Giuseppe Arena

Neurotrophins are a family of growth factors that play a key role in the development and regulation of the functioning of the central nervous system. Their use as drugs is made difficult by their poor stability, cellular permeability, and side effects. Continuing our effort to use peptides that mimic the neurotrophic growth factor (NGF), the family model protein, and specifically the N-terminus of the protein, here we report on the spectroscopic characterization and resistance to hydrolysis of the 14-membered cyclic peptide reproducing the N-terminus sequence (SSSHPIFHRGEFSV (c-NGF(1-14)). Far-UV CD spectra and a computational study show that this peptide has a rigid conformation and left-handed chirality typical of polyproline II that favors its interaction with the D5 domain of the NGF receptor TrkA. c-NGF(1-14) is able to bind Cu²⁺ with good affinity; the resulting complexes have been characterized by potentiometric and spectroscopic measurements. Experiments on PC12 cells show that c-NGF(1-14) acts as an ionophore, influencing the degree and the localization of both the membrane transporter (Ctr1) and the copper intracellular transporter (CCS). c-NGF(1-14) induces PC12 differentiation, mimics the protein in TrkA phosphorylation, and activates the kinase cascade, inducing Erk1/2 phosphorylation. c-NGF(1-14) biological activities are enhanced when the peptide interacts with Cu²⁺ even with the submicromolar quantities present in the culture media as demonstrated by ICP-OES measurements. Finally, c-NGF(1-14) and Cu²⁺ concur to activate the cAMP response element-binding protein CREB that, in turn, induces the brain-derived neurotrophic factor (BDNF) and the vascular endothelial growth factor (VEGF) release.

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.

Series of lanthanide‐containing metallic coordination complexes are frequently presented as structurally analogous, due to the similar chemical and coordinative properties of the lanthanides. In the case of chiral (Ln^III[15‐MC _N(L‐pheHA)‐5])³⁺ metallacrowns (MCs), which are well established supramolecular hosts, the formation of dimers templated by a dicarboxylate guest (muconate) in solution of neutral pH is herein shown to have a unique dependence on the identity of the MC's central lanthanide. Calorimetric data and nuclear magnetic resonance diffusion studies demonstrate that MCs containing larger or smaller lanthanides as the central metal only form monomeric host‐guest complexes whereas analogues with intermediate lanthanides (for example, Eu, Gd, Dy) participate in formation of dimeric host‐guest‐host compartments. The driving force for the dimerization event across the series is thought to be a competition between formation of highly stable MCs (larger lanthanides) and optimally linked bridging guests (smaller lanthanides).

In this work we report on the synthesis and physiochemical/biological characterization of a peptide encompassing the first thirteen residues of neurotrophin-3 (NT-3). The protein capability to promote neurite outgrowth and axonal branching by a downstream mechanism that involves the increase of the cAMP response element-binding level (CREB) was found for the NT3(1–13) peptide, thus validating its protein mimetic behaviour. Since copper ions are also involved in neurotransmission and their internalization may be an essential step in neuron differentiation and CREB phosphorylation, the peptide and its copper complexes were characterized by potentiometric and spectroscopic techniques, including UV-visible, CD and EPR. To have a detailed picture of the coordination features of the copper complexes with NT3(1–13), we also scrutinized the two peptide fragments encompassing the shorter sequences 1–5 and 5–13, respectively, showing that the amino group is the main anchoring site for Cu(II) at physiological pH. The peptide activity increased in the presence of copper ions. The effect of copper(II) addition is more marked for NT3(1–13) than the other two peptide fragments, in agreement with its higher affinity for metal ions. Confocal microscopy measurements carried out on fluorescently labelled NT3(1–13) indicated that copper ions increase peptide internalization.

Conflicting values, obtained by different techniques and often under different experimental conditions have been reported on the affinity of Zn²⁺ for amyloid-β, that is recognized as the major interaction responsible for Alzheimer’s disease. Here, we compare the approaches employed so far, i.e., the evaluation of K_d and the determination of the stability constants to quantitatively express the affinity of Zn²⁺ for the amyloid-β peptide, evidencing the pros and cons of the two approaches. We also comment on the different techniques and conditions employed that may lead to divergent data. Through the analysis of the species distribution obtained for two selected examples, we show the implications that the speciation, based on stoichiometric constants rather than on K_d, may have on data interpretation. The paper also demonstrates that the problem is further complicated by the occurrence of multiple equilibria over a relatively narrow pH range.

The supramolecular assembly [Ga₄L₆]¹²⁻ acts as a nanoscale flask to mediate the reactivity of encapsulated reactive guests and also functions as a catalyst to carry out enzyme‐like chemical transformations. The guest binding to the interior cavity and exterior of this host is difficult to untangle because multiple equilibria occur in solution, and only when refining simultaneously data obtained from different techniques, such as NMR, UV/Vis, and calorimetry, can the accurate solution thermodynamics of these host–guest systems be determined. This study reports the driving forces for the inclusion and stepwise exterior guest binding of different aliphatic quaternary ammonium guests to the [Ga₄L₆]¹²⁻ assembly. Encapsulation into the host cavity was found to be an entropy‐driven process, whereas exterior ion association is driven either by enthalpically favorable attractive forces or by the entropy gain due to desolvation, depending on guest size and character. The analysis of the energetics of reaction may help predicting and understanding the intimate role and contribution of the transition state in those rate‐accelerated reactions involving this supramolecular assembly as an enzyme‐like molecular flask.

Metallacrown complexes capable of sequestering dianions, as shown in the solid state, also exist in aqueous solution at neutral pH, as demonstrated by calorimetric and mass spectrometric data. The driving forces for the formation of these dimeric complexes in solution strongly depend on the chain length of the guest rather than its degree of unsaturation.

The program HypCal has been developed to provide a means for the simultaneous determination, from data obtained by isothermal titration calorimetry, of both standard enthalpy of reaction and binding constant values. The chemical system is defined in terms of species of given stoichiometry rather than in terms of binding models (e.g., independent or cooperative). The program does not impose any limits on the complexity of the chemical systems that can be treated, including competing ligand systems. Many titration curves may be treated simultaneously. HypCal can also be used as a simulation program when designing experiments. The use of the program is illustrated with data obtained with nicotinic acid (niacin, pyridine-3 carboxylic acid). Preliminary experiments were used to establish the rather different titration conditions for the two sets of titration curves that are needed to determine the parameters for protonation of the carboxylate and amine groups.