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Islet amyloid polypeptide (IAPP) is a hormone co‐secreted with insulin and zinc from pancreatic β‐cells. To overcome the low solubility of human IAPP, we characterized zinc complexes species formed with 1) a mutated form of rat‐IAPP(1–37; R18 H) able to mimic the human IAPP, 2) the r‐IAPP(1–37) and the IAPP(1–8) fragment. Stoichiometry, speciation and coordination features of zinc(II) complexes were unveiled by ESI‐MS, potentiometry and NMR measurements combined with DFT and free‐energy simulations. Mononuclear species start to form around pH 6; Zn2+ binds both His18 and N‐amino terminus in rat‐IAPP(1–37; R18 H). The in silico study allows us to assess not only a structured turn compact domain in r‐IAPP(1–37) and r‐IAPP(1–37; R18 H) featured by a different free energy barrier for the transition from the compact to elongated conformation upon the coordination of Zn2+, but also to bring into light a coordination shell further stabilized by noncovalent interactions. [ABSTRACT FROM AUTHOR]

The copper-binding features of rat islet amyloid polypeptide (r-IAPP) are herein disclosed through the determination of the stability constants and spectroscopic properties of its copper complex species. To mimic the metal binding sites of the human IAPP (h-IAPP), a soluble, single-point mutated variant of r-IAPP, having a histidine residue in place of Arg18, was synthesized, that is, r-IAPP(1-37; R18H). The peptide IAPP(1-8) was also characterized to have deeper insight into the N-terminus copper(II)-binding features of r-IAPP as well as of its mutated form. A combined experimental (thermodynamic and spectroscopic) and computational approach allowed us to assess the metal loading and the coordination features of the whole IAPP. At physiological pH, the N-terminal amino group is the Cu2+ main binding site both of entire r-IAPP and of its mutated form that mimics h-IAPP. The histidine residue present in this mutated polypeptide accounts for the second Cu2+ binding. We can speculate that the copper driven toxicity of h-IAPP in comparison to that of r-IAPP can be attributed to the different metal loading and the presence of a second metal anchoring site, the His18, whose role is usually invoked in the process of h-IAPP aggregation. [ABSTRACT FROM AUTHOR]

Type-2 diabetes (T2D) is considered to be a potential threat on a global level. Recently, T2D has been listed as a misfolding disease, such as Alzheimer's and Parkinson's diseases. Human islet amyloid polypeptide (hIAPP) is a molecule cosecreted in pancreatic β cells and represents the main constituent of an aggregated amyloid found in individuals affected by T2D. The trace-element serum level is significantly influenced during the development of diabetes. In particular, the dys-homeostasis of Cu2+ ions may adversely affect the course of the disease. Conflicting results have been reported on the protective role played by complex species formed by Cu2+ ions with hIAPP or its peptide fragments in vitro. The histidine (His) residue at position 18 represents the main binding site for the metal ion, but contrasting results have been reported on other residues involved in metal-ion coordination, in particular those toward the N or C terminus. Sequences that encompass regions 17-29 and 14-22 were used to discriminate between the two models of the hIAPP coordination mode. Due to poor solubility in water, poly(ethylene glycol) (PEG) derivatives were synthesized. A peptide fragment that encompasses the 17-29 region of rat amylin (rIAPP) in which the arginine residue at position 18 was substituted by a histidine residue was also obtained to assess that the PEG moiety does not alter the peptide secondary structure. The complex species formed by Cu2+ ions with Ac-PEG-hIAPP(17-29)-NH2, Ac-rIAPP(17-29)R18H-NH2, and Ac-PEG-hIAPP(14-22)-NH2 were studied by using potentiometric titrations coupled with spectroscopic methods (UV/Vis, circular dichroism, and EPR). The combined thermodynamic and spectroscopic approach allowed us to demonstrate that hIAPP is able to bind Cu2+ ions starting from the His18 imidazole nitrogen atom toward the N-terminus domain. The stability constants of copper(II) complexes with Ac-PEG-hIAPP(14-22)-NH2 were used to simulate the different experimental conditions under which aggregate formation and oxidative stress of hIAPP has been reported. Speciation unveils: 1) the protective role played by increased amounts of Cu2+ ions on the hIAPP fibrillary aggregation, 2) the effect of adventitious trace amounts of Cu2+ ions present in phosphate-buffered saline (PBS), and 3) a reducing fluorogenic probe on H2O2 production attributed to the polypeptide alone. [ABSTRACT FROM AUTHOR]

