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Dysregulated metal homeostasis is associated with many pathological conditions, including arthritic diseases. Osteoarthritis and rheumatoid arthritis are the two most prevalent disorders that damage the joints and lead to cartilage and bone destruction. Recent studies show that the levels of zinc (Zn) and copper (Cu) are generally altered in the serum of arthritis patients. Therefore, metal dyshomeostasis may reflect the contribution of these trace elements to the disease's pathogenesis and manifestations, suggesting their potential for prognosis and treatment. Carnosine (Car) also emerged as a biomarker in arthritis and exerts protective and osteogenic effects in arthritic joints. Notably, its zinc(II) complex, polaprezinc, has been recently proposed as a drug-repurposing candidate for bone fracture healing. On these bases, this review article aims to provide an overview of the beneficial roles of Cu and Zn in bone and cartilage health and their potential application in tissue engineering. The effects of Car and polaprezinc in promoting cartilage and bone regeneration are also discussed. We hypothesize that polaprezinc could exchange Zn for Cu, present in the culture media, due to its higher sequestering ability towards Cu. However, future studies should unveil the potential contribution of Cu in the beneficial effects of polaprezinc. [ABSTRACT FROM AUTHOR]

A new Cu(I) probe (OBEP-CS1), bearing an alkyl-pyridinium moiety to get complete water solubility, was synthesized. Fluorescence titrations support copper binding and high selectivity in a water solution whereas formation of aggregates was excluded by DOSY experiments. The detection capability for intracellular Cu(I), especially localized in mitochondria, was demonstrated in differentiated neuroblastoma cells by confocal microscopy. [ABSTRACT FROM AUTHOR]

Prion disorders are a group of fatal neurodegenerative conditions of mammals. The key molecular event in the pathogenesis of such diseases is the conformational conversion of prion protein, PrPC, into a misfolded form rich in -sheet structure, PrPSc, but the detailed mechanistic aspects of prion protein conversion remain enigmatic. There is uncertainty on the precise physiological function of PrPC in healthy individuals. Several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ mainly through a domain composed by four to five repeats of eight amino acids. In addition to mammals, PrP homologues have also been identified in birds, reptiles, amphibians and fish. The globular domain of protein is retained in the different species, suggesting that the protein carries out an essential common function. However, the comparison of amino acid sequences indicates that prion protein has evolved differently in each vertebrate class. The primary sequences are strongly conserved in each group, but these exhibit a low similarity with those of mammals. The N-terminal domain of different prions shows tandem amino acid repeats with an increasing amount of histidine residues going from amphibians to mammals. The difference in the sequence affects the number of copper binding sites, the affinity and the coordination environment of metal ions, suggesting that the involvement of prion in metal homeostasis may be a specific characteristic of mammalian prion protein. In this review, we describe the similarities and the differences in the metal binding of different species' prion protein, as revealed by studies carried out on the entire protein and related peptide fragments. [ABSTRACT FROM AUTHOR]

Abstract: Insulin degrading enzyme (IDE) is known to play a pivotal role on amyloidogenic peptide degradation but little is known about the changes in the proteolytic activity of the enzyme upon modification of external factors. Particularly, although it has been reported that altered ubiquitin concentration and/or hyperinsulinaemia increase the risk of developing Alzheimer''s disease (AD), the molecular mechanism involved is unclear. In this work, we study the role that ubiquitin plays on IDE capability of binding and degrading insulin molecules and the obtained results indicate that ubiquitin has an allosteric role for IDE and high ubiquitin levels impair IDE activity. [Copyright &y& Elsevier]

The 3A-deoxy-3A-(2-methylaminopyridine)-2A(S),3A(R)-β-cyclodextrin (CDampy3) was synthesized and characterized by NMR spectroscopy. The proton and copper(II) complex formation and stability of this new β-cyclodextrin derivative were determined by potentiometric and calorimetric investigations, carried out in aqueous solution at 25 °C and I=0.1 mol dm−3 (KNO3). The ΔH° and ΔS° values allowed to bring to light the role of the hydrogen bond between the amine and the hydroxyl groups in the basicity decrease of NH group of this 3-functionalized β-cyclodextrin. The comparison between the log K values of copper(II) complexes with CDampy3 and those of 6-deoxy-6-(2-methylaminopyridine)-β-cyclodextrin (CDampy6) allowed to determine the donor atoms involved in the coordination to metal ion. The high stability constant of [Cu(CDampy3)]2+ in comparison with [Cu(CDampy6)]2+ was attributed to the involvement of the OH groups in the binding to copper(II) ion. [Copyright &y& Elsevier]

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 Kd 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 Kd, 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. [ABSTRACT FROM AUTHOR]

