Stéphanie Sayen

Deprotonation of the Asp1Ala2 Peptide Bond Induces Modification of the Dynamic Copper(II) Environment in the Amyloid‐β Peptide near Physiological pH

Aggregation of the amyloid-b (Ab) peptide and the production of reactive oxygen species by aggregates are two key features in Alzheimer’s disease. Copper ions have been linked to both of these events, 3] and hence determination of the basic interaction of Cu and Ab is essential for understanding its roles in the development of the pathology. The native Ab peptides consist of 39 to 43 amino acid residues and have been shown to be strongly prone to aggregation (from a few mm concentration). However, the Cu binding site has been localized in the N-terminal part of the peptide encompassing the first 16 amino acid residues (see Scheme S1 in the Supporting Information for the peptide sequence), 5] a truncated peptide that is highly soluble. Hence, this shortened peptide is accepted as a valuable model of Cu coordination to full-length Ab and its high solubility allows classical spectroscopic methods, such as those of the present study, to be used. While most techniques aim at identifying the Cu ligands (for a review, see reference [6] and for very recent reports, see references [7, 8]), NMR spectroscopy is among the few methods also able to reveal dynamical processes in the coordination of Cu to Ab. Indeed, the paramagnetism of the Cu ion induces an enhancement of the relaxation rate of the peptide nuclei, this effect diminishing according to the inverse sixth power of the interatomic distance (for reviews, see references [9, 10]). Consequently, selective broadening of the NMR signals of nuclei spatially close to the metal-ion binding site(s) is observed. In the case of Cu, the line broadening is severe and the effect of the largely substoichiometric ratio of the paramagnetic ion is detectable in the case of fast exchange of the paramagnet between sites. This is also true for C NMR signals despite the lower sensitivity to broadening effects for this nucleus as a result of its lower gyromagnetic ratio compared to that of the proton. As concerns Cu coordination to Ab, only a few NMR studies have been reported and they are limited to H NMR or H–N heteronuclear single quantum correlation (HSQC) experiments. 14] Fast amide proton exchanges are responsible for the loss of the signals of several amino acids (including Asp1 and the three His residues) in apo–Ab peptide in the latter cases, an effect that precludes the analysis of Cu-induced signal broadening. For those reasons, herein we focus on C{H} NMR spectroscopy, which is a straightforward way to inspect the effect of Cu on Ab peptide signals. Furthermore, it is known that near physiological pH, two Cu complexes of Ab coexist, which differ in the protonation state of the peptide and their spectroscopic signatures. 15] They are referred to below as “low-pH” and “high-pH” species. We identify the amino acid residues involved in Cu binding, and give clear-cut evidence for the presence of equilibria between different ligands in both forms. We also give new insights into the dramatic change undergone by the Cu binding sites in Ab between pH values of about 6.6 and 8.7, which arises from the deprotonation and binding of the Asp1 Ala2 peptide bond amide. Figure 1 shows the evolution of the C{H} NMR spectra of the Ab peptide (sequence DAEFRHDSGYEVHHQK) upon addition of 0.1 equivalents of Cu at pH 6.6 and 8.7 (see also Figure S4 in the Supporting Information for spectral domains that concern His residues). 17] Addition of Cu leads to broadening of several signals that is more selective at high pH (right-hand spectra in Figure 1) with only Asp1, Ala2, and the side chain of His mainly affected. More precisely, at pH 6.6 peaks of the carboxylate groups from Asp1, Asp7, Glu3, Glu11, and the unprotected C terminus are significantly broadened with a slight preference for that of Asp7. Only that of Asp1 is broadened at pH 8.7. Peaks of the [*] Dr. C. Hureau, Dr. Y. Coppel, Dr. L. Sabater, Prof. Dr. P. Faller CNRS; LCC (Laboratoire de Chimie de Coordination) 205 route de Narbonne, 31077 Toulouse (France) and Universit de Toulouse; UPS, INPT; LCC 31077 Toulouse (France) Fax: (+ 33)5-6155-3003 E-mail: christelle.hureau@lcc-toulouse.fr peter.faller@lcc-toulouse.fr

X‐ray and Solution Structures of CuIIGHK and CuIIDAHK Complexes: Influence on Their Redox Properties

