into plasma (reviewed in Drakesmith et al 1). Iron homeostasis is maintained by the hepcidin-ferroportin (Fpn) axis, which controls intestinal absorption of iron, as well as internal iron recycling and systemic distribution. 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. 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. 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.
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. 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. We characterized 11 clinically relevant and 5 nonclinical Fpn mutations using stably transfected, inducible isogenic cell lines. Fpn is regulated by the hormone hepcidin, which induces Fpn endocytosis and cellular iron retention. Nonclassical ferroportin disease (FD) is a form of hereditary hemochromatosis caused by mutations in the iron transporter ferroportin (Fpn), resulting in parenchymal iron overload.
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