Gabriele Meloni

Metal swap between Zn7-metallothionein-3 and amyloid-|[beta]||[ndash]|Cu protects against amyloid-|[beta]| toxicity

Aberrant interactions of copper and zinc ions with the amyloid-beta peptide (Abeta) potentiate Alzheimers disease (AD) by participating in the aggregation process of Abeta and in the generation of reactive oxygen species (ROS). The ROS production and the neurotoxicity of Abeta are associated with copper binding. Metallothionein-3 (Zn(7)MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain and downregulated in AD. This protein protects, by an unknown mechanism, cultured neurons from the toxicity of Abeta. Here, we show that a metal swap between Zn(7)MT-3 and soluble and aggregated Abeta(1-40)-Cu(II) abolishes the ROS production and the related cellular toxicity. In this process, copper is reduced by the protein thiolates forming Cu(I)(4)Zn(4)MT-3, in which an air-stable Cu(I)(4)-thiolate cluster and two disulfide bonds are present. The discovered protective effect of Zn(7)MT-3 from the copper-mediated Abeta(1-40) toxicity may lead to new therapeutic strategies for treating AD.

Redox Silencing of Copper in Metal-linked Neurodegenerative Disorders REACTION OF Zn7METALLOTHIONEIN-3 WITH Cu2+ IONS

Dysregulation of copper and zinc homeostasis in the brain plays a critical role in Alzheimer disease (AD). Copper binding to amyloid-β peptide (Aβ) is linked with the neurotoxicity of Aβ 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 Zn7MT-3, by an unknown mechanism, protects neurons from the toxicity of Aβ. We have, therefore, used a range of complementary spectroscopic and biochemical methods to characterize the interaction of Zn7MT-3 with free Cu2+ ions. We show that Zn7MT-3 scavenges free Cu2+ ions through their reduction to Cu+ and binding to the protein. In this reaction thiolate ligands are oxidized to disulfides concomitant with Zn2+ release. The binding of the first four Cu2+ is cooperative forming a Cu(I)4-thiolate cluster in the N-terminal domain of Cu4,Zn4MT-3 together with two disulfides bonds. The Cu4-thiolate cluster exhibits an unusual stability toward air oxygen. The results of UV-visible, CD, and Cu(I) phosphorescence at 77 K suggest the existence of metal-metal interactions in this cluster. We have demonstrated that Zn7MT-3 in the presence of ascorbate completely quenches the copper-catalyzed hydroxyl radical (OH·) production. Thus, zinc-thiolate clusters in Zn7MT-3 can efficiently silence the redox-active free Cu2+ ions. The biological implication of our studies as to the protective role of Zn7MT-3 from the Cu2+ toxicity in AD and other neurodegenerative disorders is discussed.

Organization and assembly of metal-thiolate clusters in epithelium-specific metallothionein-4.

Mammalian metallothionein-4 (MT-4) was found to be specifically expressed in stratified squamous epithelia where it plays an essential but poorly defined role in regulating zinc or copper metabolism. Here we report on the organization, stability, and the pathway of metal-thiolate cluster assembly in MT-4 reconstituted with Cd2+ and Co2+ ions. Both the 113Cd NMR studies of 113Cd7MT-4 and the spectroscopic characterization of Co7MT-4 showed that, similar to the classical MT-1 and MT-2 proteins, metal ions are organized in two independent Cd4Cys11 and Cd3Cys9 clusters with each metal ion tetrahedrally coordinated by terminal and bridging cysteine ligands. Moreover, we have demonstrated that the cluster formation in Cd7MT-4 is cooperative and sequential, with the Cd4Cys11 cluster being formed first, and that a distinct single-metal nucleation intermediate Cd1MT-4 is required in the cluster formation process. Conversely, the absorption and circular dichroism features of metal-thiolate clusters in Cd7MT-4 indicate that marked differences in the cluster geometry exist when compared with those in Cd7MT-1/2. The biological implication of our studies as to the role of MT-4 in zinc metabolism of stratified epithelia is discussed.

Redox activity of α-synuclein–Cu is silenced by Zn7-metallothionein-3

The aggregation of α-synuclein (α-Syn), the major component of intracellular Lewy body inclusions in dopaminergic neurons of the substantia nigra, plays a critical role in the etiology of Parkinson disease (PD). Long-term effects of redox-active transition metals (Cu, Fe) and oxidative chemical imbalance underlie the disease progression and neuronal death. In this work, we provide evidence that a brain metalloprotein, Zn₇-metallothionein-3 (Zn₇MT-3), possesses a dynamic role in controlling aberrant protein-copper interactions in PD. We examined the properties of the α-Syn-Cu(II) complex with regard to molecular oxygen, the biological reducing agent ascorbate, and the neurotransmitter dopamine. The results revealed that under aerobic conditions α-Syn-Cu(II) possesses catalytic oxidase activity. The observed metal-centered redox chemistry significantly promotes the production of hydroxyl radicals and α-Syn oxidation and oligomerization, processes considered critical for cellular toxicity. Moreover, we show that Zn₇MT-3, through Cu(II) removal from the α-Syn-Cu(II) complex, efficiently prevents its deleterious redox activity. We demonstrate that the Cu(II) reduction by thiolate ligands of Zn₇MT-3 and the formation of Cu(I)₄Zn₄MT-3, in which an unusual oxygen-stable Cu(I)₄-thiolate cluster is present, comprise the underlying molecular mechanism by which α-Syn and dopamine oxidation, α-Syn oligomerization, and ROS production are abolished. These studies provide new insights into the bioinorganic chemistry of PD.

