Cu(II) mediates kinetically distinct, non-amyloidogenic aggregation of amyloid-β peptides

Cu(II) ions are implicated in the pathogenesis of Alzheimer disease by influencing the aggregation of the amyloid-β (Aβ) peptide. Elucidating the underlying Cu(II)-induced Aβ aggregation is paramount for understanding the role of Cu(II) in the pathology of Alzheimer disease. The aim of this study was to characterize the qualitative and quantitative influence of Cu(II) on the extracellular aggregation mechanism and aggregate morphology of Aβ(1–40) using spectroscopic, microelectrophoretic, mass spectrometric, and ultrastructural techniques. We found that the Cu(II):Aβ ratio in solution has a major influence on (i) the aggregation kinetics/mechanism of Aβ, because three different kinetic scenarios were observed depending on the Cu(II):Aβ ratio, (ii) the metal:peptide stoichiometry in the aggregates, which increased to 1.4 at supra-equimolar Cu(II):Aβ ratio; and (iii) the morphology of the aggregates, which shifted from fibrillar to non-fibrillar at increasing Cu(II):Aβ ratios. We observed dynamic morphological changes of the aggregates, and that the formation of spherical aggregates appeared to be a common morphological end point independent on the Cu(II) concentration. Experiments with Aβ(1–42) were compatible with the conclusions for Aβ(1–40) even though the low solubility of Aβ(1–42) precluded examination under the same conditions as for the Aβ(1–40). Experiments with Aβ(1–16) and Aβ(1–28) showed that other parts than the Cu(II)-binding His residues were important for Cu(II)-induced Aβ aggregation. Based on this study we propose three mechanistic models for the Cu(II)-induced aggregation of Aβ(1–40) depending on the Cu(II):Aβ ratio, and identify key reaction steps that may be feasible targets for preventing Cu(II)-associated aggregation or toxicity in Alzheimer disease

The Native Copper- and Zinc- Binding Protein Metallothionein Blocks Copper-Mediated Aβ Aggregation and Toxicity in Rat Cortical Neurons

Background: A major pathological hallmark of AD is the deposition of insoluble extracellular b-amyloid (Ab) plaques. There are compelling data suggesting that Ab aggregation is catalysed by reaction with the metals zinc and copper. Methodology/Principal Findings: We now report that the major human-expressed metallothionein (MT) subtype, MT-2A, is capable of preventing the in vitro copper-mediated aggregation of Ab1–40 and Ab1–42. This action of MT-2A appears to involve a metal-swap between Zn 7MT-2A and Cu(II)-Ab, since neither Cu 10MT-2A or carboxymethylated MT-2A blocked Cu(II)-Ab aggregation. Furthermore, Zn7MT-2A blocked Cu(II)-Ab induced changes in ionic homeostasis and subsequent neurotoxicity of cultured cortical neurons. Conclusions/Significance: These results indicate that MTs of the type represented by MT-2A are capable of protecting against Ab aggregation and toxicity. Given the recent interest in metal-chelation therapies for AD that remove metal from Ab leaving a metal-free Ab that can readily bind metals again, we believe that MT-2A might represent a different therapeuti

Designed Metal-ATCUN Derivatives: Redox- and Non-redox-Based Applications Relevant for Chemistry, Biology, and Medicine

UID/QUI/50006/2019The designed "ATCUN'' motif (amino-terminal copper and nickel binding site) is a replica of naturally occurring ATCUN site found in many proteins/peptides, and an attractive platform for multiple applications, which include nucleases, proteases, spectroscopic probes, imaging, and small molecule activation. ATCUN motifs are engineered at periphery by conjugation to recombinant proteins, peptides, fluorophores, or recognition domains through chemically or genetically, fulfilling the needs of various biological relevance and a wide range of practical usages. This chemistry has witnessed significant growth over the last few decades and several interesting ATCUN derivatives have been described. The redox role of the ATCUN moieties is also an important aspect to be considered. The redox potential of designed M-ATCUN derivatives is modulated by judicious choice of amino acid (including stereochemistry, charge, and position) that ultimately leads to the catalytic efficiency. In this context, a wide range of M-ATCUN derivatives have been designed purposefully for various redox- and non-redox-based applications, including spectroscopic probes, target-based catalytic metallodrugs, inhibition of amyloid-beta toxicity, and telomere shortening, enzyme inactivation, biomolecules stitching or modification, next-generation antibiotic, and small molecule activation.publishersversionpublishe

