Structural model of a Ni(II) complex with a 30-amino acid peptide through an NMR study

Multidimensional NMR spectroscopy is a useful tool for the calculation of structures or structural models of metal-peptide complexes in solution. We applied bidimensional NMR techniques to study the interactions of Ni(II) ions with a 30-aminoacid peptide, a fragment of the C-terminal tail of Cap 43 protein. This protein is strictly connected to nickel exposure in cells, since it seems to be specifically expressed as a response to the presence of this metal in the cellular medium and it is also related to cancer development; an abnormal level of Cap43 protein has been detected in a number of tumour tissues. The striking feature of Cap 43 is a three-repeated decapeptide sequence at its C-terminus; each 10-aminoacid fragment (TRSRSHTSEG) bearing a histidinic residue, which has been indicated as an anchoring site for metal binding in numerous cases. We previously reported that each fragment is able to coordinate a Ni(II) ion in a very effective way. Structure calculations for the peptide-metal complex were performed for a single monohistidinic fragment on the basis of the ROE crosscorrelations observed in the 2D 1H-1H ROESY spectra at pH = 10. The metal complex involves an imidazolic nitrogen of histidine residue, three amidic nitrogens of the backbone and an oxygen atom from a deprotonated serine residue which takes part to the formation of a square pyramidal structure. The structural model calculated allowed us a better understanding of the features of nickel coordination with the C-terminal region of Cap43 protein

Molecular mechanisms of nickel carcinogenesis: nickel binding to histone H4 and Cap43 protein

The carcinogenicity of nickel compounds has been confirmed and corroborated by numerous epidemiological studies in humans and carcinogenesis bioassays in animals. We also found that an excellent tumour marker recently discovered and specifically induced by nickel, Cap43 protein, has a new mono-histidinic motif consisting of ten amino acids TRSRSHTSEG repeated three times in the C-terminus which is able to bind several metal ions in a cooperative way

Improvements in understanding nickel toxicity and carcinogenesis through NMR studies: the case of nickel binding to histone H4

Although nickel has been shown to be an essential trace element involved in the metabolism of bacteria, archaea, plants and higher organisms, the carcinogenicity of certain nickel compounds has been confirmed by the combination of epidemiological evidence in humans and carcinogenesis bioassays in animals and it is probably due to alteration in gene expression rather than by direct DNA damage. We have previously reported that Ni(II) is a potent suppressor of histone H4 acetyaltion, in both yeast and mammalian cells. Here we present our recent results on the coordination ability of Ni(II) to the N-terminal tail of HIstone H4 achieved by the use of NMR techiniques like 1d, 2D Tocsy and Noesy H NMR experiments. A structural model of the peptide-Ni(II) complex has been calculated, pointing out the important structural changes occurring to the peptide upon coordination

An NMR study on nickel binding to Cap43 protein

Cap43 has been reported to be specifically induced by nickel compounds in a variety of cell lines. Its function is not yet clear, but Cap43 protein does appear to be inducec in response to an increase in intracellular concentration of Ca2+, caused by nickel ion exposure in cultured human cells. Cap43 is expressed at low levels in normal tissues. However, in a variety of cancers, it is overexpressed in cancer cells. The peculiarity of Cap43 is its mono-histidinic motif consisting of ten amino acids (TRSRHTSEG) repeated three times in the C-terminus. We have analyzed, for Ni(II) binding, the 30-amino acid C-terminal sequence of the protein, TRSRSHTSEG-TRSRTHTSEG-TRSRSHTSEG, by the use of different NMR techniques such as 1D, NOESY, TOCSY and ROESY experiments. From the data thus collected we calculated a model of the structure for the peptide-Ni(II) complex, confirming the characteristic binding of a nickel ion to each 10-amino acid fragment and showing the interesting structural changes the peptide undergoes upon metal coordination

Zinc binding in a multi-histidinic peptide fragment

A multi-histidinic peptide and its minimal models have been investigated for metal binding. We have used NMR spectroscopy to probe the binding of zinc to the three repeats (T1R2S3R4S5H6T7S8E9G10)3 and to its mono-histidinic minimal models, the 9- and 10-aminoacid fragment. 1H-1H TOCSY, 1H-13C HSQC, 1H-1H NOESY and 1H-1H ROESY multidimensional NMR techniques were performed to understand the details of metal binding sites and the conformational behaviour of the peptides at different pH values and at different ligand to metal molar ratios. Zinc coordination involves imidazole Nδ of His6 and carboxyl γ-O of Glu9 residues; interaction with peptide oxygens of the His6-Thr7 or Thr7-Ser8 bonds in a tetrahedral arrangement with the minimal model peptides, cannot be excluded. Zinc coordination involves, at physiologic pH, all the three imidazole Nδ donors of His6, His16 and His26 as well as carboxyl γ -O of Glu residues in a tetra, penta or octahedral arrangement with the three repeats, the 30-aminoacid fragment. Zinc complexation induces important structural changes with the C-terminal portion of the ligand, constraining it to leave its disordered conformation. Our results give rise to a model of the induced structure of the peptides when bound to zinc. At high pH, amide deprotonation does not take place and hydroxo or high molecular weight polymeric species may be formed

