Brain-derived neurotrophic factor (BDNF) is a neurotrophin that influences development, maintenance, survival, and synaptic plasticity of central and peripheral nervous systems. Altered BDNF signaling is involved in several neurodegenerative disorders including Alzheimer's disease. Metal ions may influence the BDNF activity and it is well known that the alteration of Cu(2+) homeostasis is a prominent factor in the development of neurological pathologies. The N-terminal domain of BDNF represents the recognition site of its specific receptor TrkB, and metal ions interaction with this protein domain may influence the protein/receptor interaction. In spite of this, no data inherent the interaction of BDNF with Cu(2+) ions has been reported up to now. Cu(2+) complexes of the peptide fragment BDNF(1-12) encompassing the sequence 1-12 of N-terminal domain of human BDNF protein were characterized by means of potentiometry, spectroscopic methods (UV/Vis, CD, EPR), parallel tempering simulations and DFT-geometry optimizations. Coordination features of the acetylated form, Ac-BDNF(1-12), were also characterized to understand the involvement of the terminal amino group. Whereas, an analogous peptide, BDNF(1-12)D3N, in which the aspartate residue was substituted by an asparagine, was synthesized to provide evidence on the possible role of carboxylate group in Cu(2+) coordination. The results demonstrated that the amino group is involved in metal binding and the metal coordination environment of the predominant complex species at physiological pH consisted of one amino group, two amide nitrogen atoms, and one carboxylate group. Noteworthy, a strong decrease of the proliferative activity of both BDNF(1-12) and the whole protein on a SHSY5Y neuroblastoma cell line was found after treatment in the presence of Cu(2+). The effect of metal addition is opposite to that observed for the analogous fragment of nerve growth factor (NGF) protein, highlighting the role of specific domains, and suggesting that Cu(2+) may drive different pathways for the BDNF and NGF in physiological as well as pathological conditions.

Mounting evidence suggests an important role of cyclodextrins in providing protection in neurodegenerative disorders. Metal dyshomeostasis is reported to be a pathogenic factor in neurodegeneration because it could be responsible for damage involving oxidative stress and protein aggregation. As such, metal ions represent an effective target. To improve the metal-binding ability of cyclodextrin, we synthesized three new 8-hydroxyquinoline-cyclodextrin conjugates with difunctionalized cyclodextrins. In particular, the 3-difunctionalized regioisomer represents the first example of cyclodextrin with two pendants at the secondary rim, resulting in a promising compound. The derivatives have significant antioxidant capacity and the powerful activity in inhibiting self-induced amyloid-β aggregation seems to be led by synergistic effects of both cyclodextrin and hydroxyquinoline. Moreover, the derivatives are also able to complex metal ions and to inhibit metal-induced protein aggregation. Therefore, these compounds could have potential as therapeutic agents in diseases related to protein aggregation and metal dyshomeostasis.

The prion protein (PrP(c)) is a copper-binding glycoprotein that can misfold into a beta-sheet-rich and pathogenic isoform (PrP(sc)) leading to prion diseases. The first non-mammalian PrP(c) was identified in chicken and it was found to keep many structural motifs present in mammalian PrP(c), despite the low sequence identity (approximately 40%) between the two primary structures. The present paper describes the synthesis and the coordination properties of some hexapeptide fragments (namely, PHNPGY , HNPGYP and NPGYPH) as well as a bishexapeptide (PHNPGYPHNPGY), which encompasses two hexarepeats. The copper(II) complexes were characterized by means of potentiometric, UV-vis, circular dichroism and electron paramagnetic resonance techniques. We also report the synthesis of three hexapeptides (PHNPGF, HNPGFP and NPGFPH), in which one tyrosine was replaced by phenylalanine as well as two bishexapeptides in which either one (PHNPGFPHNPGY and PHNPGYPHNPGF), or two tyrosines were replaced by phenylalanine, in order to check whether tyrosine was involved in copper(II) binding. Overall, the results indicate that the major copper(II) species formed by the chicken PrP dodecapeptides are stabler than the analogous species reported for the peptide fragments containing two octarepeat peptides from the mammalian prion protein. It is concluded that the presence of four prolyl residues, that are break points in copper coordination, induces the metal-assisted formation of macrochelates as well as the formation of binuclear species. Furthermore, it has been shown that the phenolic group is directly involved in the formation of copper binuclear species.

Helical polymers with switchable screw sense are versatile frameworks for chiral functional materials. In this work, we reconstructed the free energy landscape of helical poly(2,7-bis(4-tert-butylphenyl)fluoren-9-yl acrylate) [poly(BBPFA)], as its racemization is selectively driven by light without any rearrangement of chemical bonds. The chirality inversion was enforced by atomistic free energy simulations using chirality indices as reaction coordinates. The free energy landscape reproduced the experimental electronic circular dichroism spectra. We propose that the chirality inversion of poly(BBPFA) proceeds from a left-handed 31 helix via multistate free energy pathways to reach the right-handed 31 helix. The inversion is triggered by the rotation of biphenyl units with an activation barrier of 38 kcal/mol. To the best of our knowledge, this is the first report on the chiral inversion mechanism of a helical polymer determined in a quantitative way in the framework of atomistic free energy simulations.

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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.

Accumulation of neurotoxic amyloid-β peptide (Aβ) and alteration of metal homeostasis (metallostasis) in the brain are two main factors that have been very often associated with neurodegenerative diseases, such as Alzheimer's disease (AD). Aβ is constantly produced from the amyloidprecursor-protein APP precursor and immediately catabolized under normal conditions, whereas dysmetabolism of Aβ and/or metal ions seems to lead to a pathological deposition. Although insulin-degrading enzyme (IDE) is the main metalloprotease involved in Aβ degradation in the brain being up-regulated in some areas of AD brains, the role of IDE for the onset and development of AD is far from being understood. Moreover, the biomolecular mechanisms involved in the recognition and interaction between IDE and its substrates are still obscure. In spite of the important role of metals (such as copper, aluminum, and zinc), which has brought us to propose a "metal hypothesis of AD", a targeted study of the effect of metallostasis on IDE activity has never been carried out. In this work, we have investigated the role that various metal ions (i.e., Cu(2+), Cu(+), Zn(2+), Ag(+), and Al(3+)) play in modulating the interaction between IDE and two Aβ peptide fragments, namely Aβ(1-16) and Aβ(16-28). It was therefore possible to identify the direct effect that such metal ions have on IDE structure and enzymatic activity without interferences caused by metal-induced substrate modifications. Mass spectrometry and kinetic studies revealed that, among all the metal ions tested, only Cu(2+), Cu(+), and Ag(+) have an inhibitory effect on IDE activity. Moreover, the inhibition of copper(II) is reversed by adding zinc(II), whereas the monovalent cations affect the enzyme activity irreversibly. The molecular basis of their action on the enzyme is also discussed on the basis of computational investigations.

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