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-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.

WDR45 Mutation Impairs the Autophagic Degradation of Transferrin Receptor and Promotes Ferroptosis

WDR45 is an autophagy-related protein that involves in the formation of autophagosome. Mutations in WDR45 lead to the impairment of autophagy which is associated with the human β-propeller protein-associated neurodegeneration (BPAN). However, the relationship between autophagy and brain iron accumulation in patients with BPAN remains unclear. Here, we demonstrated that transferrin receptor (TfRC) which is critical for the iron import of cells was degraded via autophagy. TfRC was accumulated after the inhibition of autophagy by treatment with autophagic inhibitor chloroquine or knockdown of ATG2A. The intracellular iron content was increased in cells overexpressing TfRC or mutant WDR45, however, ferritin H (FTH) chain was decreased. Increased TfRC and simultaneously decreased FTH consequently resulted in an elevated level of ferrous iron (Fe2+) which further promoted cell ferroptosis, demonstrated by the increased lipid peroxidation and reactive oxygen species (ROS) and the decreased glutathione peroxidase 4 (GPX4) and cell viability. Taken together, these findings provide a piece of important evidence that WDR45 deficiency impairs autophagic degradation of TfRC, therefore leading to iron accumulation, and the elevated iron promotes ferroptosis which may contribute to the progression of BPAN.

The Use of Ferritin as a Carrier of Peptides and Its Application for Hepcidin

Hepcidin a 25-amino-acid and highly disulfide bonded hormone, is the central regulator of iron homeostasis. In this chapter we propose ferritin as a peptide carrier to promote the association of the hybrid hepcidin/ferritin nanoparticle with a particular cell or tissue for therapeutic or diagnostic use. Indeed, human ferritin H-chain fused directly (on its 5’end) with camel mature hepcidin was cloned into the pASK-43 plus vector and expressed using BL21 (DE3) pLys E. coli strain. The transformed E.coli produced efficiently hepcidin-ferritin construct (hepcH), consisting of 213 amino acids with a molecular weight of 24 KDa. The recovered product is a ferritin exposing hepcidin on outer surface. The hepcH monomer was characterized by immunoblotting using a monoclonal antibody specific for human ferritin and a polyclonal antibody specific for hepcidin-25. The results were also confirmed by MALDI-TOF mass spectrometry. The recombinant native human ferritin and the commercial human hepcidin-25 were used as controls in this experiment. The assembly of hepcH, as an heteropolymer molecule, was performed in presence of denatured human ferritin-H and -L chains. After cysteine oxidation of the recombinant nanoparticles, cellular binding assays were performed on mammalian cells such as mouse monocyte–macrophage cell line J774, HepG2 and COS7.

Effect of iron oxide nanoparticles on vascular function and nitric oxide production in acute stress-exposed rats

We investigated whether polyethylene glycol-coated Fe(3)O(4) nanoparticles (IONs), acute stress and their combination modifies vascular functions, nitric oxide synthase (NOS) activity, mean arterial pressure (MAP) as well as hepcidin and ferritin H gene expressions in Wistar-Kyoto rats. Rats were divided into control, ION-treated rats (1 mg Fe/kg i.v.), repeated acute air-jet stress-exposed rats and IONs-and-stress co-exposed rats. Maximal acetylcholine (ACh)-induced and sodium nitroprusside (SNP)-induced relaxations in the femoral arteries did not differ among the groups. IONs alone significantly elevated the Nω-nitro-L-arginine methyl ester (L-NAME)-sensitive component of ACh-induced relaxation and reduced the sensitivity of vascular smooth muscle cells to SNP. IONs alone also elevated NOS activity in the brainstem and hypothalamus, reduced NOS activity in the kidneys and had no effect in the liver. Acute stress alone failed to affect vascular function and NOS activities in all the tissues investigated but it elevated ferritin H expression in the liver. In the ION-and-stress group, NOS activity was elevated in the kidneys and liver, but reduced in the brainstem and hypothalamus vs. IONs alone. IONs also accentuated air-jet stress-induced MAP responses vs. stress alone. Interestingly, stress reduced ION-originated iron content in blood and liver while it was elevated in the kidneys. In conclusion, the results showed that 1) acute administration of IONs altered vascular function, increased L-NAME-sensitive component of ACh-induced relaxation and had tissue-dependent effects on NOS activity, 2) ION effects were considerably reduced by co-exposure to repeated acute stress, likely related to decrease of ION-originated iron in blood due to elevated decomposition and/or excretion.