Nickel binding sites in histone proteins

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

The Role of Y-PARK9 protein in preventing manganese-induced Parkinson's disease

A variety of metals are essential trace elements but can reach localized toxic concentrations through various disease processes or environmental exposures and have been implicated as having a role in neurodegeneration. In particular, chronic inorganic manganese exposure causes selective toxicity to the nigrostriatal dopaminergic system, resulting in a Parkinsonian-like neurological condition known as Manganism. YPK9 gene (Yeast PARK9; also known as YOR291W) encodes a transmembrane P-type transport ATPase presumably involved in metal coordination and transportation, though its substrate specificity still remains unknown. Mutations in the human homolog of YPK9, PARK9 (ATP13A2), have been linked to genetic forms of early onset parkinsonism. Recently a strong genetic interaction between YPK9 and another Parkinson's disease protein, α-synuclein, has been evidenced in multiple model systems, indicating a crucial role for YPK9 in manganese detoxification in yeast and a specific protecting effect against manganese poisoning [1,3]. With the purpose to shed light on the protective property of YPK9 in Manganese-induced Parkinsonism, we tested the binding ability of Mn(II) and other divalent cations (Cu(II), Zn(II)) towards several peptide sequences from YPK9, with a particular focus on highly conserved sequences from yeast to human. The work was carried out at different pH values and ligand/metal molar ratios by means of potentiometric and spectroscopic techniques (multidimensional and heteronuclear NMR and UV-visible), in order to evaluate and compare the coordination propensity of such fragments with Mn(II) and the other metal probes selected [4,5]

Ni(II) binding to the Human Tool Like Receptor (HTLR4)

Nickel allergy is the most frequent cause of contact hypersensitivity (burning, redness, itching, swelling and even blisters) in industrialized countries, with 30% of population being affected. Contact allergy is commonly induced by nickel ions present in nickel-containing jewelry such as rings and earrings, as well as in nickel-containing cellular telephones. Ni(II) seems to trigger an inflammatory response by activating human Toll-like-Receptor 4 (hTLR4) [1-4]. Species-specific activation, as in this case, requires distinct sequence motifs that are present in humans but not in mouse, a species not sensitive to nickel-induced allergies. A sequence containing three histidine residues, H431, and the non-conserved H456 and H458, localized in the C-terminus, could be identified as the specific region of human TLR4 responsible for nickel responses. It has been proposed that the imidazole side chain of the histidine residues H456 and H458 may provide a potential binding site for this metal because they are located at an optimal distance to interact with Ni(II) ions, whereas H431 is located further apart. The aim of our research was to verify the possibility of metal binding to the sequence containing the three histidines supposedly involved in nickel response. The chosen segment was the 32aa peptide FQH431SNLKQMSEFSVFLSLRNLIYLDISH456TH458TR, which was studied in order to understand both its binding properties and 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 multidimensional and eteronuclear NMR spectroscopy. 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

Iron chelating agents for iron overload diseases

Although iron is an essential element for life, an excessive amount may become extremely toxic both for its ability to generate reactive oxygen species, and for the lack in humans of regulatory mechanisms for iron excretion. Chelation therapy has been introduced in clinical practice in the seventies of last century to defend thalassemic patients from the effects of iron overload and, in spite of all its limitations, it has dramatically changed both life expectancy and quality of life of patients. It has to be considered that the drugs in clinical use present some disadvantages too, this makes urgent new more suitable chelating agents. The requirements of an iron chelator have been better and better defined over the years and in this paper they will be discussed in detail. As a final point the most interesting ligands studied in the last years will be presented

Interazione di ioni Cu(II) con un frammento della proteina PARK9

La Malattia di Parkinson (MP) è una patologia neurodegenerativa le cui cause non sono state ancora comprese appieno. Per questo motivo è detta anche sindrome idiopatica, cioè senza cause note o identificabili, sebbene alcuni tipi di MP possano avere una causa genetica o post-traumatica, e diversi fattori di rischio come l’esposizione ad alcuni pesticidi. Recentemente è emerso che anche l’esposizione al manganese (come capita ai minatori o ai saldatori) può causare una sindrome simile alla MP (Parkinsonismo), ed è stata scoperta una correlazione tra cause genetiche e ambientali attraverso lo studio della mutazione di una proteina chiamata Park9.1 Successivamente, una ricerca sul gene di un lievito, YPK9, che è molto simile al PARK9 umano, ha rivelato che la cancellazione di questo gene conferisce al lievito una maggiore sensibilità alla crescita in presenza diversi cationi divalenti, suggerendo che la proteina Ypk9 possa giocare un ruolo nel sequestro di metalli pesanti nelle loro forme divalenti.2 Allo stesso modo, una mutazione sul PARK9 potrebbe esporre l’uomo all’azione di queste specie cationiche. In quest’ottica, abbiamo scelto dei piccoli fragmenti della proteina Ypk9 che includono sequenze interessanti per un potenziale legame con i metalli, altamente conservate in un grosso numero di organismi e collocate in porzioni della proteina facilmente accessibili ai metalli. Abbiamo inizialmente studiato il loro comportamento nei confronti di cationi divalenti come il manganese e lo zinco, usando tecniche NMR mono- e bidimensionali, insieme alla spettroscopia EPR.3 Durante lo svolgimento di questo studio abbiamo iniziato inoltre quello relativo alla coordinazione del Cu(II), e da una serie di misure potenziometriche e spettroscopiche è emerso che anche questo metallo è in grado di legarsi efficacemente alla proteina