Effects of Zn2+, Ca2+, and Mg2+ on the Structure of Zn7Metallothionein-3: Evidence for an Additional Zinc Binding Site

Human metallothionein-3 (Zn(7)MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain, where it plays an important role in the homeostasis of the essential metal ions Cu(+) and Zn(2+). Like other mammalian metallothioneins (MT-1 and -2), the protein contains a M(II)(3)(CysS)(9) and a M(II)(4)(CysS)(11) cluster localized in two independent protein domains linked by a flexible hinge region. However, there is a substantially increased number of acidic residues in MT-3 (11 residues) compared with MT-2 (four residues) which may act as binding ligands for additional metal ions. In this study, the binding of Zn(2+), Ca(2+), and Mg(2+) to human Zn(7)MT-3 and its mutant lacking an acidic hexapeptide insert, Zn(7)MT-3(Delta55-60), was investigated and compared with the binding of Zn(7)MT-2. By using spectroscopic and spectrometric techniques, we demonstrate that one additional Zn(2+) binds with an apparent binding constant (K(app)) of approximately 100 microM to Zn(7)MT-3 and Zn(7)MT-3(Delta55-60), but not to Zn(7)MT-2. The changes in spectroscopic features of metal-thiolate clusters and gel filtration behavior reveal that the formation of Zn(8)MT-3 is immediate and is accompanied by a decrease in the Stokes radius (R(s)). The changes in the R(s) suggest a mutual approach of both protein domains. The fast binding of Zn(2+) is followed by a slow time-dependent protein dimerization. The binding of Zn(2+) to Zn(7)MT-3 is specific as in the presence of Ca(2+) and Mg(2+) only an alteration of the R(s) of Zn(7)MT-3 at substantially higher concentrations was observed. The significance of these findings for the biological role of MT-3 is discussed.

Structure and Function of Cu(I)- and Zn(II)-ATPases

Copper and zinc are micronutrients essential for the function of many enzymes while also being toxic at elevated concentrations. Cu(I)- and Zn(II)-transporting P-type ATPases of subclass 1B are of key importance for the homeostasis of these transition metals, allowing ion transport across cellular membranes at the expense of ATP. Recent biochemical studies and crystal structures have significantly improved our understanding of the transport mechanisms of these proteins, but many details about their structure and function remain elusive. Here we compare the Cu(I)- and Zn(II)-ATPases, scrutinizing the molecular differences that allow transport of these two distinct metal types, and discuss possible future directions of research in the field.

Mammalian Metallothionein-3: New Functional and Structural Insights

Metallothionein-3 (MT-3), a member of the mammalian metallothionein (MT) family, is mainly expressed in the central nervous system (CNS). MT-3 possesses a unique neuronal growth inhibitory activity, and the levels of this intra- and extracellularly occurring metalloprotein are markedly diminished in the brain of patients affected by a number of metal-linked neurodegenerative disorders, including Alzheimer’s disease (AD). In these pathologies, the redox cycling of copper, accompanied by the production of reactive oxygen species (ROS), plays a key role in the neuronal toxicity. Although MT-3 shares the metal-thiolate clusters with the well-characterized MT-1 and MT-2, it shows distinct biological, structural and chemical properties. Owing to its anti-oxidant properties and modulator function not only for Zn, but also for Cu in the extra- and intracellular space, MT-3, but not MT-1/MT-2, protects neuronal cells from the toxicity of various Cu(II)-bound amyloids. In recent years, the roles of zinc dynamics and MT-3 function in neurodegeneration are slowly emerging. This short review focuses on the recent developments regarding the chemistry and biology of MT-3.

11:Metallothionein-3, Zinc, and Copper in the Central Nervous System

Metallothionein-3 (MT-3), also known as the neuronal growth inhibitory factor, has been discovered by Uchida and coworkers in 1991 in their search for a cellular component responsible for antagonizing aberrant neuritic sprouting and increased survival of cultured neurons stimulated by Alzheimers disease (AD) brain extract. Since this initial discovery further studies showed that MT-3 possesses peculiar structural and functional properties not shared by other members of the mammalian MT family. Several lines of evidence suggest that the metal-binding protein MT-3 plays a vital role in zinc and copper homeostasis in the brain. Although far from being understood, the unusual structural properties of MT-3 are responsible for its neuronal growth inhibitory activity, involvement in trafficking of zinc vesicles in the central nervous system, protection against copper-mediated toxicity in AD and in controlling abnormal metal-protein interactions in other neurodegenerative disorders.