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Contact allergy, commonly induced by nickel, is the most frequent cause of contact hypersensitivity in industrialized countries, with 30% of population being affected. Ni(II) seems to trigger an inflammatory response by activating human Tool-like-Receptor 4 (hTLR4). Species-specific activation, as in this case, required distinct sequence motifs that are present in human but not in mouse, a species not sensitive to nickel-induced allergies. The specific region of human TLR4 responsible for nickel responses could be a sequence containing three histidine residues, H431, and the non- conserved H456 and H458, localized in the C-terminus. It has been proposed that the imidazole side chains of the histidine residues H456 and H458 provide a potential binding site for nickel because they were located at an optimal distance to interact with Ni(II) ions, whereas H431 was further apart. We decided to verify the possibility of metal binding to FQH431SNLKQMSEFSVFLSLRNLIYLDISH456TH458TR sequence, containing the three histidines supposedly involved in nickel response, in order to study the binding properties of the peptide fragment and on the thermodynamic stability of its metal complexes. Formation equilibria of Ni(II) complexes have been investigated in aqueous solution and in a wide pH range. Protonation and complex-formation constants have been potentiometrically determined; complex-formation models and species stoichiometry have been checked by means of UV-Vis absorption and CD spectroscopy and investigation through NMR is currently being carried out. The predominant species for a 1:1 peptide/Ni(II) molar ratio was obtained at physiological pH and showed an effective binding of the metal to the target sequence.

Parkinson Disease (PD) is a neurodegenerative pathology whose causes have not yet been fully clarified. In this perspective, we have chosen short fragments of Ypk9 protein that include interesting sequences for metal binding and are highly conserved in a number of different organisms, located in domains of the protein readily accessible to the metals. We studied their behaviour towards divalent cations such as manganese and zinc, using NMR mono- and bidimensional techniques and EPR spectroscopy. As this study was going on, we also started the investigation on Cu(II) coordination, showing by a series of potentiometric and spectroscopic measurements that this metal too is able to effectively bind the chosen sequence. Here we would like to report our latest findings.

It has been found that anionic species of metal salts can lead to different geometries and stoichiometries in the synthesis of coordination compounds. Therefore, using the same cation but different polyatomic anion groups such as oxalate, sulfate, acetate, oxamate etc. [1], it is possible to obtain metal complexes with various structural and stoichiometric features upon coordination. In fact, these anionic groups can behave themselves, generally, as mono-, bidentate and bridgedbidentate ligands towards metal ions. In the last years, with the aim to design a multi-dentate ligand capable of supporting simultaneous coordination to three copper atoms and to mimic the multicopper active sites of blue copper oxidases (e.g., laccase, ascorbate oxidase and ceruloplasmin), we focused our studies on the synthesis and structural characterization of copper(II) complexes of compartmental acyclic bis(salicylhydrazone) ligands derived from iminoand methyl-iminodiacetic acid, finding a different behaviour using copper(II) sulfate, perchlorate and acetate salts. Thus, we have obtained, with a 1:3 (ligand-to-metal) molar ratio, a trinuclear coordination polymer from Cu(II) perchlorate [2], a sulfato-bridged hexanuclear dimer [3] and a mononuclear complex using Cu(II) acetate [4]. Microbiological investigations showed a good activity of the sulfato and perchlorato complexes. Studies concerning DNA binding of these compounds are now in progress.

It is known that rich repeat domains in peptides can be of interest as the models for the study of molecular phenomena related to metal ion binding in proteins involved in neurodegenerative disorders. Imbalances in transition metal ions are assumed to contribute to the conversion of the multi-histidinic amyloid β-peptide (Aβ) from its soluble form to an amyloidogenic form, and to Aβ deposition. Of these ions, it has been reported that manganese binding to PrP is detrimental and causes a conformational change in the protein, suggesting that manganese binding could potentially play a role in prion disease progression in vivo. It appears that PrP is less stable on binding manganese and quickly converts to a misfolded form. The binding of manganese to PrP potentially results in the conversion of the protein to an abnormal isoform with properties reminiscent of PrPsc. In particular, although PrP can bind the same number of manganese atoms as of copper atoms, the resulting protein becomes proteinase resistant, forms fibrils and loses function.[1,2] Regarding cobalt, a novel low-affinity binding site for Co(II) was discovered between PrP residues 104 and 114, with residue His111 being the key amino acid for coordinating Co(II).[3] Thus, despite the interest in manganese and cobalt binding to PrP, a thorough analysis of the interaction of both metals with proteins related to brain pathies has not yet been reported. The (T1R2S3R4S5H6T7S8E9G10)3 fragment from Cap43 protein, which is induced by metal ions, is characterized by a decarepeat domain comprising three decapeptide units with one histidine and one glutamate residue in each repeat. Therefore the study of the interaction of the 30-aminoacid peptide from Cap43 protein with metal ions can contribute to the understanding of the crucial role of multi-imidazol and glutamate sites in the protein coordination processes and the possible role of divalent metal ions in the pathogenesis of prion disease and other related protein pathies.[4-8] Here we present our recent results on the Cobalt(II) and Manganese(II) complexes of terminally protected mono- and multi-histidine-glutamate peptides studied by combination of potentiometric measurements and spectroscopic techniques (NMR, UV-Vis and EPR). Metal complexation induces important structural changes with the C-terminal portion of the ligand, constraining it to leave its disordered conformation and promoting side chain orientation. Our results give rise to a molecular model of the induced structure for the peptides complexed with cobalt and manganese.

