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

Metal swap between Zn7metallothionein–3 and amyloid–β–Cu protects against amyloid–β toxicity

Aberrant interactions of copper and zinc ions with the amyloid–β peptide (Aβ) potentiate Alzheimer disease (AD) by participating in the aggregation process of Aβ and in the generation of reactive oxygen species (ROS). The ROS production and the neurotoxicity of Aβ are associated with copper binding. Metallothionein–3 (Zn7MT–3), an intra– and extracellularly occurring metalloprotein, is highly expressed in the brain and down–regulated in AD. This protein protects, by an unknown mechanism, cultured neurons from the toxicity of Aβ. Herein, we show that a metal swap between Zn7MT–3 and soluble and aggregated Aβ1–40–Cu(II) abolishes the ROS production and the related cellular toxicity. In this process, copper is reduced by the protein thiolates forming Cu(I)4Zn4MT–3 in which an air stable Cu(I)4–thiolate cluster and two disulfide bonds are present. The discovered protective effect of Zn7MT–3 from the copper–mediated Aβ1–40 toxicity may lead to new therapeutic strategies in treating AD

Pleomorphic copper coordination by Alzheimer's disease amyloid-beta peptide

Numerous conflicting models have been proposed regarding the nature of the Cu coordination environment of the amyloid β (Aβ) peptide, the causative agent of Alzheimer's disease. This study used multifrequency CW-EPR spectroscopy to directly resolve the superhyperfine interactions between Cu and the ligand nuclei of Aβ, thereby avoiding ambiguities associated with introducing point mutations. Using a library of Aβ16 analogues with site-specific N-labeling at Aspl, His6, His13, and His14, numerical simulations of the superhyperfine resonances delineated two independent 3N1O Cu coordination modes, {N ,O,N ,N H} (component la) and {N ,O,NH,NH1 } (component Ib), between pH 6-7. A third coordination mode (component II) was identified at pH 8.0, and simulation of the superhyperfine resonances indicated a 3N1O coordination sphere involving nitrogen ligation by His6, His13, and His14. No differences were observed upon O- labeling of the phenolic oxygen of Tyr10, confirming it is not a key oxygen ligand in the physiological pH range. Hyperfine sublevel correlation (HYSCORE) spectroscopy, in conjunction with site-specific N-labeling, provided additional support for the common role of His6 in components la and Ib, and for the assignment of a {O, NH,N H,NH} coordination sphere to component II. HYSCORE studies of a peptide analogue with selective C-labeling of Asp1 revealed C cross-peaks characteristic of equatorial coordination by the carboxylate oxygen of Asp1 in component Ia/b coordination. The direct resolution of Cu ligand interactions, together with the key finding that component I is composed of two distinct coordination modes, provides valuable insight into a range of conflicting ligand assignments and highlights the complexity of Cu /Aβ interactions