Trehalose-Carnosine Prevents the Effects of Spinal Cord Injury Through Regulating Acute Inflammation and Zinc(II) Ion Homeostasis.

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), (ß-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.

Carnosine, Zinc and Copper: A Menage a Trois in Bone and Cartilage Protection.

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.

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.

Mono- and dialdehyde of trehalose: new synthons to prepare trehalose bio-conjugates.

Trehalose, a non-reducing disaccharide of glucose, is a natural bioactive and non-toxic sugar. It is found in many organisms that synthesise it when their cells are exposed to stress conditions. While not produced by mammalian cells, this disaccharide and also some of its derivatives have been shown to have a number of interesting properties that indicate their importance in the treatment of certain human diseases. Differentiating the two glucosyl moieties in the trehalose molecule has often been a synthetic challenge. We report here an easy way to obtain the monoaldehyde of trehalose, as well as the relevant symmetrical dialdehyde. The reactivity of the aldehyde functionalities involved in the molecular structure of these synthons allows the easy preparation of the corresponding amino or carboxy derivatives of trehalose, as well the synthesis of some new trehalose conjugates useful for diagnostic or therapeutic purposes.

Nerve Growth Factor Peptides Bind Copper(II) with High Affinity: A Thermodynamic Approach to Unveil Overlooked Neurotrophin Roles.

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 (Aß) 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 Aß 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 Aß, with consequences on the metal cellular uptake and therefore be among causes of the Alzheimer's disease onset.

Ionophore Ability of Carnosine and Its Trehalose Conjugate Assists Copper Signal in Triggering Brain-Derived Neurotrophic Factor and Vascular Endothelial Growth Factor Activation In Vitro.

l-carnosine (ß-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.

Zinc Interactions with a Soluble Mutated Rat Amylin to Mimic Whole Human Amylin: An Experimental and Simulation Approach to Understand Stoichiometry, Speciation and Coordination of the Metal Complexes.

Islet amyloid polypeptide (IAPP) is a hormone co-secreted with insulin and zinc from pancreatic ß-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.

Membrane Binding Strongly Affecting the Dopamine Reactivity Induced by Copper Prion and Copper/Amyloid-ß (Aß) Peptides. A Ternary Copper/Aß/Prion Peptide Complex Stabilized and Solubilized in Sodium Dodecyl Sulfate Micelles.

The combination between dyshomeostatic levels of catecholamine neurotransmitters and redox-active metals such as copper and iron exacerbates the oxidative stress condition that typically affects neurodegenerative diseases. We report a comparative study of the oxidative reactivity of copper complexes with amyloid-ß (Aß40) and the prion peptide fragment 76-114 (PrP76-114), containing the high-affinity binding site, toward dopamine and 4-methylcatechol, in aqueous buffer and in sodium dodecyl sulfate micelles, as a model membrane environment. The competitive oxidative and covalent modifications undergone by the peptides were also evaluated. The high binding affinity of Cu/peptide to micelles and lipid membranes leads to a strong reduction (Aß40) and quenching (PrP76-114) of the oxidative efficiency of the binary complexes and to a stabilization and redox silencing of the ternary complex CuII/Aß40/PrP76-114, which is highly reactive in solution. The results improve our understanding of the pathological and protective effects associated with these complexes, depending on the physiological environment.

Interaction between Hemin and Prion Peptides: Binding, Oxidative Reactivity and Aggregation.