The Gly-His-Lys (GHK) peptide and the Asp-Ala-His-Lys (DAHK) sequences are naturally occurring high-affinity copper(II) chelators found in the blood plasma and are hence of biological interest. A structural study of the copper complexes of these peptides was conducted in the solid state and in solution by determining their X-ray structures, and by using a large range of spectroscopies, including EPR and HYSCORE (hyperfine sub-level correlation), X-ray absorption and (1)H and (13)C NMR spectroscopy. The results indicate that the structures of [Cu(II)(DAHK)] in the solid state and in solution are similar and confirm the equatorial coordination sphere of NH(2), two amidyl N and one imidazole N. Additionally, a water molecule is bound apically to Cu(II) as revealed by the X-ray structure. As reported previously in the literature, [Cu(II)(GHK)], which exhibits a dimeric structure in the solid state, forms a monomeric complex in solution with three nitrogen ligands: NH(2), amidyl and imidazole. The fourth equatorial site is occupied by a labile oxygen atom from a carboxylate ligand in the solid state. We probe that fourth position and study ternary complexes of [Cu(II)(GHK)] with glycine or histidine. The Cu(II) exchange reaction between different DAHK peptides is very slow, in contrast to [Cu(II)(GHK)], in which the fast exchange was attributed to the presence of a [Cu(II)(GHK)(2)] complex. The redox properties of [Cu(II)(GHK)] and [Cu(II)(DAHK)] were investigated by cyclic voltammetry and by measuring the ascorbate oxidation in the presence of molecular oxygen. The measurements indicate that both Cu(II) complexes are inert under moderate redox potentials. In contrast to [Cu(II)(DAHK)], [Cu(II)(GHK)] could be reduced to Cu(I) around -0.62 V (versus AgCl/Ag) with subsequent release of the Cu ion. These complete analyses of structure and redox activity of those complexes gave new insights with biological impact and can serve as models for other more complicated Cu(II)-peptide interactions.

Copper Coordination to Native N-Terminally Modified versus Full-Length Amyloid-β: Second-Sphere Effects Determine the Species Present at Physiological pH

Alzheimers disease is characterized by senile plaques in which metallic ions (copper, zinc, and iron) are colocalized with amyloid-β peptides of different sequences in aggregated forms. In addition to the full-length peptides (Aβ1-40/42), N-terminally truncated Aβ3-40/42 forms and their pyroglutamate counterparts, Aβp3-40/42, have been proposed to play key features in the aggregation process, leading to the senile plaques. Furthermore, they have been shown to be more toxic than the full-length Aβ, which made them central targets for therapeutic approaches. In order to better disentangle the possible role of metallic ions in the aggregation process, copper(II) coordination to the full-length amyloid peptides has been extensively studied in the last years. However, regarding the N-terminally modified forms at position 3, very little is known. Therefore, copper(I) and copper(II) coordination to those peptides have been investigated in the present report using a variety of complementary techniques and as a function of pH. Copper(I) coordination is not affected by the N-terminal modifications. In contrast, copper(II) coordination is different from that previously reported for the full-length peptide. In the case of the pyroglutamate form, this is due to preclusion of N-terminal amine binding. In the case of the N-terminally truncated form, alteration in copper(II) coordination is caused by second-sphere effects that impact the first binding shell and the pH-dependent repartition of the various [Cu(peptide)] complexes. Such second-sphere effects are anticipated to apply to a variety of metal ions and peptides, and their importance on changing the first binding shell has not been fully recognized yet.

Aging effect on the copper sorption on a vineyard soil: column studies and SEM-EDS analysis.

The uptake of copper by a vineyard soil in fixed bed column systems was investigated in order to study the influence of the aging time on the soil retention capacity. The application of copper by means of several additions, as in field conditions, increases the retention capacity of the soil relative to a single one application of the metal. The aging effect is responsible for this phenomenon, since its increase enhances the amount of adsorbed copper. Moreover, aging the soil reduces the amount of available copper during a Ca(NO(3))(2) leaching of the soil columns which suggests a redistribution of a weakly-bound copper fraction to a more strongly-bound fraction. Scanning electron microscopy combined with energy dispersive X-ray spectroscopy (SEM-EDS) indicates that copper is heterogeneously distributed within the soil sample. Nevertheless, copper is preferentially associated with fractions containing Fe and Al atoms.

A novel copper(II) mononuclear complex with the non-steroidal anti-inflammatory drug diclofenac: Structural characterization and biological activity

A copper(II) complex with the non-steroidal anti-inflammatory drug (NSAID) diclofenac has been for the first time synthesized and characterized without any co-organic ligand. Its biological activity against four human cell lines underlined a higher activity of the monomeric complex than the parent molecule in the case of tumoral cell lines. A ternary Cu-diclofenac-albumin complex was suspected to be the reactive species in biological medium.

Zn impacts Cu coordination to amyloid-β, the Alzheimer's peptide, but not the ROS production and the associated cell toxicity

Combined coordination of Zn(II) and Cu(I) or Cu(II) to the amyloid-β peptide has been investigated using XANES, EPR and NMR spectroscopies. While Zn(II) does alter Cu(II) binding to Aβ, this has no effect on (Aβ)Cu induced ROS production and associated cell toxicity.

