title: 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.
abstract: 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) and Ac-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 with confocal microscopy. A significant role of copper ions was found in the modulation 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 the metal-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 peptides able to activate the NGF-TrkA signaling cascade. Metal ions modulated the activity of both NGF protein and the NGF-mimicking peptides. Such findings demonstrated that NGF(1-14) sequence can reproduce the signal transduction of whole protein, therefore representing a very promising drug candidate for further pre-clinical studies.
authors.affiliation: Endocrinology, Department of Clinical and Experimental Medicine, Garibaldi-Nesima Medical Center, University of CataniaCatania, Italy; Institute of Biostructures and Bioimages - Catania, National Research CouncilCatania, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pandini
authors.affiliation: Department of Chemical Sciences, University of CataniaCatania, Italy; Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi BiologiciBari, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Satriano
authors.affiliation: Department of Health Sciences, University of Catanzaro Catanzaro, Italy.
authors.forename: Adriana
authors.initials: A
authors.lastname: Pietropaolo
authors.affiliation: Endocrinology, Department of Clinical and Experimental Medicine, Garibaldi-Nesima Medical Center, University of CataniaCatania, Italy; Institute of Biostructures and Bioimages - Catania, National Research CouncilCatania, Italy.
authors.forename: Fiorenza
authors.initials: F
authors.lastname: Gianì
authors.affiliation: Centre for Neural Science, New York University New York, NY, USA.
authors.forename: Alessio
authors.initials: A
authors.lastname: Travaglia
authors.affiliation: Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi BiologiciBari, Italy; Department of Pharmacy, University of PisaPisa, Italy.
authors.forename: Diego
authors.initials: D
authors.lastname: La Mendola
authors.affiliation: Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi BiologiciBari, Italy; Section of Medical Biochemistry, Department of Biomedical and Biotechnological Sciences (BIOMETEC), University of CataniaCatania, Italy.
authors.forename: Vincenzo G
authors.initials: VG
authors.lastname: Nicoletti
authors.affiliation: Institute of Biostructures and Bioimages - Catania, National Research CouncilCatania, Italy; Department of Chemical Sciences, University of CataniaCatania, Italy; Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi BiologiciBari, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Rizzarelli
keywords: Alzheimer's disease; BDNF; CREB; ionophore; metal ions; nanomedicine; neurotrophins; peptidomimetics