Cellular Catabolism of the Iron-Regulatory Peptide Hormone Hepcidin

Gloria Cuevas Preza,Elizabeta Nemeth,Tomas Ganz,Rogelio Pinon,Tracey Rouault

doi:10.1371/journal.pone.0058934

Gloria Cuevas Preza, Elizabeta Nemeth + Show 3 more

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https://doi.org/10.1371/journal.pone.0058934

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Abstract

Hepcidin, a 25-amino acid peptide hormone, is the principal regulator of plasma iron concentrations. Hepcidin binding to its receptor, the iron exporter ferroportin, induces ferroportin internalization and degradation, thus blocking iron efflux from cells into plasma. The aim of this study was to characterize the fate of hepcidin after binding to ferroportin. We show that hepcidin is taken up by ferroportin-expressing cells in a temperature- and pH-dependent manner, and degraded together with its receptor. When Texas red-labeled hepcidin (TR-Hep) was added to ferroportin-GFP (Fpn-GFP) expressing cells, confocal microscopy showed co-localization of TR-Hep with Fpn-GFP. Using flow cytometry, we showed that the peptide was almost completely degraded by 24 h after its addition, but that lysosomal inhibitors completely prevented degradation of both ferroportin and hepcidin. In addition, using radio-labeled hepcidin and HPLC analysis we show that hepcidin is not recycled, and that only degradation products are released from the cells. Together these results show that the hormone hepcidin and its receptor ferroportin are internalized together and trafficked to lysosomes where both are degraded.

Highlights

The interaction between the iron-regulatory hormone hepcidin and its receptor, the cellular iron exporter ferroportin, is central to mammalian iron homeostasis
Hepcidin binding to ferroportin was pH dependent, with 50% binding seen at pH 6 and highest binding at pH 9 (Figure 1b)
As disulfide exchange is promoted at alkaline pH, this observation is consistent with our previous work showing thiol-disulfide interaction between hepcidin and ferroportin [7,8]

Summary

Introduction

The interaction between the iron-regulatory hormone hepcidin and its receptor, the cellular iron exporter ferroportin, is central to mammalian iron homeostasis. Hepcidin is a 25 amino acid hepatic peptide hormone that controls the delivery of iron to blood plasma so as to meet cellular iron needs but avoid iron excess and toxicity. Endocytosed receptors may be degraded or recycled but their ligands may dissociate during endocytosis and may or may not follow the fate of their receptor [3]. Previous studies have shown that binding of hepcidin to ferroportin results in ubiquitination of the receptor which leads to its internalization and subsequent degradation in lysosomes [1,4,5,6]. We examine the formation of the hepcidin-ferroportin receptor-ligand complex and follow the cellular fate of the receptor-bound hepcidin

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