New Insight in Copper-Ion Binding to Human Islet Amyloid: The Contribution of Metal-Complex Speciation To Reveal the Polypeptide Toxicity

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

Rectal bleeding by Dieulafoy-like lesion: successful endoscopic treatment

A 81-year old woman affected by chronic renal failure, non insulin-dependent diabetes mellitus (NIDM) and hypertension, had an severe anemia massive hematochezia. The colonoscopy could not localize the bleeding site except some blood spots in the rectum. The patient was readmitted after 1 month with hypovolemic shock by massive hematochezia and required several blood transfusions. The endoscopic examination showed an important arterial bleeding treated successfully with epinephrine and bipolar elettro-coagulation (BICAP). We suggested that the patient presented a Dieulafoy-like lesion; this is an uncommon gastrointestinal cause of bleeding due to a defect of a submucosal artery without evidence of atherosclerosis or vasculitis. Both chronic renal failure and age could be considered as predisponent factors in this patient. Hematochezia is the most important sign and is often complicated by haemorrhagic shock. The diagnosis was delayed due to the difficulty in localizing the bleeding site; moreover, the patient needed several blood transfusions. The arteriographic diagnosis associated to endoscopic treatment by epinephrine and BICAP enabled a successful therapy

Coordination environment of Cu(II) ions bound to N-terminal peptide fragments of angiogenin protein

Angiogenin (Ang) is a potent angiogenic factor, strongly overexpressed in patients affected by different types of cancers. The specific Ang cellular receptors have not been identified, but it is known that Ang–actin interaction induces changes both in the cell cytoskeleton and in the extracellular matrix. Most in vitro studies use the recombinant form (r-Ang) instead of the form that is normally present in vivo (“wild-type”, wt-Ang). The first residue of r-Ang is a methionine, with a free amino group, whereas wt-Ang has a glutamic acid, whose amino group spontaneously cyclizes in the pyro-glutamate form. The Ang biological activity is influenced by copper ions. To elucidate the role of such a free amino group on the protein–copper binding, we scrutinized the copper(II) complexes with the peptide fragments Ang(1–17) and AcAng(1–17), which encompass the sequence 1–17 of angiogenin (QDNSRYTHFLTQHYDAK-NH2), with free amino and acetylated N-terminus, respectively. Potentiometric, ultraviolet-visible (UV-vis), nuclear magnetic resonance (NMR) and circular dichroism (CD) studies demonstrate that the two peptides show a different metal coordination environment. Confocal microscopy imaging of neuroblastoma cells with the actin staining supports the spectroscopic results, with the finding of different responses in the cytoskeleton organization upon the interaction, in the presence or not of copper ions, with the free amino and the acetylated N-terminus peptides

A rapid and non-invasive method for authenticating the origin of pistachio samples by NIR spectroscopy and chemometrics

In this study, near-infrared spectroscopy coupled to chemometrics is used to build an analytical protocol to authenticate the origin of pistachio nuts (Pistacia vera L.), a high value-added food product. In particular, 483 samples from six different origins (Sicily, India, Iran, Syria, Turkey and U.S.A.) were analyzed by NIR spectroscopy. Spectra were recorded on half seeds cut longitudinally in reflectance mode. Spectral data were then processed by chemometrics to build classification models by SIMCA and PLS-DA. The discriminant approach resulted in classification accuracies higher than 90% for most of the classes. On the other hand, SIMCA built class-models with high sensitivity and specificities, the only exception being the two categories Turkey and Iran, whose heterogeneity resulted in a poorer specificity (anyway higher than 80%). In particular, the results obtained for the samples coming from Bronte (Sicily), the only PDO pistachio production in Europe 95.5% non-error rate in PLS-DA, 90% sensitivity and 97% specificity in SIMCA, as evaluated on the external test set are very promising from the viewpoint of the authentication of this product. In general, the results show that the coupling of NIR spectroscopy to chemometric classification techniques can be a valuable tool for tracing the origin of pistachio nuts, providing a reliable authentication in a rapid, relatively cheap and non-invasive way.Vitale, R.; Bevilacqua, M.; Bucci, R.; Magrì, A.; Magri, A.; Marini, F. (2013). A rapid and non-invasive method for authenticating the origin of pistachio samples by NIR spectroscopy and chemometrics. Chemometrics and Intelligent Laboratory Systems. 121:90-99. doi:10.1016/j.chemolab.2012.11.019S909912

Neurotrophic Activity and Its Modulation by Zinc Ion of a Dimeric Peptide Mimicking the Brain-Derived Neurotrophic Factor N-Terminal Region

Brain-derived neurotrophic factor (BDNF) is a neurotrophin (NT) essential for neuronal development and synaptic plasticity. Dysregulation of BDNF signaling is implicated in different neurological disorders. The direct NT administration as therapeutics has revealed to be challenging. This has prompted the design of peptides mimicking different regions of the BDNF structure. Although loops 2 and 4 have been thoroughly investigated, less is known regarding the BDNF N-terminal region, which is involved in the selective recognition of the TrkB receptor. Herein, a dimeric form of the linear peptide encompassing the 1-12 residues of the BDNF N-terminal (d-bdnf) was synthesized. It demonstrated to act as an agonist promoting specific phosphorylation of TrkB and downstream ERK and AKT effectors. The ability to promote TrkB dimerization was investigated by advanced fluorescence microscopy and molecular dynamics (MD) simulations, finding activation modes shared with BDNF. Furthermore, d-bdnf was able to sustain neurite outgrowth and increase the expression of differentiation (NEFM, LAMC1) and polarization markers (MAP2, MAPT) demonstrating its neurotrophic activity. As TrkB activity is affected by zinc ions in the synaptic cleft, we first verified the ability of d-bdnf to coordinate zinc and then the effect of such complexation on its activity. The d-bdnf neurotrophic activity was reduced by zinc complexation, demonstrating the role of the latter in tuning the activity of the new peptido-mimetic. Taken together our data uncover the neurotrophic properties of a novel BDNF mimetic peptide and pave the way for future studies to understand the pharmacological basis of d-bdnf action and develop novel BDNF-based therapeutic strategies

The role of copper(II) in the aggregation of human amylin

Amylin is the 37-residue peptide hormone produced by the islet β-cells in the pancreas and the formation of amylin aggregates is strongly associated with β-cells degeneration in type 2 diabetes, as demonstrated by more than 95% of patients exhibiting amylin amyloid upon autopsy. It is widely recognized that metal ions such as copper(II) have been implicated in the aggregation process of amyloidogenic peptides such as Aβ and α-synuclein and there is evidence that also amylin self-assembly is largely affected by copper(II). For this reason, in this work, the role of copper(II) in the aggregation of amylin has been investigated by several different experimental approaches. Mass spectrometric investigations show that copper(II) induces significant changes in the amylin structure which decrease the protein fibrillogenesis as observed by ThT measurements. Accordingly, solid-state NMR experiments together with computational analysis carried out on a model amylin fragment confirmed the non fibrillogenic nature of the copper(II) induced aggregated structure. Finally, the presence of copper(II) is also shown to have a major influence on amylin proneness to be degraded by proteases and cytotoxicity studies on different cell cultures are reported

A Deeper Insight in Metal Binding to the hCtr1 N-terminus Fragment: Affinity, Speciation and Binding Mode of Binuclear Cu2+ and Mononuclear Ag+ Complex Species

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

UV-Vis spectrophotometric method for the quantitation of all the components of Italian general denaturant and its application to check the conformity of alcohol samples

A simple, fast and relatively inexpensive spectrophotometric method for the identification and the quantification of the individual components of the Italian general denaturant in alcohol samples is proposed