Copper(II) binding properties of hepcidin

Abstract

Hepcidin is a peptide hormone that regulates the homeostasis of iron metabolism. The N-terminal domain of hepcidin is conserved amongst a range of species and is capable of binding CuII and NiII through the amino terminal copper–nickel binding motif (ATCUN). It has been suggested that the binding of copper to hepcidin may have biological relevance. In this study we have investigated the binding of CuII with model peptides containing the ATCUN motif, fluorescently labelled hepcidin and hepcidin using MALDI-TOF mass spectrometry. As with albumin, it was found that tetrapeptide models of hepcidin possessed a higher affinity for CuII than that of native hepcidin. The log K1 value of hepcidin for CuII was determined as 7.7. CuII binds to albumin more tightly than hepcidin (log K1 = 12) and in view of the serum concentration difference of albumin and hepcidin, the bulk of kinetically labile CuII present in blood will be bound to albumin. It is estimated that the concentration of CuII-hepcidin will be less than one femtomolar in normal serum and thus the binding of copper to hepcidin is unlikely to play a role in iron homeostasis. As with albumin, small tri and tetra peptides are poor models for the metal binding properties of hepcidin.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-016-1342-2) contains supplementary material, which is available to authorized users.

Intersection of Iron and Copper Metabolism in the Mammalian Intestine and Liver

“…It was suggested that copper was required for hepcidin activity, suggesting that perturbations in copper status could potentially alter iron metabolism via changes in hepcidin function. Another recent study, however, concluded that the level of interaction between HEPC and copper was likely insufficient to influence the ability of HEPC to regulate iron homeostasis (161). Moreover, the copper-binding properties of hepcidin-25 were recently utilized in the development of a novel detection assay for human hepcidin in serum (156).…”

Copper chelation and interleukin-6 proinflammatory cytokine effects on expression of different proteins involved in iron metabolism in HepG2 cell line

“…As consequence, TfR1-mediated iron uptake is also reduced. Some studies have reported that hepcidin activity can be dependent on copper availability [77]; in fact, it has an “ATCUN” (amino-terminal Cu-Ni)-binding motif in the N-terminal of the mature protein capable to bind copper and nickel, even if a recent study has questioning this possibility [77–79]. Tselepis et al highlighted that the incapacity of hepcidin to bind copper, drastically reduce the capacity of hepcidin to induce ferroportin degradation [77].…”

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

“…Also, the decrease in hepcidin signal at molar ratio 1:5 and 1:10 coincides with significantly increased amounts of a new species identified further by MS/MS as the complex of hepcidin with two copper ions. This species was briefly mentioned previously in the literature [30], but no clear significance was attributed to the complex. To our best knowledge, this is the first reported chromatographic separation and MS/MS analysis of the peptide complex with two copper ions.…”

“…Analyzing the solution of hepcidin-Cu 2+ (molar ratio 1:10) revealed a hepcidin-25 species complexed with two Cu 2+ -ions. This compound was previously reported in literature using MALDI-TOF analysis [30], but never separated and characterized using LC-MS/MS. The doubly, triply and quadruply charged quasi-molecular ions were observed at m/z 1456.1, 971.0 and 728.5, respectively, but the signal intensity was lower compared to the other two species (see Supplementary Material Fig.…”

“…The dissociation constant of the hepcidin-25-copper(II) complex was reported to be << 10 -6 M. Kulprachakarn et al further characterized the affinity of Hep-25 for copper using MALDI-MS, with a reported dissociation constant of 10 -7.7 M. However, the measurements lacking an internal standard provide a poor peptide quantitation which lead to a higher uncertainty in affinity determination. Recently, Plonka et al employed potentiometric titration and UV-Vis spectroscopy, which are widely recognized as the standard procedure for binding constant calculations [22,29], and characterized the flexible N-terminal hexapeptide of hepcidin-25 (DTHFPI) as the strongest ATCUN motif ever reported with a dissociation constant of 10 -14.66 M, which is even higher than the affinity of albumin for copper (KD=10 -12 M) [30]. Moreover, this study showed a rapid transfer of Cu 2+ from HSA to the 6-residue Nterminus [31].…”