Le complexe CuII Amyloïde-bêta lié à la Maladie d'Alzheimer :<br />Etude structurale, thermodynamique et réactivité

A peptide called amyloid-beta (Aβ) seems to play a key role in Alzheimer's disease (AD). Aβ is present in healthy humans in a soluble form, but forms aggregates in AD (amyloid plaques). According to the amyloid cascade hypothesis, these aggregates are toxic to neurons and hence lead to their degeneration and the development of AD. The degeneration occurs via the production of reactive oxygen species (ROS). Copper ions play an important role in these processes, because they can bind to Aβ and they are highly accumulated in the plaques. Moreover, copper ions influence the aggregation of amyloid-beta and are supposed to be involved in the ROS production.Our studies focused on the soluble complex CuII-Aβ. They revealed new insights : (i) Isothermal Titration Calorimetry (ITC) showed two CuII binding sites, with an apparent Kd of 10-7 M and 10-5 M, respectively. (ii) It is know that the three histidine (position 6,13,14) are involved in the complex. EPR showed a 3N/1O environment and 1H-NMR suggests that the Asp in position 1 is involved in the ligation to CuII. So, we propose the carboxylate of the Asp1 as the fourth ligand of copper. (iii) We have shown than CuII-Aβ peptide could generate hydroxyl radicals HO• in the presence of ascorbate. This production has been linked to the redox potentials of complexes. (iv) The influence of copper on the aggregation (kinetic, oligomers formation, type of aggregates) was also studied.Nos études portent sur le complexe soluble CuII-Aβ. (i) Nous avons montré en titration isotherme calorimétrique que le peptide Aβ possède 2 sites de fixation pour le cuivre avec des constantes de dissociation espectives de 100nM et 10µM. (ii) A pH 7,4 les complexes sont hétérogènes. La RPE et la RMN proposent que la forme majoritaire fait intervenir les 3 Histidines de la séquence (en position 6, 13 et 14) et la fonction carboxylique de l'Asp1. (iii) Nous avons également montré que les complexes CuII-Aβ sont capables de produire des radicaux hydroxyles HO•. La quantité de HO• générée a par ailleurs été corrélée aux potentiels d'oxydo-réduction des complexes. (iv) L'influence des métaux sur l'agrégation (vitesse, formation d'oligomères, type d'agrégats formés) a été analysée

Le complexe CuII-Amyloïde-bêta lié à la Maladie d Alzheimer (étude structurale, thermodynamique et réactivité)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T(9)TKE(12) sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K(d) = 25±6 nM to K(d) = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K(d) = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν(as)(P-O) and ν(s)(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν(as)(UO(2))(2+) vibration (from 923 cm(-1) to 908 cm(-1)) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH

Water Quality and Brain Function

In the United States, regulations are in place to ensure the quality of drinking water. Such precautions are intended to safeguard the health of the population. However, regulatory guidelines may at times fail to achieve their purpose. This may be due to lack of sufficient data regarding the health hazards of chronic low dose exposure to contaminants or the introduction of new substances that pose a health hazard risk that has yet to be identified. In this review, examples of different sources of contaminants in drinking water will be discussed, followed by an evaluation of some select individual toxicants with known adverse neurological impact. The ability of mixtures to potentially cause additive, synergistic, or antagonistic neurotoxic responses will be briefly addressed. The last section of the review will provide examples of select mechanisms by which different classes of contaminants may lead to neurological impairments. The main objective of this review is to bring to light the importance of considering trace amounts of chemicals in the drinking water and potential brain abnormalities. There is continued need for toxicology studies to better understand negative consequences of trace amounts of toxins and although it is beyond the scope of this brief overview it is hoped that the review will underscore the paucity of studies focused on determining how long-term exposure to minute levels of contaminants in drinking water may pose a significant health hazard