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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

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-con...

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Autores principales: Boumaiza, Mohamed, Fhoula, Imene, Carmona, Fernando, Poli, Maura, Asperti, Michela, Gianoncelli, Alessandra, Bertuzzi, Michela, Arosio, Paolo, Marzouki, Mohamed Nejib
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8929011/
https://www.ncbi.nlm.nih.gov/pubmed/35723388
http://dx.doi.org/10.3390/cimb44010009
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author Boumaiza, Mohamed
Fhoula, Imene
Carmona, Fernando
Poli, Maura
Asperti, Michela
Gianoncelli, Alessandra
Bertuzzi, Michela
Arosio, Paolo
Marzouki, Mohamed Nejib
author_facet Boumaiza, Mohamed
Fhoula, Imene
Carmona, Fernando
Poli, Maura
Asperti, Michela
Gianoncelli, Alessandra
Bertuzzi, Michela
Arosio, Paolo
Marzouki, Mohamed Nejib
author_sort Boumaiza, Mohamed
collection PubMed
description 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.
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spelling pubmed-89290112022-06-04 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 Boumaiza, Mohamed Fhoula, Imene Carmona, Fernando Poli, Maura Asperti, Michela Gianoncelli, Alessandra Bertuzzi, Michela Arosio, Paolo Marzouki, Mohamed Nejib Curr Issues Mol Biol Article 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. MDPI 2021-12-28 /pmc/articles/PMC8929011/ /pubmed/35723388 http://dx.doi.org/10.3390/cimb44010009 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Boumaiza, Mohamed
Fhoula, Imene
Carmona, Fernando
Poli, Maura
Asperti, Michela
Gianoncelli, Alessandra
Bertuzzi, Michela
Arosio, Paolo
Marzouki, Mohamed Nejib
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
title 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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_short 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
title_sort 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
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8929011/
https://www.ncbi.nlm.nih.gov/pubmed/35723388
http://dx.doi.org/10.3390/cimb44010009
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