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Biochemical, Biophysical and Functional Characterization of an Insoluble Iron Containing Hepcidin–Ferritin Chimeric Monomer Assembled Together with Human Ferritin H/L Chains at Different Molar Ratios

Authors :
Mohamed Boumaiza
Imene Fhoula
Fernando Carmona
Maura Poli
Michela Asperti
Alessandra Gianoncelli
Michela Bertuzzi
Paolo Arosio
Mohamed Nejib Marzouki
Source :
Current Issues in Molecular Biology, Vol 44, Iss 9, Pp 117-127 (2022), Current Issues in Molecular Biology; Volume 44; Issue 1; Pages: 117-127
Publication Year :
2021
Publisher :
MDPI AG, 2021.

Abstract

Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin–ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery.

Details

ISSN :
14673045
Volume :
44
Database :
OpenAIRE
Journal :
Current Issues in Molecular Biology
Accession number :
edsair.doi.dedup.....58fea2c9e4a51aef23f5f95cd91be74a
Full Text :
https://doi.org/10.3390/cimb44010009