Unravelling the molecular basis for Alzheimer’s disease (AD) represents one of the main scientific challenges we face when it comes to treating and – most importantly – preventing the onset of this devastating condition. The amyloid-beta peptide (Aβ) is a key element around which the metabolic pathways linked to sporadic AD forms have been investigated. The right balance between production and degradation of Aβ is fundamental to the physiological activity of the peptide and to preventing the formation of toxic aggregated species. Whilst the metabolic pathways for the formation of Aβ from the amyloid precursor protein (APP) have been unveiled, the mechanism involved in the control of Aβ levels through enzymatic systems (UPS, lysosomes and Aβ-degrading enzymes), both in vitro and in vivo , is underexplored. Overwhelming evidence clearly indicates that the abnormal aggregation of Aβ is not the main – or even the only – biochemical event that characterizes the onset and progression of AD, which can be also driven by the alteration of metallostasis (metal homeostasis) of d-block metal ions, such as copper(II) and zinc(II). The “Metal Hypothesis” in AD also concerns the effects of these metal ions on the formation and activity of Aβ fragments. Once Aβ is processed by the Aβ-degrading systems, the amyloid fragments can lose, maintain or modify the metal binding properties, unlike the full-length peptide. In addition, the physiological and/or pathological functions of the peptide fragments and their metal complexes can be different from those exerted by Aβ and its metal complex systems. We will outline the Aβ fragments detected in human fluids and tissues from both healthy and AD patients, as well as the structural and biochemical features of copper(II) and zinc(II) complexes with the naturally-occurring Aβ fragments. Then we will delve into the essential features and functionalities of the enzymatic systems involved in the clearance and degradation of Aβ and the effects of Cu 2+ and Zn 2+ in their activity. Finally, we will explain how we to match the cleavage sites of the Aβ-degrading enzymes to the natural-occurring cleavage sites of Aβ. [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI) leads to long-term and permanent motor dysfunctions, and nervous system abnormalities. Injury to the spinal cord triggers a signaling cascade that results in activation of the inflammatory cascade, apoptosis, and Zn(II) ion homeostasis. Trehalose (Tre), a nonreducing disaccharide, and l-carnosine (Car), (β-alanyl-l-histidine), one of the endogenous histidine dipeptides have been recognized to suppress early inflammatory effects, oxidative stress and to possess neuroprotective effects. We report on the effects of the conjugation of Tre with Car (Tre–car) in reducing inflammation in in vitro and in vivo models. The in vitro study was performed using rat pheochromocytoma cells (PC12 cell line). After 24 h, Tre–car, Car, Tre, and Tre + Car mixture treatments, cells were collected and used to investigate Zn2+ homeostasis. The in vivo model of SCI was induced by extradural compression of the spinal cord at the T6–T8 levels. After treatments with Tre, Car and Tre–Car conjugate 1 and 6 h after SCI, spinal cord tissue was collected for analysis. In vitro results demonstrated the ionophore effect and chelating features of l-carnosine and its conjugate. In vivo, the Tre–car conjugate treatment counteracted the activation of the early inflammatory cascade, oxidative stress and apoptosis after SCI. The Tre–car conjugate stimulated neurotrophic factors release, and influenced Zn2+ homeostasis. We demonstrated that Tre–car, Tre and Car treatments improved tissue recovery after SCI. Tre–car decreased proinflammatory, oxidative stress mediators release, upregulated neurotrophic factors and restored Zn2+ homeostasis, suggesting that Tre–car may represent a promising therapeutic agent for counteracting the consequences of SCI. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Carnosinases are Xaa-His dipeptidases that play diverse functions throughout all kingdoms of life. Human isoforms of carnosinase (CN1 and CN2) under appropriate conditions catalyze the hydrolysis of the dipeptides carnosine (β-alanyl-L-histidine) and homocarnosine (γ-aminobutyryl-L-histidine). Alterations of serum carnosinase (CN1) activity has been associated with several pathological conditions, such as neurological disorders, chronic diseases and cancer. For this reason the use of carnosinase levels as a biomarker in cerebrospinal fluid (CSF) has been questioned. The hydrolysis of imidazolerelated dipeptides in prokaryotes and eukaryotes is also catalyzed by aminoacyl-histidine dipeptidases like PepD (EC 3.4.13.3), PepV (EC 3.4.13.19) and anserinase (EC 3.4.13.5). The review deals with the structure and function of this class of enzymes in physiological and pathological conditions. The main substrates of these enzymes, i.e., carnosine, homocarnosine and anserine (β-alanyl-3-methyl-L-histidine) will also be described. [ABSTRACT FROM AUTHOR]

Abstract: Carnosine (β-alanyl-l-histidine) is an endogenous dipeptide, extensively studied owing to its multifunctional activity exhibited in tissues of several animal species. This natural compound may act as a physiological buffer, ion-chelating agent (especially for copper(II) and zinc(II)), antioxidant and antiglycating agent. The main limit for the therapeutical uses of carnosine is the rapid hydrolysis mostly in human plasma by carnosinase. The chemical derivatization of carnosine is a promising strategy to improve the bioavailability of the dipeptide and facilitating the site-specific transport to different tissues. On this basis, a new carnosine derivative with biotin was synthesized and structurally characterized by NMR and MS measurements, with aim of exploiting the avidin–biotin technology that offers a universal system for selective delivery of any biotinylated agent. The stability of the new carnosine derivative towards the hydrolytic action of serum carnosinase as well as the copper(II) binding ability of the carnosine–biotin conjugate were also assessed. The binding affinity of the new molecular entity to avidin and streptavidin, investigated by a spectrophotometric assay, was exploited to functionalize avidin– and streptavidin–gold nanoparticles with the carnosine–biotin conjugate. [Copyright &y& Elsevier]

Carnosine (β-alanyl- l-histidine) is an endogenous dipeptide widely and abundantly distributed in the muscle and nervous tissues of several animal species. Many functions have been proposed for this compound because of its antioxidant and metal ion-chelator properties. Many potential therapeutic properties have been recognized especially related to the antioxidant activity, but the therapeutic uses are strongly limited by the mechanism governing its homeostasis. This fact has been the main reason for developing the synthesis of carnosine derivatives with interesting potentiality, but until now there have been very few applications. These derivatives could represent the future drugs for many pathologies related to oxidative stress and metal ion dyshomeostasis. [ABSTRACT FROM AUTHOR]