The fragments of rat amylin rIAPP(17–29) (Ac-VRSSNNLGPVLPP-NH2), rIAPP(17–22) (Ac-VRSSNN-NH2), rIAPP(19–22) (Ac-SSNN-NH2) and rIAPP(17–20) (Ac-VRSS-NH2) together with the related mutant peptides (Ac-VASS-NH2and Ac-VRAA-NH2) have been synthesized and their copper(ii) complexes studied by potentiometric, UV-Vis, CD and EPR spectroscopic methods. Despite the lack of any common strongly coordinating donor functions some of these fragments are able to bind copper(ii) ions in the physiological pH range. The longest fragment rat amylin(17–29) keeps one equivalent copper(ii) ion in solution in the whole pH range, while two other peptides Ac-VRSSNN-NH2and Ac-SSNN-NH2are also able to interact with copper(ii) ions in the slightly alkaline pH range. According to the spectral parameters of the complexes, the peptides can be classified into two different categories: (i) the tetrapeptides Ac-VRSS-NH2, Ac-VASS-NH2and Ac-VRAA-NH2can interact with copper(ii) only under strongly alkaline conditions (pH > 10.0) and the formation of only one species with four amide nitrogen coordination can be detected; (ii) the peptides Ac-VRSSNNLGPVLPP-NH2, Ac-VRSSNN-NH2and Ac-SSNN-NH2can form complexes above pH 6.0 with the major stoichiometries [CuH−2L], [CuH−3L]−and [CuH−4L]2−. These data support that rIAPP(17–29) can interact with copper(ii) ions under physiological conditions and the SSNN tetrapeptide fragment can be considered as the shortest sequence responsible for metal binding. Density functional theory (DFT) calculations provide some information on the possible coordination modes of Ac-SSNN-NH2towards the copper(ii) ion and suggest that for [CuH−2L], [CuH−3L]−and [CuH−4L]2−, the binding of two, three and four deprotonated amide nitrogens, with NH−of the side chain of asparagine as anchoring group, is probable. Moreover, these data reveal that peptides can be effective metal binding ligands even in the absence of anchoring groups, if more polar side chains are present in a specific sequence. [ABSTRACT FROM AUTHOR]

Nerve growth factor (NGF) is a protein essential to neurons survival, which interacts with its receptor as a non-covalent dimer. Peptides belonging to NGF N-terminal domain are able to mimic the activity of the whole protein. Such activity is affected by the presence of copper ions. The metal is released in the synaptic cleft where proteins, not yet identified, may bind and transfer to human copper transporter 1 (hCtr1), for copper uptake in neurons. The measurements of the stability constants of copper complexes formed by amyloid beta and hCtr1 peptide fragments suggest that beta-amyloid (Aβ) can perform this task. In this work, the stability constant values of copper complex species formed with the dimeric form of N-terminal domain, sequence 1–15 of the protein, were determined by means of potentiometric measurements. At physiological pH, NGF peptides bind one equivalent of copper ion with higher affinity of Aβ and lower than hCtr1 peptide fragments. Therefore, in the synaptic cleft, NGF may act as a potential copper chelating molecule, ionophore or chaperone for hCtr1 for metal uptake. Copper dyshomeostasis and mild acidic environment may modify the balance between metal, NGF, and Aβ, with consequences on the metal cellular uptake and therefore be among causes of the Alzheimer's disease onset. [ABSTRACT FROM AUTHOR]

The vascular endothelial growth factor A (VEGF-A) is a potent angiogenic factor, its activity may be influenced by the presence of copper(II) ions. To mimic the interaction between copper(II) and VEGF (Vascular Endotelial Growth Factor), the N- and C-terminally blocked peptide fragments VEGF73-101 and VEGF84-101, owing to VEGF165 protein, have been synthesized. These protein domains represent a specific recognition site with the VEGF receptor (VEGFR). Copper(II) complexes with VEGF73-101 and VEGF84-101 were investigated by means of potentiometry and UV-Vis, ESI-MS, CD, EPR spectroscopic methods. Both peptides have three histidine residues and display a binding high affinity for copper(II) ions. The proliferative activity of the peptides in the absence and presence of copper(II) ions as well as of VEGF-165 protein was also tested on HUVEC cells (Human Umbilical Vein Endothelial Cells). The VEGF73-101 showed a dose-dependent anti-proliferative activity, while the shorter peptide VEGF84-101 did not affect HUVEC proliferation, both in the presence and in the absence of VEGF. [ABSTRACT FROM AUTHOR]

