The Inorganic Side of NGF: Copper(II) and Zinc(II) Affect the NGF Mimicking Signaling of the N-Terminus Peptides Encompassing the Recognition Domain of TrkA Receptor

The nerve growth factor(NGF)N-terminus peptide, NGF(1–14,and its acetylated form, Ac-NGF(1–14), were investigated to scrutinize the ability of this neurotrophin domain to mimic the whole protein. Theoretical calculations demonstrated that non-covalent forces assist the molecular recognition of TrkA receptor by both peptides. Combined parallel tempering/docking simulations discriminated the effect of the N-terminal acetylation on the recognition of NGF(1–14)by the domain 5 of TrkA(TrkA-D5). Experimental findings demonstrated that both NGF(1–14)andAc-NGF(1–14)activate TrkA signaling pathways essential for neuronal survival. The NGF-induced TrkA internalization was slightly inhibited in the presence of Cu2+ and Zn2+ ions, whereas the metal ions elicited the NGF(1–14)-induced internalization of TrkA and no significant differences were found in the weak Ac-NGF(1–14)-induced receptor internalization. The crucial role of the metals was confirmed by experiments with the metal-chelator bathocuproine disulfonic acid, which showed different inhibitory effects in the signaling cascade, due to different metal affinity of NGF,NGF(1–14)and Ac-NGF(1–14). The NGF signaling cascade, activated by the two peptides,induced CREB phosphorylation, but the copper addition further stimulated the Akt, ERK and CREB phosphorylation in the presence of NGF and NGF(1–14)only. A dynamic and quick influx of both peptides into PC12 cells was tracked by live cell imaging withconfocal microscopy. A significant role of copper ions was found in themodulation of peptide sub-cellular localization, especially at the nuclear level. Furthermore, a strong copper ionophoric ability of NGF(1–14)was measured. The Ac-NGF(1–14)peptide,which binds copper ions with a lower stability constant than NGF(1–14), exhibited a lower nuclear localization with respect to the total cellular uptake. These findings were correlated to themetal-induced increase of CREB and BDNF expression caused by NGF(1–14)stimulation. In summary, we here validated NGF(1–14)and Ac-NGF(1–14) as first examples of monomer and linear