Abstract
Ctr1 regulates copper uptake and its intracellular distribution. The first 14 amino acid sequence of the Ctr1 ectodomain Ctr1(1-14) encompasses the characteristic Amino Terminal Cu2+ and Ni2+ binding motif (ATCUN) as well as the bis-His binding motif (His5 and His6). We report a combined thermodynamic and spectroscopic (UV-vis, CD, EPR) study dealing with the formation of Cu2+ homobinuclear complexes with Ctr1(1-14), the percentage of which is not negligible even in the presence of a small Cu2+ excess and clearly prevails at a M/L ratio of 1.9. Ascorbate fails to reduce Cu2+ when bound to the ATCUN motif, while it reduces Cu2+ when bound to the His5-His6 motif involved in the formation of binuclear species. The histidine diade characterizes the second binding site and is thought to be responsible for ascorbate oxidation. Binding constants and speciation of Ag+ complexes with Ctr1(1-14), which are assumed to mimic Cu+ interaction with N-terminus of Ctr1(1-14), were also determined. A preliminary immunoblot assay evidences that the anti-Ctr1 extracellular antibody recognizes Ctr1(1-14) in a different way from the longer Ctr1(1-25) that encompasses a second His and Met rich domain.
Highlights
Published: 8 March 2022Copper is an essential element for life that is needed for all cellular systems of both prokaryotic and eukaryotic organisms [1] Cu+ /Cu2+ redox process can be detrimental to cells, as it may result in the production of different reactive oxygen, nitrogen and carbon species that are thought to be the cause of different disorders [2,3].A defective absorption of the metal ion is linked to a series of pathologies, while an excessive copper accumulation may cause degenerative disorders [4,5].it is not a surprise that copper homeostasis is strictly controlled in all eukaryotic organisms
In an attempt to answer the questions above, we report a combined thermodynamic and spectroscopic (UV-vis, Circular Dichroism (CD), EPR) study dealing with the formation of binuclear species of Cu2+ with copper transporter 1 (Ctr1)(1-14) as well as the binding constants and speciation of Ag+ complexes with Ctr1(1-14) that are assumed to mimic Cu+ interaction with the N-terminus of Ctr1(1-14)
Stefaniak et al have explored a 1:1 Cu2+ /Ctr1(1-14) ratio and obtained the stability constant values and the related speciation supporting the ability of the Nterminus of Human Ctr1 (hCtr1) to sequester Cu2+ from human serum albumin [17]
Summary
Copper is an essential element for life that is needed for all cellular systems of both prokaryotic and eukaryotic organisms [1] Cu+ /Cu2+ redox process can be detrimental to cells, as it may result in the production of different reactive oxygen, nitrogen and carbon species that are thought to be the cause of different disorders [2,3]. It is not a surprise that copper homeostasis is strictly controlled in all eukaryotic organisms. To this end, a protein network is devoted to the regulation of intracellular Cu+ levels. The membrane copper transporter 1 (Ctr1), which belongs to the SLC31 family of solute carriers [6] and possesses high specificity and affinity for Cu+ , is the most important protein transporting copper into the cell [7] Ctr in concert with.
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