Abstract: Amyloid β-peptides (Aβs) are involved in several neuropathological conditions such as Alzheimer''s disease and considerable experimental evidences have emerged indicating that different proteases play a major role in regulating the accumulation of Aβs in the brain. Particularly, insulin-degrading enzyme (IDE) has been shown to degrade Aβs at different cleavage sites, but the experimental results reported in the literature and obtained by mass spectrometry methods are somehow fragmentary. The detection of Aβs is often complicated by solubility issues, oxidation artifacts and spontaneous aggregation/cleavage and, in order to rationalize the different reported results, we analyzed Aβs solutions by three different MS approaches: matrix assisted laser desorption ionization-time of flight (MALDI-TOF), atmospheric pressure (AP) MALDI ion trap and electrospray ionization (ESI) ion trap. Differences in the obtained results are discussed and ESI is chosen as the most suitable MS method for Aβs detection. Finally, cleavage sites produced by interaction of Aβs with IDE are identified, two of which had never been reported in the literature. [Copyright &y& Elsevier]

Spectroscopic (UV-Vis and EPR) and voltammetric studies have been carried out on the copper(II) complexes with the Ac-PEG11-(PHGGGWGQ)4-NH2 (L) polypeptide. In the ratios Cu :L 3 : 1 and 4 : 1, the two [Cu3(L)H-6] and [Cu4(L)H-8] complex species have been characterized at neutral pH values. All the copper atoms occupy similar coordination sites formed by imidazole, peptidic nitrogen atoms and carbonyl oxygen atoms in a square base pyramidal geometry. Voltammetric measurements on these systems point out the cooperativity in the electron transfer processes among the copper(II) sites during their reduction. NO interaction with these polynuclear copper species is characterized by the reduction of the copper sites through the formation of two different intermediate complex species.When an excess of the Ac-PEG11-(PHGGGWGQ)4-NH2 ligand is considered, frozen solution EPR parameters and UV-Vis spectroscopic data identify the [Cu(Nim)4]2+ chromophore, which does not interact with NO. [ABSTRACT FROM AUTHOR]

Spectroscopic (UV-Vis and EPR) and voltammetric studies have been carried out on the copper(ii) complexes with the Ac-PEG11-(PHGGGWGQ)4-NH2(L) polypeptide. In the ratios Cu : L 3 : 1 and 4 : 1, the two [Cu3(L)H−6] and [Cu4(L)H−8] complex species have been characterized at neutral pH values. All the copper atoms occupy similar coordination sites formed by imidazole, peptidic nitrogen atoms and carbonyl oxygen atoms in a square base pyramidal geometry. Voltammetric measurements on these systems point out the cooperativity in the electron transfer processes among the copper(ii) sites during their reduction. NO interaction with these polynuclear copper species is characterized by the reduction of the copper sites through the formation of two different intermediate complex species. When an excess of the Ac-PEG11-(PHGGGWGQ)4-NH2ligand is considered, frozen solution EPR parameters and UV-Vis spectroscopic data identify the [Cu(Nim)4]2+chromophore, which does not interact with NO. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinases (MMPs) are cell-secreted soluble and membrane-tethered enzymes that are capable of degrading extracellular matrix proteins, but also can process a number of bioactive molecules. They are involved in the cleavage of cell surface receptors, but are also thought to play a major role on cell behaviors as well as in diverse physiological and pathological processes, including embryonic development, wound repair, inflammatory diseases, and cancer. For these reasons, it is obvious that a control over MMPs activity is highly desirable. Consequently, the frantic search for new inhibitors has been coupled to the development of high-throughput methods able to rapidly screen the effect of possible MMP inhibitors on the activity of these enzymes. In this scenario, solid-state–based methods play a major role because of their compatibility with array formats that are able to extract more information from smaller sample volumes and offer some important advantages that are not available in the standard solution assays. In this work, the catalytic domain of MMP-12 was immobilized on a gold substrate and the surface coverage was measured by FT-SPR experiments. A new experimental procedure was developed to freeze-dry the anchored molecules and their activity was measured by ESI-MS. The kinetics parameters obtained for the immobilized enzyme are in good accordance with those reported for similar systems in solution. Inhibition of the immobilized molecules was also carried out, demonstrating the applicability of the method for rapid screening of MMP inhibitors. [Copyright &y& Elsevier]

The functionalized cyclodextrin 3A-[1-(2-amino)ethylamino]-3A-deoxy-2A(S),3A(R)-β-cyclodextrin was synthesized and an NMR, EPR, pH-metric and calorimetric investigation was carried out in aqueous solution in order to ascertain its behavior towards proton and copper(II) complexation. The NMR spectra show the involvement of functionalizing moiety in H-bond formation, in keeping with the ΔH° and ΔS° values. The potentiometry and EPR results display the involvement of secondary OHs, particularly of 2-OH in the complexation of copper(II). [Copyright &y& Elsevier]

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]

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]