We investigate the interaction of hemin with four fragments of prion protein (PrP) containing from one to four histidines (PrP106-114, PrP95-114, PrP84-114, PrP76-114) for its potential relevance to prion diseases and possibly traumatic brain injury. The binding properties of hemin-PrP complexes have been evaluated by UV-visible spectrophotometric titration. PrP peptides form a 1:1 adduct with hemin with affinity that increases with the number of histidines and length of the peptide; the following log K1 binding constants have been calculated: 6.48 for PrP76-114, 6.1 for PrP84-114, 4.80 for PrP95-114, whereas for PrP106-114, the interaction is too weak to allow a reliable binding constant calculation. These constants are similar to that of amyloid-ß (Aß) for hemin, and similarly to hemin-Aß, PrP peptides tend to form a six-coordinated low-spin complex. However, the concomitant aggregation of PrP induced by hemin prevents calculation of the K2 binding constant. The turbidimetry analysis of [hemin-PrP76-114] shows that, once aggregated, this complex is scarcely soluble and undergoes precipitation. Finally, a detailed study of the peroxidase-like activity of [hemin-(PrP)] shows a moderate increase of the reactivity with respect to free hemin, but considering the activity over long time, as for neurodegenerative pathologies, it might contribute to neuronal oxidative stress.

Increased Thyroid Cancer Incidence in Volcanic Areas: A Role of Increased Heavy Metals in the Environment?

Thyroid cancer incidence is significantly increased in volcanic areas, where relevant non-anthropogenic pollution with heavy metals is present in the environment. This review will discuss whether chronic lifelong exposure to slightly increased levels of metals can contribute to the increase in thyroid cancer in the residents of a volcanic area. The influence of metals on living cells depends on the physicochemical properties of the metals and their interaction with the target cell metallostasis network, which includes transporters, intracellular binding proteins, and metal-responsive elements. Very little is known about the carcinogenic potential of slightly increased metal levels on the thyroid, which might be more sensitive to mutagenic damage because of its unique biology related to iodine, which is a very reactive and strongly oxidizing agent. Different mechanisms could explain the specific carcinogenic effect of borderline/high environmental levels of metals on the thyroid, including (a) hormesis, the nonlinear response to chemicals causing important biological effects at low concentrations; (b) metal accumulation in the thyroid relative to other tissues; and (c) the specific effects of a mixture of different metals. Recent evidence related to all of these mechanisms is now available, and the data are compatible with a cause-effect relationship between increased metal levels in the environment and an increase in thyroid cancer incidence.

Zn2+ Interaction with Amyloid-Β: Affinity and Speciation.

Conflicting values, obtained by different techniques and often under different experimental conditions have been reported on the affinity of Zn2+ for amyloid-ß, 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-ß 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.

Transcriptome analysis reveals an altered expression profile of zinc transporters in colorectal cancer.

Zinc is a transition metal and catalytic cofactor involved in many biological processes including proliferation, development, differentiation, and metabolism. Zinc transporters (ZnTs) play a fundamental role in cellular zinc homeostasis. ZnTs are responsible of zinc efflux and are encoded by 10 genes belonging to solute carrier family 30A (SLC30A1-10), while zinc-regulated transporter (ZRT)/iron-regulated transporter (IRT)-like protein (ZIP) transporters are responsible for the influx of zinc into the cytoplasm and are encoded by 14 genes belonging to solute carrier family 39A (SLC39A1-14). In this study, we analyzed, by transcriptome analysis, the microRNA levels of ZnT-encoding and ZIP-encoding genes in colorectal cancer (CRC) samples matched to normal colon tissues and in CRC cell lines. Results revealed an upregulation of specific ZnT and ZIP transcripts in CRC. Upregulation of SLC30A5, SLC30A6, SLC30A7 transcripts, encoding zinc efflux transporters ZnT5, ZnT6, ZnT7, localized on endoplasmic reticulum membranes, might be part of a coordinated transcriptional program associated to the increased activity of the early secretory pathway, while transcriptional upregulation of several specific ZIP transporters (SLC39A6, SLC39A7, SLC39A9, SLC39A10, and SLC39A11) could contribute in meeting the increased demand of zinc in cancer cells. Moreover, exon-level analysis of SLC30A9, a nuclear receptor coactivator involved in the transcriptional regulation of Wnt-responsive genes, revealed the differential expression of alternative transcripts in CRC and normal colonic mucosa.

A New Ratiometric Lysosomal Copper(II) Fluorescent Probe To Map a Dynamic Metallome in Live Cells.