Nickel compounds are well known as human carcinogens, though the molecular events that are responsible for this are not well understood. It has been proposed that a crucial element in the mechanism of carcinogenesis is the binding of Ni(II) ions within the cell nucleus. It is known that DNA polymer binds Ni(II) only weakly, leaving the proteins of the cell nucleus as the likely Ni(II) targets. Being histone proteins the most abundant among them, they can be considered the primary sites for nickel binding. Here we describe the interactions of nickel with histone H4, core tetramer (H3-H4)2 and several peptide fragments which have been selected as the candidates for specific binding sites in the histone octamer. The results allowed us to propose several mechanisms of nickel induced damage resulting from metal coordination, including structural changes of histone proteins, as well as nucleobase oxidation and sequence-specific histone hydrolysis. The aim of the present work is to provide a comprehensive overview of literature dealing with nickel coordination to histone proteins and its link with nickel involvement in toxicity and carcinogenicity.

E' noto come l'accumulo dei metalli nel cervello possa portare, o quantomeno contribuire, allo sviluppo di malattie neurodegenerative, attraverso meccanismi che solo ora cominciano a essere lentamente chiariti. La malattia di Parkinson, cosiddetta “idiopatica” in quanto non presenta alcuna causa apparente, è stata messa di recente in correlazione con l'esposizione o l'avvelenamento da manganese. Studi epidemiologici condotti da diverse università degli Stati Uniti hanno evidenziato come gli abitanti in zone urbane con alte concentrazioni di questo metallo avessero una probabilità di sviluppare il Parkinson quasi due volte più alta rispetto agli abitanti in zone meno inquinate, o inquinate da altri metalli (quali ad esempio il rame). Altri studi apparsi di recente in letteratura correlano l'esposizione al manganese con alcune modificazioni di un gene legato alla sinucleina, proteina presente con diverse funzioni in tutte le malattie neurodegenerative. Lo studio è stato effettuato su una proteina di un lievito la YPK9, al 58% simile e al 38% uguale all'analoga umana PARK9, la cui mutazione causa appunto lo sviluppo di una forma ereditaria di Parkinson. Silenziando il gene YPK9 nei lieviti si è notato che in assenza della relativa proteina questi mostravano disturbi nella crescita se sottoposti all'azione di diversi metalli, mentre in presenza del manganese la crescita era particolarmente ridotta. Veniva quindi dimostrata l'azione protettiva della YPK9 nei confronti dei cationi bivalenti, specialmente del manganese. Pare dunque possibile che una modifica sull'analogo umano, il PARK9, sia in grado di inficiare i normali meccanismi con cui il nostro organismo si protegge da ioni metallici dannosi, quali il manganese, e dando il via a una serie di processi che portano allo sviluppo della malattia neurodegenerativa. Abbiamo pertanto voluto verificare l'effettiva propensione di tale proteina a interagire con ioni Mn(II), selezionando sulla sequenza della YPK9 dei frammenti promettenti per il legame con il metallo e investigando la possibilità di una interazione efficace di questi frammenti con diversi cationi bivalenti, tra cui appunto il manganese, ma anche il calcio e lo zinco. I risultati preliminari, ottenuti attraverso alcune tecniche spettroscopiche quali l'NMR mono- e bidimensionale e l'EPR, verranno esposti in questa comunicazione.

Da diversi anni il nostro gruppo di ricerca si occupa di interazioni tra metalli e alcune proteine contenenti residui istidinici correlate con lo sviluppo di diverse patologie. Una di queste proteine, la Cap43, si è dimostrata molto promettente come ‘palestra’ per lo studio di tali interazioni, sia perché contiene una sequenza molto interessante per il legame con i metalli, un decapaptide monoistidinico ripetuto per tre volte consecutive, sia perché contenendo più di un residuo istidinico può fungere da modello nella ricerca sulle cosiddette ‘multihistidinic proteins’ che recentemente sono state identificate come responsabili di malattie neurodegenerative, o sono comunque coinvolte nel loro sviluppo. La Cap43, oltre a essere presente in numerose cellule tumorali, viene espressa anche dalle cellule sane quando si trovino in presenza di alti livelli di nichel, e in misura minore di cobalto. Abbiamo verificato che la sequenza di legame è estremamente efficace nel legame col nichel (lega fino a tre ioni metallici contemporaneamente), ipotizzando dunque un suo ruolo come agente detossificante nei confronti di metalli tossici. In qualità di residuo multi-istidinico abbiamo voluto testare anche la sua interazione con altri metalli di interesse biologico, quali il rame e lo zinco, evidenziando come invece questi ioni abbiano la tendenza a legarsi utilizzando un diverso pattern coordinativo. Lo sviluppo naturale della nostra ricerca è stato pertanto quello di estenderla ad altri due metalli, il cobalto, in quanto anch'esso è capace di indurre l‟espressione della Cap43 nelle cellule sane, e il manganese, recentemente identificato come il responsabile di alcuni parkinsonismi. Vogliamo pertanto presentare qui i nostri ultimi risultati, ottenuti prevalentemente attraverso l‟uso di tecniche spettroscopiche quali l'NMR multidimensionale, l'UV-Vis e l'EPR.