Many neurodegenerative proteinopathies are characterized by ubiquitin (Ub)-containing intraneuronal inclusion bodies. Recent reports have shown that Ub is able to bind CuII and ZnII, the dyshomeostasis of which is a hallmark of neurodegeneration. Here we use complementary techniques like potentiometry, circular dichroism-visible, and electron spin resonance to unveil the Ub/metal species that form, at neutral pH, their binding constants and structural features. Next, we show that both ZnII and CuII ions hinder the interactions between Ub and Ub-conjugating E2 enzymes and inhibit significantly both Lys48 and Lys63 self-polyubiquitination reactions in a cell-free medium. The effects of ZnII and CuII on Lys63 and Lys48 polyUb chain synthesis are compatible with the hypothesis that metal binding to His68 modifies the Ile44 hydrophobic patch of Ub and makes the protein less available for polyUb. These findings contribute to further arguments for a close relationship between metal dyshomeostasis and abnormal protein degradative pathways in the upstream events, triggering neurodegeneration [ABSTRACT FROM AUTHOR]

Abstract: GHHPH is the peptide repeat present in histidine–proline rich glycoprotein (HPRG), a plasma glycoprotein involved in angiogenesis process. The copper(II) ions interaction with mono (Ac-GHHPHG-NH2) and its bis-repeat (Ac-GHHPHGHHPHG-NH2) was investigated by means of potentiometric and spectroscopic techniques. To single out the copper(II) coordination environments of different species formed with Ac-GHHPHG-NH2, three single point mutated peptides were also synthesized and their ability to coordinate Cu2+ investigated. Ac-GHHPHG-NH2 binds Cu2+ by the imidazole side chain and the amide nitrogen deprotonation that takes place towards the N-terminus. The bis-repeat is able to bind Cu2+ more efficiently than Ac-GHHPHG-NH2. This difference is not only due to the number of His residues in the sequence but also to the different binding sites. In fact, the comparison of the potentiometric and spectroscopic data of the copper(II) complexes with a bis-repeatPeg construct Ac-(GHHPHG)-Peg-(GHHPHG)-NH2 and those of the metal complexes with Ac-HGHH-NH2, indicates that the central HGHH amino acid sequence is the main copper(II) binding site. [Copyright &y& Elsevier]

Abstract: Numerous conflicting values have been proposed regarding the affinity of Cu2+ for amyloid-β (Aβ) peptide, the causative agent of Alzheimer''s disease. In the present review, we critically compare the two approaches employed so far (the K d and the stability constant approach) to express the affinity of copper(II) for the amyloid-β (Aβ) peptide and highlight the limits and the advantages of the two approaches. We also analyze the conditions employed for some experiments, which we have taken as examples, highlighting some of the points that may have generated the deriving divergent propositions. Through the analysis of the species distribution, we show the implications that a correct speciation may have on data interpretation as well as on experiment planning. By doing so, this review aims at shifting the perspective on the binding issue from the classic K d approach, based on low and high affinity binding sites – often referred to as component I and II, or form I and II – to stoichiometry determination and, as a consequence, to the speciation of Cu–Aβ complexes. Additionally, this review has the purpose of demonstrating that a quantitative assessment of the coordination sphere is complicated by the variety of equilibria often occurring over a relatively narrow pH range. [Copyright &y& Elsevier]

Abstract: Nickel(II) complexes of the peptide fragments of human prion protein containing histidyl residues both inside and outside the octarepeat domain have been studied by the combined application of potentiometric, UV–visible and circular dichroism spectroscopic methods. The imidazole-N donor atoms of histidyl residues are the exclusive metal binding sites below pH 7.5, but the formation of stable macrochelates was characteristic only for the peptide HuPrP(76–114) containing four histidyl residues. Yellow colored square planar complexes were obtained above pH 7.5–8 with the cooperative deprotonation of three amide nitrogens in the [Nim,N−,N−,N−] coordination mode. It was found that the peptides can bind as many nickel(II) ions as the number of independent histidyl residues. All data supported that the complex formation processes of nickel(II) are very similar to those of copper(II), but with a significantly reduced stability for nickel(II), which shifts the complex formation reactions into the slightly alkaline pH range. The formation of coordination isomers was characteristic of the mononuclear complexes with a significant preference for the nickel(II) binding at the histidyl sites outside the octarepeat domain. The results obtained for the two-histidine fragments of the protein, HuPrP(91–115), HuPrP(76–114)H85A and HuPrP(84–114)H96A, made it possible to compare the binding ability of the His96 and His111 sites. These data reveal a significant difference in the nickel(II) and copper(II) binding sites of the peptides: His96 was found to predominate almost completely for nickel(II) ions, while the opposite order, but with comparable concentrations, was reported for copper(II). [Copyright &y& Elsevier]