Copper is involved in several biological processes. The static and labile copper pools are controlled by means of a network of influx and efflux transporters, storage proteins, chaperones, transcription factors and small molecules as glutathione (GSH), which contributes to the cell reducing environment. To follow the fate of intracellular copper labile pool, a variant of human apocarbonic anhydrase has been proposed as fluorescent probe to monitor cytoplasmic Cu2+. Aware that in this cellular compartment copper ion is present as Cu+, electron spin resonance technique (ESR) was used to ascertain whether (bovine or human) carbonic anhydrase (CA) was able to accommodate Cu+ in the same sites occupied by Cu2+, in the presence of naturally occurring reducing agents such as ascorbate and GSH. Our ESR results on Cu2+ complexes with CA allow for a complete characterization of the two metal binding sites of the protein in solution. The use of the reported affinity constants of zinc in the catalytic site and of Cu2+ in the peripheral and catalytic site, allow us to obtain the speciation of copper species mimicking the spectroscopic study conditions. The different Cu2+ coordination features in the catalytic and the peripheral (the N-terminus cleft mouth) binding sites influence the chemical reduction effect of the two main naturally occurring reductants. Ascorbate reversibly reduces the Cu2+ complex with CA, while glutathione irreversibly induces the formation of Cu2+ complex with its oxidized form (GSSG). Our results questioned the use of CA as intracellular Cu2+ sensor. Furthermore, translating these findings to intracellular environment, the conversion of GSH in GSSG can significantly alter the metallostasis. Human and bovine carbonic anhydrase forms mono- and dinuclear copper(II) species; the two binding sites were characterized by electron spin resonance spectroscopy. This technique was also used to test the ability of carbonic anhydrase to retain the two metal centres upon reduction with ascorbate (Asc) or glutathione (GSH). Unlabelled Image • Human and bovine carbonic anhydrase forms mono- and dinuclear copper(II) complexes. • Peripheral N-terminal and catalytic sites are characterized by ESR. • Both Cu2+ centres are reduced in the presence of ascorbate or glutathione (GSH). • Re-oxidation following ascorbate treatment restores original dicopper species. • GSH fully reduces both metal ions and Cu(II)-GSSH forms in the presence of dioxygen. [ABSTRACT FROM AUTHOR]

Islet transplantation is a valid therapeutic option for type 1 diabetes treatment. However, in this procedure one of the major problems is the oxidative stress produced during pancreatic islet isolation. The aim of our study was to evaluate potential protective effects of L-carnosine and its isomer D-carnosine against oxidative stress. We evaluated the carnosine effect on cell growth, cell death, insulin production, and the main markers of oxidative stress in rat and murine stressed beta cell lines as well as in human pancreatic islets. Both isomers clearly inhibited hydrogen peroxide induced cytotoxicity, with a decrease in intracellular reactive oxygen and nitrogen species, prevented hydrogen peroxide induced apoptosis/necrosis, nitrite production, and reduced glucose-induced insulin secretion. In addition, NF-κB expression/translocation and nitrated protein induced in stressed cells was significantly reduced. Furthermore, both isomers improved survival and function, and decreased reactive oxygen and nitrogen species, and nitrite and nitrotyrosine production in human islets cultured for 1, 3, and 7 days. These results seem to indicate that both L and D-carnosine have a significant cytoprotective effect by reducing oxidative stress in beta cell lines and human islets, suggesting their potential use to improve islet survival during the islet transplantation procedure. [ABSTRACT FROM AUTHOR]

The role of copper in cancer progression has been established since decades. Additionally, copper is able to stimulate angiogenesis through the control of VEGF expression and activity in endothelial cells. In this paper a tetrapeptide, belonging to the histidine–proline-rich glycoprotein (HPRG) and encompassing four repeats of the sequence GHHPH (named TetraHPRG), was synthesized and its copper(ii) complex species were characterized by means of potentiometry, UV-vis, circular dichroism (CD), electron paramagnetic resonance (EPR) and electron spray ionization mass spectrometry (ESI-MS). Moreover, a peptide covalently bound through an amidic bond to trehalose (TH-TetraHPRG) was designed and synthesized as a prodrug system. The activity of both TetraHPRG and TH-TetraHPRG molecules on copper and VEGF induced angiogenic responses in endothelial cells was assessed. The two peptides show a similar and effective anti-angiogenic activity on both molecular and cellular responses. Since the trehalose derivative has a higher resistance to enzymatic degradation, it can be further exploited as a potential drug delivery system with anti-angiogenic activity. [ABSTRACT FROM AUTHOR]

A novel water-soluble ionophore based on the thiocarbohydrazone moiety conjugated with glucose ( GluTch ) was synthesized through a simple two-step procedure. Structural elucidation was carried out in water solution by means of various spectroscopic techniques (NMR, UV–Vis, and CD), electrospray ionization mass spectrometry and density functional theory calculations. The flexible nature of the thiocarbohydrazone moiety of the new glycoderivative compound induced both different coordination motifs and stoichiometry towards copper and zinc. Cytotoxicity assays of the ligands on the human normal keratinocyte NCTC-2544, MDA-MB-231 breast cancer and PC-3 human prostate adenocarcinoma cell lines demonstrated that i) higher activity on cancer cells growth inhibition compared to a normal cell line; ii) the introduction of the glucose unit does not alter the cytotoxic activity of the underivatized ionophore ligand and iii) the presence of copper ion improves the activity of the thiocarbohydrazones. [ABSTRACT FROM AUTHOR]

Summary: Alzheimer's disease (AD) is a progressive neurodegenerative disorder associated with synaptic dysfunction, pathological accumulation of β‐amyloid (Aβ), and neuronal loss. The self‐association of Aβ monomers into soluble oligomers seems to be crucial for the development of neurotoxicity (J. Neurochem., 00, 2007 and 1172). Aβ oligomers have been suggested to compromise neuronal functions in AD by reducing the expression levels of the CREB target gene and brain‐derived neurotrophic factor (BDNF) (J. Neurosci., 27, 2007 and 2628; Neurobiol. Aging, 36, 2015 and 20406 Mol. Neurodegener., 6, 2011 and 60). We previously reported a broad neuroprotective activity of physiological Aβ monomers, involving the activation of type‐1 insulin‐like growth factor receptors (IGF‐IRs) (J. Neurosci., 29, 2009 and 10582, Front Cell Neurosci., 9, 2015 and 297). We now provide evidence that Aβ monomers, by activating the IGF‐IR‐stimulated PI3‐K/AKT pathway, induce the activation of CREB in neurons and sustain BDNF transcription and release. [ABSTRACT FROM AUTHOR]

