The molecular mechanism for human metallothionein-3 to protect against the neuronal cytotoxicity of Aβ1–42 with Cu ions

Aggregation and cytotoxicity of Aβ with redox-active metals in neuronal cells have been implicated in the progression of Alzheimer disease. Human metallothionein (MT) 3 is highly expressed in the normal human brain and is downregulated in Alzheimer disease. Zn7MT3 can protect against the neuronal toxicity of Aβ by preventing copper-mediated Aβ aggregation, abolishing the production of reactive oxygen species (ROS) and the related cellular toxicity. In this study, we intended to decipher the roles of single-domain proteins (α/β) and the α–β domain–domain interaction of Zn7MT3 to determine the molecular mechanism for protection against the neuronal cytotoxicity of Aβ1–42 with copper ions. With this in mind, the α and β single-domain proteins, heterozygous β(MT3)–α(MT1), and a linker-truncated mutant ∆31–34 were prepared and characterized. In the presence/absence of various Zn7MT3 proteins, the Aβ1–42–Cu2+-mediated aggregation, the production of ROS, and the cellular toxicity were investigated by transmission electron microscopy, ROS assay by means of a fluorescent probe, and SH-SY5Y cell viability, respectively. The β domain cannot abolish Aβ1–42–Cu2+-induced aggregation, and neither the β domain nor the α domain can quench the production of ROS because of the redox cycling of Aβ–Cu2+. Similarly to wild-type Zn7MT3, the heterozygous β(MT3)–α(MT1) possesses the characteristic of alleviating Aβ1–42 aggregation and oxidative stress to neuronal cells. Therefore, the two domains through the linker Lys-Lys-Ser form a cooperative unit, and each of them is indispensable in conducting its bioactivity. The α domain plays an important role in modulating the stability of the metal–thiolate cluster, and the α–β domain–domain interaction through the linker is critical for its protective role in the brain.