Daniela Valensin

Bioinorganic Chemistry of Parkinson's Disease: Structural Determinants for the Copper-Mediated Amyloid Formation of Alpha-Synuclein

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinsons disease (PD). A central, unresolved question in the pathophysiology of PD relates to the role of AS-metal interactions in amyloid fibril formation and neurodegeneration. Our previous works established a hierarchy in alpha-synuclein-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. Two independent, non-interacting copper-binding sites were identified at the N-terminal region of AS, with significant difference in their affinities for the metal ion. In this work we have solved unknown details related to the structural binding specificity and aggregation enhancement mediated by Cu(II). The high-resolution structural characterization of the highest affinity N-terminus AS-Cu(II) complex is reported here. Through the measurement of AS aggregation kinetics we proved conclusively that the copper-enhanced AS amyloid formation is a direct consequence of the formation of the AS-Cu(II) complex at the highest affinity binding site. The kinetic behavior was not influenced by the His residue at position 50, arguing against an active role for this residue in the structural and biological events involved in the mechanism of copper-mediated AS aggregation. These new findings are central to elucidate the mechanism through which the metal ion participates in the fibrillization of AS and represent relevant progress in the understanding of the bioinorganic chemistry of PD.

The dimeric and tetrameric octarepeat fragments of prion protein behave differently to its monomeric unit

Potentiometric and spectroscopic data have shown that octarepeat dimer and tetramer are much more effective ligands for Cu(II) ions than simple octapeptide. Thus, the whole N-terminal segment of prion protein due to cooperative effects, could be more effective in binding of Cu(II) than simple peptides containing a His residue. The gain of the Cu(II) binding by longer octarepeat peptides derives from the involvement of up to four imidazoles in the coordination of the first Cu(II) ion. This type of binding increases the order of the peptide structure, which allows successive metal ions for easier coordination.

fac-{Ru(CO)3}2+ Selectively Targets the Histidine Residues of the β-Amyloid Peptide 1-28. Implications for New Alzheimer's Disease Treatments Based on Ruthenium Complexes

The reaction of the ruthenium(II) complex fac-[Ru(CO)(3)Cl(2)(N(1)-thz)] (I hereafter; thz = 1,3-thiazole) with human beta-amyloid peptide 1-28 (Abeta(28)) and the resulting {Ru(CO)(3)}(2+) peptide adduct was investigated by a variety of biophysical methods. (1)H NMR titrations highlighted a selective interaction of {Ru(CO)(3)}(2+) with Abeta(28) histidine residues; circular dichroism revealed the occurrence of a substantial conformational rearrangement of Abeta(28); electrospray ionization mass spectrometry (ESI-MS) suggested a prevalent 1:1 metal/peptide stoichiometry and disclosed the nature of peptide-bound metallic fragments. Notably, very similar ESI-MS results were obtained when I was reacted with Abeta(42). The implications of the above findings for a possible use of ruthenium compounds in Alzheimers disease are discussed.

Copper Binding to the Neurotoxic Peptide PrP106–126: Thermodynamic and Structural Studies

The human prion protein fragment PrP106–126 is a highly fibrillogenic peptide, resistant to proteinases and toxic to neurons; it derives from the normal prion protein (PrPC), with which it can interact, thus inhibiting its superoxide dismutase‐like activity. The same properties are also shown by the abnormal isoform of the prion protein (PrPSc), and this similarity makes PrP106–126 an interesting model for the neurotoxic action of PrPSc. A role for copper in PrP106–126 aggregation and toxicity has recently been evidenced, and the interaction of terminal Lys, His and Met residues with the copper ion at neutral pH has been suggested. In order to shed more light on the complex‐formation equilibria of PrP106–126 with the copper ion, a thorough investigation has been carried out by means of several experimental techniques: potentiometry, solution calorimetry, VIS spectrophotometry, circular dichroism, EPR and NMR spectroscopy. A shorter and more soluble fragment—PrP106–113, which lacks the hydrophobic C‐terminal domain of PrP106–126 but contains all the potential donor groups—has also been considered for the sake of comparison. The involvement of terminal amino, imidazolic and amido nitrogens in complex formation has been confirmed, while no evidence was found for the interaction of side chains of Met and Lys residues with the copper ion. Solution structures for the main complexes are suggested.

Identification of a novel high affinity copper binding site in the APP(145–155) fragment of amyloid precursor protein

The copper(II) binding features of the APP(145-155) and APP(145-157) fragments of the amyloid precursor protein, Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-NH2 and Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-Glu-Thr-NH2 were studied by NMR spectroscopy and NMR findings were supported by UV-vis, CD and EPR spectra. Potentiometric measurements were performed only for the more soluble Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-Glu-Thr-NH2 peptide fragment. The following was shown: (i) the imidazole rings of all the three His residues are involved in metal coordination; (ii) metal binding induces ionisation of Leu-148 and His-149 amide nitrogens that complete the donor set to copper(II) in the species dominant at neutral pH; (iii) the unusual coordination scheme of the His-Xxx-His-Xxx-His consensus sequence justifies the high specificity for Cu(II) when compared to SOD-like or albumin-like peptides or even in amyloid Abeta fragments. The present findings may represent the key for interpreting the observed requirement of His residues conservation for the redox cycling between Cu(II) and Cu(I) by soluble APP.