Gold nanoparticles (AuNPs) have unique anti-angiogenic properties and have been applied in a variety of cancers studies. ‘Naked’ gold nanoparticles directly bound to angiogenic factors have been demonstrated to inhibit growth factor-mediated signalling in vitro and vascular endothelial growth factor-induced angiogenesis in vivo. Angiogenin (Ang) is a protein physiologically constituent the human plasma but also a pathological marker of different cancer types. Herein, the peptide Ang 60–68 , encompassing the putative cellular binding site of the protein Ang, has been synthesized and characterized in the interaction with spherical AuNPs of 12 nm of diameter. The Ang mimicking activity of the peptide was assessed in terms of cell cytoskeleton rearrangements visualized by staining of actin, which is an important target of the whole protein. The hybrid assemblies obtained by physical adsorption of the peptide molecules at the surface of the gold nanoparticles were scrutinised by UV–visible spectroscopy, to characterize with titration experiments the changes in the plasmonic properties of AuNPs as well as the peptide spectral features. The latter were obtained by using the fluorescent analogous peptide, Fam-Ang 59–68 , incorporating the carboxyfluorescein (Fam) moiety, through an amidic bond of the N-terminal residue. The hydrodynamic size of the peptide-Au systems were determined by dynamic light scattering (DLS) analyses. Proof-of work experiments with human neuroblastoma cells line demonstrated the non-toxicity of the Ang-mimicking peptide functionalised gold nanoparticles. Moreover, laser scanning confocal microscopy (LSM) experiments showed the localization of the peptide-nanoparticles at the cell membrane and their sub-cellular distribution. These data reveal a promising new platform for imaging and therapeutic activities in cancer therapy. [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]

Increasing evidence is accumulating, showing that neurodegenerative disorders are somehow associated with the toxicity of amyloid aggregates, metal ion dyshomeostasis as well as with products generated by oxidative stress. Within the biological oxidation products, acrolein does have a prominent role. A promising strategy to deal with the above neurogenerative disorders is to use multi-functions bio-molecules. Herein, we show how a class of bio-conjugates takes advantage of the antiaggregating, antioxidant and antiglycating properties of trehalose and carnosine. Their ability to sequester acrolein and to inhibit both self- and metal-induced aggregation is here reported. The copper(II) coordination properties of a new trehalose-carnosine conjugate and the relative antioxidant effects have also been investigated. [ABSTRACT FROM AUTHOR]

Many biochemical pathways involving nerve growth factor (NGF), a neurotrophin with copper(II) binding abilities, are regulated by the ubiquitin (Ub) proteasome system. However, whether NGF binds Ub and the role played by copper(II) ions in modulating their interactions have not yet been investigated. Herein NMR spectroscopy, circular dichroism, ESI-MS, and titration calorimetry are employed to characterize the interactions of NGF with Ub. NGF1-14, which is a short model peptide encompassing the first 14 N-terminal residues of NGF, binds the copper-binding regions of Ub ( KD=8.6 10−5 m). Moreover, the peptide undergoes a random coil-polyproline type II helix structural conversion upon binding to Ub. Notably, copper(II) ions inhibit NGF1-14/Ub interactions. Further experiments performed with the full-length NGF confirmed the existence of a copper(II)-dependent association between Ub and NGF and indicated that the N-terminal domain of NGF was a valuable paradigm that recapitulated many traits of the full-length protein. [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]

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. [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]

The intracellular tracking of zinc and copper, metals essential for life, is nowadays pivotal to unravel the complex mechanism that involves the physiological or pathological role of such elements. Traditional methods to determine cellular copper and zinc levels, including those based on the use of fluorescent probes, are aimed at scrutinizing the metallome, to identify both the individual species and their concentrations. The metallome, however, is also a nonstatic concept, as it responds to environmental perturbations in biologically relevant pathways, with highly dynamic spatiotemporal changes. Through an overview of improvements and limits of the state of the art of synthetic fluorescent probes for the detection of intracellular zinc and copper, we report here new routes for the design and the synthesis of novel metal coordination compounds able to overcome the present weaknesses for a new concept of dynamic metallostasis. [ABSTRACT FROM AUTHOR]

Copper is a catalytic cofactor required for the normal function of many enzymes involved in fundamental biological processes but highly cytotoxic when in excess. Therefore its homeostasis and distribution is strictly regulated by a network of transporters and intracellular chaperones. ATOX1 (antioxidant protein 1) is a copper chaperone that plays a role in copper homeostasis by binding and transporting cytosolic copper to ATPase proteins in the trans -Golgi network. In the present study the Caco-2 cell line, a colon carcinoma cell line, was used as an in vitro model to evaluate if ATOX1 deficiency could affect sensitivity to experimentally induced copper dyshomeostasis. Silencing of ATOX1 increased toxicity of a short treatment with a high concentration of Cu 2 + . Copper ionophores, such as 5-chloro-8-hydroxyquinoline, induced a copper-dependent cell toxicity which was significantly potentiated after ATOX1 silencing. The copper chelator TPEN (N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine) produced a form of cell toxicity that was reversed by the addition of Cu 2 + . ATOX1 silencing increased Caco-2 cell sensitivity to TPEN toxicity. Our results suggest the possibility of a therapy with copper-chelating or ionophore drugs in subtypes of tumors showing specific alterations in ATOX1 expression. [ABSTRACT FROM AUTHOR]