Copper, Nickel and Zinc Speciation in a Biosolid-Amended Soil: pH Adsorption Edge, μ-XRF and μ-XANES Investigations

Metal solid phase speciation plays an important role in the control of the long-term stability of metals in biosolid-amended soils. The present work used pH-adsorption edge experiments and synchrotron-based spectroscopy techniques to understand the solid phase speciation of copper, nickel and zinc in a biosolid-amended soil. Comparison of metal adsorption edges on the biosolid-amended soil and the soil sample showed that Cu, Ni, and Zn can be retained by both soil and biosolid components such as amorphous iron phases, organic matter and clay minerals. These data are combined with microscopic results to obtain structural information about the surface complexes formed. Linear combination fitting of K-edge XANES spectra of metal hot-spots indicated consistent differences in metal speciation between metals. While organic matter plays a dominant role in Ni binding in the biosolid-amended soil, it was of lesser importance for Cu and Zn. This study suggests that even if the metals can be associated with soil components (clay minerals and organic matter), biosolid application will increase metals retention in the biosolid-amended soil by providing reactive organic matter and iron oxide fractions. Among the studied metals, the long-term mobility of Ni could be affected by organic matter degradation while Cu and Zn are strongly associated with iron oxides.

Influence of initial pesticide concentrations and plant population density on dimethomorph toxicity and removal by two duckweed species.

Aquatic plants take up, transform and sequester organic contaminants and may therefore be used in phytoremediation for the removal of pollutants from wastewaters. A better understanding of factors affecting the rate of contaminant uptake by aquatic plants is needed to improve engineered systems for removal of pollutants from wastewaters. This work focused on the influence of initial concentrations of pesticide and population density of plants on toxicity and uptake of the fungicide dimethomorph by two duckweed species. An increased sensitivity to dimethomorph was observed with increasing duckweed population density. Less light, due to crowding, may explain this higher sensitivity and reduced removal rate. A positive relationship was also found between toxicity or contaminant uptake and initial pesticide concentration with a maximal removal of 41 and 26 microg g(-1) fresh weight of dimethomorph (at 600 microg L(-1) of dimethomorph and an initial density of 0.10g E-flask(-1)) by Lemna minor and Spirodela polyrhiza, respectively. This research also indicated that these aquatic plants can efficiently eliminate organic contaminants and may ultimately serve as phytoremediation agents in the natural environment.

Zinc(II) modulates specifically amyloid formation and structure in model peptides

Metal ions such as zinc and copper can have dramatic effects on the aggregation kinetics of and the structures formed by several amyloidogenic peptides/proteins. Depending on the identity of the amyloidogenic peptide/protein and the conditions, Zn(II) and Cu(II) can promote or inhibit fibril formation, and in some cases these metal ions have opposite effects. To better understand this modulation of peptide aggregation by metal ions, the impact of Zn(II) binding to three amyloidogenic peptides (Aβ14-23, Aβ11-23, and Aβ11-28) on the formation and structure of amyloid-type fibrils was investigated. Zn(II) was able to accelerate fibril formation for all three peptides as measured by thioflavin T fluorescence and transmission electron microscopy. The effects of Zn(II) on Aβ11-23 and Aβ11-28 aggregation were very different compared with the effects of Cu(II), showing that these promoting effects were metal-specific. X-ray absorption spectroscopy suggested that the Zn(II) binding to Aβ11-23 and Aβ11-28 is very different from Cu(II) binding, but that the binding is similar in the case of Aβ14-23. A model is proposed in which the different coordination chemistry of Zn(II) compared with Cu(II) explains the metal-specific effect on aggregation and the difference between peptides Aβ14-23 and Aβ11-23/Aβ11-28.

Zinc(II) Binding Site to the Amyloid-β Peptide: Insights from Spectroscopic Studies with a Wide Series of Modified Peptides

The Zn(II) ion has been linked to Alzheimer’s disease (AD) due to its ability to modulate the aggregating properties of the amyloid-β (Aβ) peptide, where Aβ aggregation is a central event in the etiology of the disease. Delineating Zn(II) binding properties to Aβ is thus a prerequisite to better grasp its potential role in AD. Because of (i) the flexibility of the Aβ peptide, (ii) the multiplicity of anchoring sites, and (iii) the silent nature of the Zn(II) ion in most classical spectroscopies, this is a difficult task. To overcome these difficulties, we have investigated the impact of peptide alterations (mutations, N-terminal acetylation) on the Zn(Aβ) X-ray absorption spectroscopy fingerprint and on the Zn(II)-induced modifications of the Aβ peptides’ NMR signatures. We propose a tetrahedrally bound Zn(II) ion, in which the coordination sphere is made by two His residues and two carboxylate side chains. Equilibria between equivalent ligands for one Zn(II) binding position have also been observed, the predominant site being made by the side chains of His6, His13 or His14, Glu11, and Asp1 or Glu3 or Asp7, with a slight preference for Asp1.