title: A convenient luminescence assay of ferroportin internalization to study its interaction with hepcidin.
abstract: Hepcidin is a liver-secreted small disulfide-rich peptide that plays a key role in iron homeostasis by binding and mediating the internalization and degradation of the only iron efflux transporter so far known, ferroportin (Fpn). To study hepcidin-Fpn interactions, in the present study we established a convenient luminescence assay for the quantitative measurement of hepcidin-induced Fpn internalization by fusing a small nanoluciferase (NanoLuc, 171 amino acids) at the Fpn C-terminus. Once the NanoLuc-tagged Fpn was internalized, the measured luminescence was significantly decreased when assayed with the intact transiently transfected cells and an inducible expression system. Through the coexpression of a NanoLuc-tagged Fpn and an enhanced green fluorescent protein (EGFP)-tagged Fpn by the use of an inducible bidirectional promoter, we could measure the hepcidin-induced Fpn internalization qualitatively and quantitatively on the basis of the fluorescence of the tagged EGFP and the luminescence of the tagged NanoLuc. Thus, our present study provides a novel and convenient assay for measuring the hepcidin-Fpn interaction qualitatively and quantitatively. Through coexpression of a NanoLuc-tagged wild-type Fpn and an EGFP-tagged hepcidin-insensitive mutant [C326S]Fpn, we demonstrated that the mutant Fpn had no effect on hepcidin-induced internalization of wild-type Fpn, suggesting that wild-type Fpn and mutant Fpn are functionally independent.
authors: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors: Ge
authors: G
authors: Song
authors: Qian
authors: Q
authors: Jiang
authors: Ting
authors: T
authors: Xu
authors: Ya-Li
authors: YL
authors: Liu
authors: Zeng-Guang
authors: ZG
authors: Xu
authors: Zhan-Yun
authors: ZY
authors: Guo
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000737:chemistry*; D027682:Cation Transport Proteins / Q000737:chemistry*; D002463:Cell Membrane Permeability; D057809:HEK293 Cells; D064451:Hepcidins; D006801:Humans; D008163:Luminescent Measurements; D014162:Transfection; D000098742:Ferroportin
chemical_list: D023181:Antimicrobial Cationic Peptides; D027682:Cation Transport Proteins; C578702:HAMP protein, human; D064451:Hepcidins; D000098742:Ferroportin