The present study addresses the reconstruction of the free-energy landscapes of amyloid-beta1-42 (Aβ 42 ) coordinated respectively with one and two zinc ions, to scrutinize whether different Aβ–zinc complex species, i.e., mononuclear and dinuclear metal complexes, induce different Aβ conformation features. We found a subtle switch of intramolecular interactions, depending both on the zinc coordination environment and on the peptide to zinc stoichiometric ratio. On the one side, hairpin-like structures are predominant in mononuclear complexes, where a salt-bridge that involves Lys28-Glu22 and Lys16-Asp23 is stabilized. On the other side, elongated conformations are instead stabilized in the dinuclear zinc complexes. Experimental studies of atomic force microscopy as well as of zinc–Aβ complex species distribution diagrams provide evidence that the theoretical calculations can be rationalized in terms of the correlation between the increased amount of amorphous aggregates and the Aβ/Zn 2 + ratio. [ABSTRACT FROM AUTHOR]

Prion diseases are a group of neurodegenerative diseases based on the conformational conversion of the normal form of the prion protein (PrPC) to the disease-related scrapie isoform (PrPSc). Copper(II) coordination to PrPC has attracted considerable interest for almost 20 years, mainly due to the possibility that such an interaction would be an important event for the physiological function of PrPC. In this work, we report the copper(II) coordination features of the peptide fragment Ac(PEG11)3PrP(60-114) [Ac=acetyl] as a model for the whole N-terminus of the PrPC metal-binding domain. We studied the complexation properties of the peptide by means of potentiometric, UV/Vis, circular dichroism and electrospray ionisation mass spectrometry techniques. The results revealed that the preferred histidyl binding sites largely depend on the pH and copper(II)/peptide ratio. Formation of macrochelate species occurs up to a 2:1 metal/peptide ratio in the physiological pH range and simultaneously involves the histidyl residues present both inside and outside the octarepeat domain. However, at increased copper(II)/peptide ratios amide-bound species form, especially within the octarepeat domain. On the contrary, at basic pH the amide-bound species predominate at any copper/peptide ratio and are formed preferably with the binding sites of His96 and His111, which is similar to the metal-binding-affinity order observed in our previous studies. [ABSTRACT FROM AUTHOR]

Angiogenin (ANG), a member of the secreted ribonuclease family, is a potent angiogenesis stimulator that interacts with endothelial cells inducing a wide range of responses. Metal ions dyshomeostasis play a fundamental role in the onset of neurodegenerative diseases, in particular copper that is also involved in angiogenesis processes. It is known that vascular pathologies are present in neurodegenerative diseases and Angiogenin is down-regulated in Alzheimer and Parkinson diseases, as well as it has been found as one of the mutated genes in amyotrophic lateral sclerosis (ALS). Copper (II) induces an increase of Angiogenin binding to endothelial cells but, so far, the relationship between copper-ANG and angiogenesis induction remain unclear. Herein, the effects of copper (II) ions on Angiogenin activity and expression were evaluated. The binding of copper was demonstrated to affect the intracellular localization of the protein decreasing its nuclear translocation. Moreover, the ANG-copper (II) system negatively affects the protein-induced angiogenesis, as well as endothelial cells migration. Surprisingly, copper also reveals the ability to modulate the Angiogenin transcription. These results highlight the tight relationship between copper and Angiogenin, pointing out the biological relevance of ANG-copper system in the regulation of endothelial cell function, and revealing a possible new mechanism at the basis of vascular pathologies. [ABSTRACT FROM AUTHOR]

Heptapeptide Semax, encompassing the sequence 4-7 of N-terminal domain of the adrenocorticotropic hormone (ACTH) and a C-terminal Pro-Gly-Pro tripeptide, belongs to a short regulatory peptides family. This compound has been found to affect learning processes and to exert marked neuroprotective activities on cognitive brain functions. Dys-homeostasis of metal ions is involved in several neurodegenerative disorders and growing evidences have showed that brain is a specialized organ able to concentrate metal ions. In this work, the metal binding ability and protective activity of Semax and its metal complexes were studied. The equilibrium study clearly demonstrated the presence of three complex species. Two minor species [CuL] and [CuLH − 1 ] − co-exist together with the [CuLH − 2 ] 2 − in the pH range from 3.6 to 5. From pH 5 the [CuLH − 2 ] 2 − species becomes predominant with the donor atoms around copper arranged in a 4 N planar coordination mode. Noteworthy, a reduced copper induced cytotoxicity was observed in the presence of Semax by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay on a SHSY5Y neuroblastoma and RBE4 endothelial cell lines. [ABSTRACT FROM AUTHOR]

Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases, involving post-translational modifications of the cellular prion protein. Epidemiological studies on Creutzfeldt-Jakob disease, a prototype prion disorder, show a majority of cases being sporadic, while the remaining occurrences are either genetic or iatrogenic. The molecular mechanisms by which PrP C is converted into its pathological isoform have not yet been established. While point mutations and seeds trigger the protein to cross the energy barriers, thus causing genetic and infectious transmissible spongiform encephalopathies, respectively, the mechanism responsible for sporadic forms remains unclear. Since prion diseases are protein-misfolding disorders, we investigated prion protein folding and stability as functions of different milieus. Using spectroscopic techniques and atomistic simulations, we dissected the contribution of major structural determinants, also defining the energy landscape of prion protein. In particular, we elucidated (i) the essential role of the octapeptide region in prion protein folding and stability, (ii) the presence of a very enthalpically stable intermediate in prion-susceptible species, and (iii) the role of the disulfide bridge in prion protein folding. [ABSTRACT FROM AUTHOR]

