Metal swap between Zn7metallothionein-3 and amyloid-β-Cu protects against amyloid-β toxicity Meloni, G; Sonois, V; Delaine, T; Guilloreau , L; Gillet, A; Teissie, J; Faller, P; Vasak, M Meloni, G; Sonois, V; Delaine, T; Guilloreau , L; Gillet, A; Teissie, J; Faller, P; Vasak, M (2008). Metal swap between Zn7metallothionein-3 and amyloid-β-Cu protects against amyloid-β toxicity. Nature Chemical Biology, 4 Metal swap between Zn7metallothionein-3 and amyloid-β-Cu protects against amyloid-β toxicity Abstract 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 thegeneration of reactive oxygen species (ROS). The ROS production and the neurotoxicity of Aβ are associated with copper binding. Metallothionein-3 (Zn7MT-3), an intra-andextracellularly 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 newtherapeutic strategies in treating AD.Metal swap between Zn 7 metallothionein-3 and amyloid-β-Cu protects against amyloid-β toxicity residue Aβ peptide, a proteolytic fragment generated from the amyloid precursor protein (APP)by β-and γ-secretases 1 . There is significant evidence indicating that the Aβ peptides can interact with metal ions such as Zn(II) and Cu(II), thereby participating in their aggregation and in the production of ROS 1, 2 . Whereas the copper-induced Aβ aggregation is related to the ROS production and neurotoxicity 3 , the zinc-induced Aβ aggregation is considered to be neuroprotective 4 . The ROS are generated by Aβ-Cu(II) through the redox cycling of copper which requires its reduction by biological components such as ascorbate (1), glutathione (2), dopamine (3), and cholesterol (4) 5,6 . A dysregulation of metal ion homeostasis, as occurs in AD, may foster an environment that promotes such degenerative conditions. The modulation of brain metal ion homeostasis, the reduction of aberrant metal-protein interactions by MPAC (metal-

Zinc is an essential micronutrient for all living organisms, required for signaling and proper function of a range of proteins involved in e.g. DNA-binding and enzymatic catalysis 1 . In prokaryotes and photosynthetic eukaryotes Zn 2+ -transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn 2+ and related elements 2,3 . Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2.P i ) of ZntA from Shigella sonnei, determined at 3.2 and 2.7 Å resolution, respectively. The structures reveal a similar fold as the Cu + -ATPases with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn 2+ ions. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including Cys392, Cys394 and Asp714. The pathway closes in the E2.P i state where Asp714 interacts with the conserved Lys693, which possibly stimulates Zn 2+ release as a built-in counter-ion, as also proposed for H + -ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter-

Dysregulation of copper and zinc homeostasis in the brain plays a critical role in Alzheimer disease (AD). Copper binding to amyloid-␤ peptide (A␤) is linked with the neurotoxicity of A␤and free radical damage. Metallothionein-3 (MT-3) is a small cysteine-and metal-rich protein expressed in the brain and found down-regulated in AD. This protein occurs intra-and extracellularly, and it plays an important role in the metabolism of zinc and copper. In cell cultures Zn 7 MT-3, by an unknown mechanism, protects neurons from the toxicity of A␤. We have, therefore, used a range of complementary spectroscopic and biochemical methods to characterize the interaction of Zn 7 MT-3 with free Cu 2؉ ions. We show that Zn 7 MT-3 scavenges free Cu Essential transition metals like copper and zinc play a critical role in neurobiology. The homeostasis of both metals is tightly regulated and essential for brain physiology (1). In vitro evidence suggests that in physiological conditions micromolar concentrations of zinc and copper are actively released from neurons during neurotransmission processes into pre-and postsynaptic clefts. However, at present it is not known whether the released copper is present in a chemically exchangeable form (2). Dysregulated metal metabolism (zinc and copper) occurs in neurodegenerative disorders such as Alzheimer (3, 4), Parkinson (5), and prion (6) diseases. In these diseases increased extracellular copper concentrations and free-radical production have been found. There is now direct evidence that copper is bound to amyloid-␤ peptide (A␤) 2 in senile plaque of Alzheimer disease (AD) (7). Copper is also linked with the neurotoxicity of A␤ and free radical damage (8).Because of its redox-active nature (Cu ϩ /Cu 2ϩ ), its reactivity with molecular oxygen (O 2 ) generates the reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radicals (9, 10). The redox cycling of copper requires its reduction by biological components, including ascorbate, which actively accumulates in the brain at concentrations between 0.5 and 10 mM (11). Hence, copper chelation and its redox-silencing may represent critical events in preventing the progression of neurodegenerative diseases. In recent years a metal chelation therapy has emerged as a promising tool to attenuate abnormal metal-protein interactions that lead to increased free-radical toxicity (10).The natural metal chelator metallothionein-3 (MT-3), also known as the growth inhibitory factor, is a small non-inducible cysteine-and metal-rich protein mainly expressed in the brain. In the brain its expression was found in zinc-enriched neurons. Like other mammalian metallothioneins (MTs), MT-3 binds with a high affinity essential monovalent and divalent d 10 metal ions Cu(I) and Zn(II). Recently, the interaction of Zn 7 MT-3 with the small-GTPase Rab3a, which is strictly linked to the exo-endocytotic cycle of synaptic vesicles, has been demonstrated. It has been suggested that Zn 7 MT-3 actively participates in synaptic cycle of zinc vesicles...

Metallothioneins (MTs) are a class of ubiquitously occurring low molecular mass, cysteine- and metal-rich proteins containing sulfur-based metal clusters formed with Zn(II), Cd(II), and Cu(I) ions. In mammals, four distinct MT isoforms designated MT-1 through MT-4 exist. The first discovered MT-1/MT-2 are widely expressed isoforms, whose biosynthesis is inducible by a wide range of stimuli, including metals, drugs, and inflammatory mediators. In contrast, MT-3 and MT-4 are noninducible proteins, with their expression primarily confined to the central nervous system and certain squamous epithelia, respectively. MT-1 through MT-3 have been reported to be secreted, suggesting that they may play different biological roles in the intracellular and extracellular space. Recent reports established that these isoforms play an important protective role in brain injury and metal-linked neurodegenerative diseases. In the postgenomic era, it is becoming increasingly clear that MTs fulfill multiple functions, including the involvement in zinc and copper homeostasis, protection against heavy metal toxicity, and oxidative damage. All mammalian MTs are monomeric proteins, containing two metal-thiolate clusters. In this review, after a brief summary of the historical milestones of the MT-1/MT-2 research, the recent advances in the structure, chemistry, and biological function of MT-3 and MT-4 are discussed.