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.

The Copper(II)-Assisted Connection between NGF and BDNF by Means of Nerve Growth Factor-Mimicking Short Peptides

“…Finally, we validated the previous hypothesized copper ability to inhibit tyrosine phosphatase activity [21]. [38] was used.…”

“…To assay the TrkA receptor internalization, PC12 cells in RPMI medium supplemented with 0.1% BSA were stimulated for 30 min with 50 ng/mL NGF or 50 µM NGF peptides in the absence or presence of CuSO 4 (1 µM) or BCS pre-treated medium (50 µM, 24 h), as previously described [21]. Briefly, the PC12 cells rinsed with cold phosphate buffered saline (PBS) were incubated with 1.5 mg/mL EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL, USA) in biotinylation buffer (10 mM boric acid, 154 mM NaCl, 7.2 mM KCl, 1.8 mM CaCl 2 , pH 8.4) for 30 min.…”

“…A total scan time of 40 min with scan intervals of 30 s was set; the acquisition parameters were kept constant for all the experiments. After the peptide addition, the solution was vigorously mixed to ensure a homogeneous peptide concentration in the whole liquid volume inside the Petri dish to avoid artefacts in the real time live cell imaging owing to different diffusion rates of the peptide molecules related to concentration gradients, as previously described [21]. For quantification of intracellular copper, the cells were treated for 15 min with 10 µM peptides in the absence or the presence of 1 µM CuSO 4 .…”

“…Recently, small peptides encompassing the 1-14 sequence of the human NGF have been shown to activate NGF-TrkA receptor kinase cascade in PC12 cells with effects comparable with those NGF-induced [20,21]. In addition, NGF mimicking peptides triggered the phosphorylation of the transcription factor cAMP response element-binding protein (CREB), which represents a key transcriptional mediator of neuronal responses to neurotrophins [22].…”

“…The NGF mimetic activity of NGF (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) peptides has been shown to be modulated by copper and zinc ions [21], although conflicting results have been reported on the role of these metal ions to regulate the NTs signaling [23]. Zinc and copper ions have been reported to have positive effects in several in vitro and in vivo models.…”