Abstract: Homocarnosine is an endogenous dipeptide distributed in cerebral regions and cerebrospinal fluid. Homocarnosine may serve as an antioxidant, free radical scavenger, neurotransmitter, buffering system and metal chelating agent, especially for copper(II) and zinc(II). The homeostasis of homocarnosine is regulated by carnosinases; the serum-circulating isoform of these metallodipeptidases partially hydrolyses homocarnosine in the blood. The enzyme activity is also inhibited by homocarnosine itself in a dose-dependent manner. We synthesized a new multifunctional homocarnosine derivative with trehalose, a disaccharide that possesses several beneficial properties, among which the inhibition of protein aggregation (i.e. Aβ amyloid and polyglutamine proteins) involved in widespread neurodegenerative disorders. We studied the copper(II) binding features of the new conjugate by means of potentiometric and spectroscopic techniques (UV–visible and circular dichroism) and the superoxide dismutase-like activity of the copper(II) complexes with homocarnosine and its trehalose conjugate was evaluated. The inhibitory effect of the new homocarnosine derivative on the carnosinase activity and its effects on Aβ aggregation were also investigated. [Copyright &y& Elsevier]

A histamine derivative of β-cyclodextrin functionalised at the secondary rim was synthesised and characterised by optical and NMR spectroscopy. Its binary systems both with proton and copper(II) were thermodynamically characterised through pH-metric potentiometry. In addition, the ternary systems with the enantiomers of tryptophan, phenylalanine and alanine were investigated. Thermodynamic stereoselectivity was observed for both the tryptophan and phenylalanine enantiomers. The properties of the synthesised cyclodextrin derivative as a chiral selector were verified in chiral ligand-exchange capillary electrophoresis (CLECE) towards the enantiomeric pairs of some amino acids. [ABSTRACT FROM AUTHOR]

Abstract: Copper(II) and zinc(II) complexes with two hexapeptides encompassing HExxH and HxxEH motif were characterized by means of a combined experimental and theoretical approach. Parallel tempering and density functional theory (DFT) investigations show the presence of different hydrogen bonding networks between the copper(II) and zinc(II) complexes with the two peptides, suggesting a significant contribution of these non-covalent interactions to the stability constant values. The glutamate carboxylate group has a direct role in metal ion binding. The location of this amino acid along the sequence of the investigated peptides is critical to determine thermodynamic and spectroscopic features of the copper(II) complex species, whereas is less relevant in the zinc(II) complexes formation. Electrospray ionization mass spectrometry (ESI-MS) characterization of the zinc(II) complex species show that in the [ZnH−2L] two deprotonated amide nitrogen atoms are involved in the metal coordination environment, an uncommon behavior in zinc(II) complexes for multi-histidine ligands. [Copyright &y& Elsevier]

Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for neuronal differentiation, growth, and survival; it is involved in memory formation and higher cognitive functions. The N-terminal domain of BDNF is crucial for the binding selectivity and activation of its specific TrkB receptor. Zn2+ ion binding may influence BDNF activity. Zn2+ complexes with the peptide fragment BDNF(1-12) encompassing the sequence 1-12 of the N-terminal domain of BDNF were studied by means of potentiometry, electrospray mass spectrometry, NMR, and density functional theory (DFT) approaches. The predominant Zn2+ complex species, at physiological pH, is [ZnL] in which the metal ion is bound to an amino, an imidazole, and two water molecules (NH2, NIm, and 2Owater) in a tetrahedral environment. DFT-based geometry optimization of the zinc coordination environment showed a hydrogen bond between the carboxylate and a water molecule bound to zinc in [ZnL]. The coordination features of the acetylated form [AcBDNF(1-12)] and of a single mutated peptide [BDNF(1-12)D3N] were also characterized, highlighting the role of the imidazole side chain as the first anchoring site and ruling out the direct involvement of the aspartate residue in the metal binding. Zn2+ addition to the cell culture medium induces an increase in the proliferative activity of the BDNF(1-12) peptide and of the whole protein on the SHSY5Y neuroblastoma cell line. The effect of Zn2+ is opposite to that previously observed for Cu2+ addition, which determines a decrease in the proliferative activity for both peptide and protein, suggesting that these metals might discriminate and modulate differently the activity of BDNF. [ABSTRACT FROM AUTHOR]

The synthesis of ( E)-2-cyano-3-(5′-piperidin-1-yl-2,2′-bithien-5-yl)acrylic acid, a novel amyloid aggregation fluorescent probe, is reported. This new probe is able to monitor soluble oligomeric aggregates after 24 h, at which time Thioflavin T emission, commonly used to monitor amyloid fibril formation, remains unchanged. Atomic force microscopy, native polyacrylamide gel electrophoresis, and dynamic light scattering confirm that the earlier stages of aggregation are prefibrillar oligomeric species not possessing the amyloid structure. This new molecular scaffold expands the toolbox of fluorescent probes for the identification of prefibrillar oligomers, which is needed in studies aimed at the early detection of the soluble toxic aggregates that characterize the so-called protein misfolding diseases. [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]