We synthesized a new ratiometric fluorescent Cu2+ probe, bearing a morpholine moiety for selective binding to lysosomes and two picolylamine arms for the specific chelation of divalent copper ions. The probe capability to detect lysosomal Cu2+ was demonstrated in human differentiated neuroblastoma cells by confocal microscopy.

From Peptide Fragments to Whole Protein: Copper(II) Load and Coordination Features of IAPP.

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.

Prion Peptides Are Extremely Sensitive to Copper Induced Oxidative Stress.

Copper(II) binding to prion peptides does not prevent Cu redox cycling and formation of reactive oxygen species (ROS) in the presence of reducing agents. The toxic effects of these species are exacerbated in the presence of catecholamines, indicating that dysfunction of catecholamine vesicular sequestration or recovery after synaptic release is a dangerous amplifier of Cu induced oxidative stress. Cu bound to prion peptides including the high affinity site involving histidines adjacent to the octarepeats exhibits marked catalytic activity toward dopamine and 4-methylcatechol. The resulting quinone oxidation products undergo parallel oligomerization and endogenous peptide modification yielding catechol adducts at the histidine binding ligands. These modifications add to the more common oxidation of Met and His residues produced by ROS. Derivatization of Cu-prion peptides is much faster than that undergone by Cu-ß-amyloid and Cu-α-synuclein complexes in the same conditions.

Fluorescent Copper Probe Inhibiting Aß1-16-Copper(II)-Catalyzed Intracellular Reactive Oxygen Species Production.

A variety of fluorescent probes are proposed to monitor the intracellular copper content. So far, none of the probes have been evaluated for their potential to inhibit copper-associated intracellular oxidative stress. Herein, we studied the ability of a fluorescent copper probe, OBEP-CS1, to inhibit intracellular oxidative stress associated with an amyloid ß (Aß) peptide-copper complex. The data showed that OBEP-CS1 completely inhibits the copper-catalyzed oxidation as well as decarboxylation/deamination of Aß1-16. Moreover, the cell imaging experiments confirmed that OBEP-CS1 can inhibit Aß-CuII-catalyzed reactive oxygen species production in SH-SY5Y cells. We also demonstrated that Aß1-16 peptide can bind intracellular copper and thereby exert oxidative stress.

Ac-LPFFD-Th: A Trehalose-Conjugated Peptidomimetic as a Strong Suppressor of Amyloid-ß Oligomer Formation and Cytotoxicity.

The inhibition of amyloid formation is a promising therapeutic approach for the treatment of neurodegenerative diseases. Peptide-based inhibitors, which have been widely investigated, are generally derived from original amyloid sequences. Most interestingly, trehalose, a nonreducing disaccharide of α-glucose, is effective in preventing the aggregation of numerous proteins. We have determined that the development of hybrid compounds could provide new molecules with improved properties that might synergically increase the potency of their single moieties. In this work, the ability of Ac-LPFFD-Th, a C-terminally trehalose-conjugated derivative, to slow down the Aß aggregation process was investigated by means of different biophysical techniques, including thioflavin T fluorescence, dynamic light scattering, ESI-MS, and NMR spectroscopy. Moreover, we demonstrate that Ac-LPFFD-Th modifies the aggregation features of Aß and protects neurons from Aß oligomers' toxic insult.

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 ß 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 Cu(2+) ions may adversely affect the course of the disease. Conflicting results have been reported on the protective role played by complex species formed by Cu(2+) 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 Cu(2+) 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 Cu(2+) 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 Cu(2+) ions on the hIAPP fibrillary aggregation, 2) the effect of adventitious trace amounts of Cu(2+) ions present in phosphate-buffered saline (PBS), and 3) a reducing fluorogenic probe on H2 O2 production attributed to the polypeptide alone.

Ubiquitin Associates with the N-Terminal Domain of Nerve Growth Factor: The Role of Copper(II) Ions.

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.

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.