[Show abstract][Hide abstract] ABSTRACT: The human islet polypeptide (hIAPP) or amylin is a 37-residue peptide hormone secreted by β-cells of the islet of Langerhans in the pancreas. Unlike the rat variant of IAPP (rIAPP), human amylin is highly amyloidogenic and is found as amyloid deposits in nearly 95% of patients afflicted with type 2 diabetes mellitus (T2DM). Human and rat IAPP have nearly identical primary sequence differing at only six positions which are encompassed within the 17-29 aminoacid region. Using Circular Dichroism (CD), Dynamic Light Scattering (DLS) and ThT-fluorescence (Th-T), we examined the aggregation properties of both full-length hIAPP1-37 and the related peptide fragment hIAPP17-29. For the sake of comparison, similar experiments were carried out on the respective rat variants rIAPP1-37 and rIAPP17-29. These studies were conducted at physiological pH in buffered solution not containing fluorinated co-solvents as well as in the presence of model membranes (LUV). In addition, the cytotoxic activity of the investigated peptides was determined toward different pancreatic β-cell lines. All the peptide studied in this work resulted cytotoxic despite β-sheet structure being observed, in vitro, for the hIAPP1-37 only. This suggests that β-sheet conformational transition that generally precedes the fibril formation, is not a prerequisite for toxicity towards β-cells. Interestingly, confocal microscopy indicated that the IAPP peptides can enter the cell and might exert their toxic action at an intracellular level.
European Journal of Medicinal Chemistry 05/2014; 81C:442-455. DOI:10.1016/j.ejmech.2014.05.038 · 3.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Novel conjugated G-quadruplex-forming d(TG3AG) oligonucleotides, linked to hydrophobic groups through phosphodiester bonds at 5'-end, have been synthesized as potential anti-HIV aptamers, via a fully automated, online phosphoramidite-based solid-phase strategy. Conjugated quadruplexes showed pronounced anti-HIV activity with some preference for HIV-1, with inhibitory activity invariably in the low micromolar range. The CD and DSC monitored thermal denaturation studies on the resulting quadruplexes, indicated the insertion of lipophilic residue at the 5'-end, conferring always improved stability to the quadruplex complex (20<ΔTm<40°C). The data suggest no direct functional relationship between the thermal stability and anti-HIV activity of the folded conjugated G-quartets. It would appear that the nature of the residue at 5' end of the d(TG3AG) quadruplexes plays an important role in the thermodynamic stabilization but a minor influence on the anti-HIV activity. Moreover, a detailed CD and DSC analyses indicate a monophasic behaviour for sequences I and V, while for ODNs (II-IV) clearly show that these quadruplex structures deviate from simple two-state melting, supporting the hypothesis that intermediate states along the dissociation pathway may exist.
[Show abstract][Hide abstract] ABSTRACT: The peptide sequence PHSRN is the second cell binding site of the human fibronectin protein, a glycoprotein which plays a critical adhesive role during development, tissue repair and angiogenesis. The copper(II) complexes with the peptide fragment PHSRN were characterized by potentiometric and UV-visible, CD, EPR spectroscopic methods. Thermodynamic and spectroscopic evidences indicate that at physiological pH, only one copper(II) complex species, [CuLH(-2)], is present and the metal ion is bound to one imidazole and two amide nitrogen atoms (N(Im), 2N(-)) in a tetrahedral distorted square planar coordination. Two new β-cyclodextrin-ethylendiamino derivatives with the PHSRN covalently attached were synthesized as multitargeting molecules, able to have a site-specific recognition sequence, to interact with copper(II) ions and to be a potential carrier of hydrophobic drugs. Copper(II) complexes with these β-cyclodextrin derivatives were characterized by means of potentiometric and spectroscopic techniques. The comparison of the experimental parameters determined at different pH values with those obtained for the parent peptide complex species, shows that at physiological pH the ethylendiamino-β-CD moiety does not influence the peptide interaction with copper ions and the β-CD hydrophobic cavity is not blocked, being available to host hydrophobic drugs such as naproxen.