l-carnosine (β-alanyl-l-histidine) (Car hereafter) is a natural dipeptide widely distributed in mammalian tissues and reaching high concentrations (0.7–2.0 mM) in the brain. The molecular features of the dipeptide underlie the antioxidant, anti-aggregating and metal chelating ability showed in a large number of physiological effects, while the biological mechanisms involved in the protective role found against several diseases cannot be explained on the basis of the above-mentioned properties alone, requiring further research efforts. It has been reported that l-carnosine increases the secretion and expression of various neurotrophic factors and affects copper homeostasis in nervous cells inducing Cu cellular uptake in keeping with the key metal-sensing system. Having in mind this l-carnosine ability, here we report the copper-binding and ionophore ability of l-carnosine to activate tyrosine kinase cascade pathways in PC12 cells and stimulate the expression of BDNF. Furthermore, the study was extended to verify the ability of the dipeptide to favor copper signaling inducing the expression of VEGF. Being aware that the potential protective action of l-carnosine is drastically hampered by its hydrolysis, we also report on the behavior of a conjugate of l-carnosine with trehalose that blocks the carnosinase degradative activity. Overall, our findings describe a copper tuning effect on the ability of l-carnosine and, particularly its conjugate, to activate tyrosine kinase cascade pathways. [ABSTRACT FROM AUTHOR]

Characterization of the copper(II) complexes formed with the tetraoctarepeat peptide at low and high metal-to-ligand ratios and in a large pH range, would provide a breakthrough in the interpretation of biological relevance of the different metal complexes of copper(II)-tetraoctarepeat system. In the present work, the potentiometric, UV/Vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) studies were carried out on copper(II) complexes with a PEG-ylated derivative of the tetraoctarepeats peptide sequence (Ac-PEG27-(PHGGGWGQ)4-NH2) and the peptide Ac-(PHGGGWGQ)2-NH2. Conjugation of tetraoctarepeat peptide sequence with polyethyleneglycol improved the solubility of the copper(II) complexes. The results enable a straightforward explanation of the conflicting results originated from the underestimation of all metal-ligand equilibria and the ensuing speciation. A complete and reliable speciation is therefore obtained with the released affinity and binding details of the main complexes species formed in aqueous solution. The results contribute to clarify the discrepancies of several studies in which the authors ascribe the redox activity of copper(II)-tetraoctarepeat system considering only the average effects of several coexisting species with very different stoichiometries and binding modes. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribose) polymerases (PARPs) are recognized as key regulators of cell survival or death. PARP-1 is essential to the repair of DNA single-strand breaks via the base excision repair pathway. The enzyme may be overactivated in response to inflammatory cues, thus depleting cellular energy pools and eventually causing cell death. Accordingly, PARP-1 inhibitors, acting by competing with its physiological substrate NAD, have been proposed to play a protective role in a wide range of inflammatory and ischemia/reperfusion-associated diseases. Recently, it has also been reported that PARP-1 regulates proinflammatory mediators, including cytokines, chemokines, adhesion molecules, and enzymes (e.g., iNOS). Furthermore, PARP-1 has been shown to act as a coactivator of NF-κB- and other transcription factors implicated in stress/inflammation, as AP-1, Oct-1, SP-1, HIF, and Stat-1. To further substantiate this hypothesis, we tested the biomolecular effects of PARP-1 inhibitors DPQ and PJ-34 on human glioblastoma cells, induced to a proinflammatory state with lipopolysaccharide and Interferon-γ. PARP-1 expression was evaluated by laser scanning confocal microscopy immunofluorescence (LSM); nitrite production, LDH release and cell viability were also determined. LSM of A-172, SNB-19 and CAS-1 cells demonstrated that DPQ and PJ-34 downregulate PARP-1 expression; they also cause a decrease of LDH release and nitrite production, while increasing cell viability. Similar effects were caused in all three cell lines by N-mono-methyl-arginine, a well known iNOS inhibitor, and by l-carnosine and trehalose, two antioxidant molecules. These results demonstrate that, similar to other well characterized drugs, DPQ and PJ-34 reduce cell inflammation and damage that follow PARP-1 overexpression, while they increase cell survival: this suggests their potential exploitation in clinical Medicine. [ABSTRACT FROM AUTHOR]

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 Cu2+ 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 Cu2+ ions has been reported up to now. Cu2+ 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 Cu2+ 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 Cu2+. 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 Cu2+ may drive different pathways for the BDNF and NGF in physiological as well as pathological conditions. [ABSTRACT FROM AUTHOR]

Abstract: Insulin degradation is a finely tuned process that plays a major role in controlling insulin action and most evidence supports IDE (insulin-degrading enzyme) as the primary degradative agent. However, the biomolecular mechanisms involved in the interaction between IDE and its substrates are often obscure, rendering the specific enzyme activity quite difficult to target. On the other hand, biometals, such as copper, aluminum and zinc, have an important role in pathological conditions such as Alzheimer''s disease or diabetes mellitus. The metabolic disorders connected with the latter lead to some metallostasis alterations in the human body and many studies point at a high level of interdependence between diabetes and several cations. We have previously reported (Grasso et al., Chem. Eur. J. 17 (2011) 2752–2762) that IDE activity toward Aβ peptides can be modulated by metal ions. Here, we have investigated the effects of different metal ions on the IDE proteolytic activity toward insulin as well as a designed peptide comprising a portion of the insulin B chain (B20–30), which has a very low affinity for metal ions. The results obtained by different experimental techniques clearly show that IDE is irreversibly inhibited by copper(I) but is still able to process its substrates when it is bound to copper(II). [Copyright &y& Elsevier]