Impact of the peptide sequence on the coordination abilities of albumin-like tripeptides towards Cu2+, Ni2+ and Zn2+ ions. Potential albumin-like peptide chelators

Thermodynamic and spectroscopic studies have shown that the insertion of α-hydroxylmethylserine (HmS) residues into the N-terminal peptide motif of human serum albumin results in a very powerful chelating agent for Cu2+ and Ni2+ ions. The insertion of two HmS residues results in the HmS–HmS–His–OH/NH2 sequence, which is the most effective chelating agent based on an albumin-like sequence for both studied metal ions, especially when the C-terminal carboxylate is protected by an amide function.

Copper(I)-α-Synuclein Interaction: Structural Description of Two Independent and Competing Metal Binding Sites

The aggregation of α-synuclein (αS) is a critical step in the etiology of Parkinsons disease. Metal ions such as copper and iron have been shown to bind αS, enhancing its fibrillation rate in vitro. αS is also susceptible to copper-catalyzed oxidation that involves the reduction of Cu(II) to Cu(I) and the conversion of O(2) into reactive oxygen species. The mechanism of the reaction is highly selective and site-specific and involves interactions of the protein with both oxidation states of the copper ion. The reaction can induce oxidative modification of the protein, which generally leads to extensive protein oligomerization and precipitation. Cu(II) binding to αS has been extensively characterized, indicating the N terminus and His-50 as binding donor residues. In this study, we have investigated αS-Cu(I) interaction by means of NMR and circular dichroism analysis on the full-length protein (αS(1-140)) and on two, designed ad hoc, model peptides: αS(1-15) and αS(113-130). In order to identify and characterize the metal binding environment in full-length αS, in addition to Cu(I), we have also used Ag(I) as a probe for Cu(I) binding. Two distinct Cu(I)/Ag(I) binding domains with comparable affinities have been identified. The structural rearrangements induced by the metal ions and the metal coordination spheres of both sites have been extensively characterized.

The C terminus of HspA—a potential target for native Ni(II) and Bi(III) anti-ulcer drugs

HspA, a protein crucial for nickel homeostasis in Helicobacter pylori (H. pylori), has a unique histidine- and cysteine-rich domain at the C terminus. In this work, we compared the coordination of nickel (the natural co-factor) and bismuth (inhibitor) to this domain (Ac-ACCHDHKKH-NH(2)) and to a reference peptide (Ac-CHCH-NH(2)). Potentiometric, CD, UV-Vis spectroscopic and NMR methods have shown that bismuth binds incomparably stronger than nickel; the same data shows the impact of histidines on such a binding. Our results are in good agreement with earlier biological data and suggest that HspA can be a potential target of the bismuth anti-ulcer drug against H. pylori.

NMR studies on Cu(II)–peptide complexes: exchange kinetics and determination of structures in solution

The interaction of copper(II) with histidine containing peptides has recently acquired renewed interest following the established link between abnormal protein behaviour in neurodegenerative processes and unpaired copper homeostasis. Five peptide sequences taken from the amyloid precursor protein and the prion protein were considered. Addition of paramagnetic Cu(II) ions to solutions of such peptides was not found to severely affect the appearance of NMR spectra, thus limiting the usual approach for structural determination. Exchange kinetics was shown to play a major role in determining the observed paramagnetic spin-lattice relaxation rates. Two independent methods were suggested for evaluating the exchange rates of His-containing peptides from the copper-coordination sphere and to calculate copper-proton distances. In such a way NMR was demonstrated to have the potential of providing detailed structures of the Cu(II)-peptide complexes in solution.

Metal binding ability of cysteine-rich peptide domain of ZIP13 Zn2+ ions transporter.

The coordination modes and thermodynamic stabilities of the complexes of the cysteine-rich N-terminal domain fragment of the ZIP13 zinc transporter (MPGCPCPGCG-NH(2)) with Zn(2+), Cd(2+), Bi(3+), and Ni(2+) have been studied by potentiometric, mass spectrometric, NMR, CD, and UV-vis spectroscopic methods. All of the studied metals had similar binding modes, with the three thiol sulfurs of cysteine residues involved in metal ion coordination. The stability of the complexes formed in solution changes in the series Bi(3+) ≫ Cd(2+) > Zn(2+) > Ni(2+), the strongest being for bismuth and the weakest for nickel. The N-terminal fragment of the human metalothionein-3 (MDPETCPCP-NH(2)) and unique histidine- and cysteine-rich domain of the C-terminus of Helicobacter pyroli HspA protein (Ac-ACCHDHKKH-NH(2)) have been chosen for the comparison studies. It confirmed indirectly which groups were the anchoring ones of ZIP13 domain. Experimental data from all of the used techniques and comparisons allowed us to propose possible coordination modes for all of the studied ZIP13 complexes.