[Show abstract][Hide abstract] ABSTRACT: The doppel protein (Dpl) is the first homologue of the prion protein (PrP(C)) to be discovered; it is overexpressed in transgenic mice that lack the prion gene, resulting in neurotoxicity. The whole prion protein is able to inhibit Dpl neurotoxicity, and its N-terminal domain is the determinant part of the protein function. This region represents the main copper(II) binding site of PrP(C). Dpl is able to bind at least one copper ion, and the specific metal-binding site has been identified as the histidine residue at the beginning of the third helical region. However, a reliable characterization of copper(II) coordination features has not been reported. In a previous paper, we studied the copper(II) interaction with a peptide that encompasses only the loop region potentially involved in metal binding. Nevertheless, we did not find a complete match between the EPR spectroscopic parameters of the copper(II) complexes formed with the synthesized peptide and those reported for the copper(II) binding sites of the whole protein. Herein, the synthesis of the human Dpl peptide fragment hDpl(122-139) (Ac-KPDNKLHQQVLWRLVQEL-NH(2)) and its copper(II) complex species are reported. This peptide encompasses the third alpha helix and part of the loop linking the second and the third helix of human doppel protein. The single-point-mutated peptide, hDpl(122-139)D124N, in which aspartate 124 replaces an asparagine residue, was also synthesized. This peptide was used to highlight the role of the carboxylate group on both the conformation preference of the Dpl fragment and its copper(II) coordination features. NMR spectroscopic measurements show that the hDpl(122-139) peptide fragment is in the prevailing alpha-helix conformation. It is localized within the 127-137 amino acid residue region that represents a reliable conformational mimic of the related protein domain. A comparison with the single-point-mutated hDpl(122-139)D124N reveals the significant role played by the aspartic residue in addressing the peptide conformation towards a helical structure. It is further confirmed by CD measurements. Potentiometric titrations were carried out in aqueous solutions to obtain the stability constant values of the species formed by copper(II) with the hDpl peptides. Spectroscopic studies (EPR, NMR, CD, UV/Vis) were performed to characterize the coordination environments of the different metal complexes. The EPR parameters of the copper(II) complexes with hDpl(122-139) match those of the previously reported copper(II) binding sites of the whole hDpl. Addition of the copper(II) ion to the peptide fragment does not alter the helical conformation of hDpl(122-139), as shown by CD spectra in the far-UV region. The aspartate-driven preorganized secondary structure is not significantly modified by the involvement of Asp124 in the copper(II) complex species that form in the physiological pH range. To elaborate on the potential role of copper(II) in the recently reported interaction between the PrP(C) and Dpl, the affinity of the copper(II) complexes towards the prion N terminus domain and the binding site of Dpl was reported.
Chemistry - A European Journal 06/2010; 16(21):6212-23. DOI:10.1002/chem.200902405 · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The complexes between copper(II) and the synthetic octapeptide fragments of the prion protein Ac-GWGQPHGG-NH2 (1), Ac-PHGGGWGQ-NH2 (3) and the cyclic analogue c-(GWGQPHGG) (2) have been comparatively investigated by circular dichroism (CD), absorption (UV–Vis), and electron paramagnetic resonance (EPR) spectroscopic methods.The results suggest a similar copper(II) coordination behaviour of the two linear peptides. In both cases two major complex species were spectroscopically detected. The first one, existing in the range of pH 7–9, showed spectroscopic parameters attributable to a 3N complex species, while the 4N complex was the main species at strongly alkaline pH values. Copper(II) binding appears to be confined within the aminoacid sequence HGG.Cyclisation of the main chain, as in the peptide 2, was found to have remarkable effects on the copper(II) complex speciation especially at pH 7–8 where the 3N species predominated in the linear counterparts. By contrast the spectroscopic data obtained at pH 11 provided evidence of the restoration of the same set of donor atoms as in the linear peptides.
[Show abstract][Hide abstract] ABSTRACT: The formation of complexes of HGGGHGHGGGHG (HG12) with copper(II) and nickel(II) have been studied in aqueous solution under various experimental conditions, including different pH and metal to ligand ratios. The study has been carried out using visible absorption, circular dichroism and electron paramagnetic resonance spectroscopic methods. Moreover, electrospray ionisation mass spectrometry has been used to directly determine the stoichiometry of the copper(II) complexes. The results indicate that HG12 can easily accommodate two metal ions in as many binding sites. The solution structure of the main complex species formed in the reaction of copper(II) with HG12 has been inferred by comparison with the copper(II) complexes formed with the shorter peptide fragments HGGGHG–NH2
(AcHG6) and Ac–HGGG–NH2
New Journal of Chemistry 01/2002; 26(5):593-600. DOI:10.1039/b110655d · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The synthesis of the new cyclodextrin derivative, 6-deoxy-6-[(N,N′-bis(phenylalanyl)-1,3-propandiamine]-β-cyclodextrin (CDPheNN3) is reported. The intra- and inter-molecular interactions in aqueous solution occurring for this molecule are highly dependent on concentration. The presence of a competitive guest (1-adamantanol), a competitive host (α-cyclodextrin and β-cyclodextrin), and the metal ion [zinc(II) or copper(II)] all strongly influence the conformation of the CDPheNN3. Both NMR and CD were used to carry out this investigation.
Journal of Supramolecular Chemistry 05/2001; 1(3):117–124. DOI:10.1016/S1472-7862(01)00017-X
[Show abstract][Hide abstract] ABSTRACT: The two epimers of β-cyclodextrin 6-functionalized with l-or d-cystein (6-S-(l)-cysteine-6-deoxy-β-cyclodextrin and (6-S-d-cysteine-6-deoxy-β-cyclodextrin) were synthesized. Their binary and ternary copper(II) complexes with amino acids were characterized by electronic, circular dichroism and ESR spectroscopies. The binary copper(II) complexes were used as eluents in ligand exchange chromatography to test their ability to resolve racemate mixtures of unmodified amino acids. High performance liquid chromatography separation of l/d-TrpO− was achieved only when the complex of the d-cysteine derivative was used as the eluent.