Aggregation of the amyloid β-peptide (Aβ) into insoluble fibrils is a key pathological event in Alzheimer's Disease (Aβ). There is now compelling evidence that metal binding to Aβ is involved in AD pathogenesis. The amino acid region 1-16 is widely considered as the metal binding domain of Aβ. In this work, we used a combined potenliometric, NMR, and electrospray ionization mass speclrometry (ESI-MS) approach to study the zinc(ll) binding to a new polyethylene glycol (PEG)-conjugated peptide fragmenl encompassing the 1-16 amino acid sequence of Aβ (Aβ(1-16)PEG). Our results demonstrate for the first time that the Aβ(1 -16) is able to coordinate up to three zinc ions, all the histidyl residues acting as independent anchor sites. The study was complemented by systematically investigating the zinc(ll) complexes of a series of shorter peplide tragmenls related to the Aβ(1-16) sequence, namely, (1-4), Aβ(1-6), AcAβ(1-6), AcAβ(8-16)Y10A. The comparison of the whole results allowed the identification of the zinc(II) preferred binding sites within the longer Aβ(1-16) amino acid sequence. Unlike copper(ll) that prefers the N-terminal amino group as the main binding site, the zinc(ll) is preferentially placed in the 8-16 amino acidic region of Aβ(1-16). [ABSTRACT FROM AUTHOR]

Abstract: Copper(II) complexes of the peptide fragment (Dpl122–130) encompassing the sequence 122–130 of human doppel protein were characterized by potentiometric, UV–Visible, CD and EPR spectroscopic methods. An analogous peptide, in which the aspartate residue was substituted by an asparagine amino acid, was synthesized in order to provide evidence on the possible role of carboxylate group in copper(II) coordination. It was found that the carboxylic group is directly involved in copper(II) coordination at acidic pH, forming the CuLH2 species with Dpl122–130. This copper(II) complex displayed EPR parameters very similar to those of the analogous complex with the whole doppel protein. At pH higher than 7, the complexes showed magnetic parameters similar to those of the major species of protein formed in the pH range 7–8, with the metal coordination environment consisting of one imidazole and three amide nitrogen atoms. The comparison of Cu-Dpl122–130 binding constant values with those of the prion peptide fragments (PrP106–114), showed that doppel peptide had a higher metal binding affinity at acidic pH whereas the prion peptide fragment binds the metal tightly at physiological pH. [Copyright &y& Elsevier]

Abstract: Potentiometric and spectroscopic (UV–Vis, CD and EPR) studies were carried out on copper(II) complexes with chicken prion protein N-terminal fragments, Ac-(PHNPGY)4-NH2, and the mutated residue, Ac-(PHNPGF)4-NH2, to assess the role of tyrosine in the copper coordination. Both thermodynamic and spectroscopic results indicate that chicken prion fragments are not able to bind more than two copper ions and only with the involvement of side chain tyrosine groups. The prevailing complex shows one copper ion bound to four imidazole nitrogen atoms in the 1:1 metal to ligand ratio systems. The superoxide dismutase (SOD)-like activity of copper(II) complexes with the avian peptides and mammal analogue, Ac-(PHGGGWGQ)4-NH2, was also investigated by means of Pulse radiolysis. The copper(II) complexes with avian peptides do not display SOD-like activity, while very low activity has been detected for the copper(II) complexes with mammalian tetraoctarepeat. [Copyright &y& Elsevier]

Alzheimer's disease (AD) is becoming a rapidly growing health problem, as it is one of the main causes of dementia in the elderly. Interestingly, copper(II) (together with zinc and iron) ions are accumulated in amyloid deposits, suggesting that metal binding to Aβ could be involved in AD pathogenesis. In Aβ, the metal binding is believed to occur within the N-terminal region encompassing the amino acid residues 1–16. In this work, potentiometric, spectroscopic (UV–vis, circular dichroism, and electron paramagnetic resonance), and electrospray ionization mass spectrometry (ESI-MS) approaches were used to investigate the copper(II) coordination features of a new polyethylene glycol (PEG)-conjugated Aβ peptide fragment encompassing the 1–16 amino acid residues of the N-terminal region (Aβ(1–16)PEG). The high water solubility of the resulting metal complexes allowed us to obtain a complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 4:1. Potentiometric and ESI-MS data indicate that Aβ(1–16)PEG is able to bind up to four copper(II) ions. Furthermore, in order to establish the coordination environment at each metal binding site, a series of shorter peptide fragments of Aβ, namely, Aβ(1–4), Aβ(1–6), AcAβ(1–6), and AcAβ(8–16)Y10A, were synthesized, each encompassing a potential copper(II) binding site. The complexation properties of these shorter peptides were also comparatively investigated by using the same experimental approach. [ABSTRACT FROM AUTHOR]