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

Aggregation and cytotoxicity of A beta 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. Zn(7)MT3 can protect against the neuronal toxicity of A beta by preventing copper-mediated A beta 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 (alpha/beta) and the alpha-beta domain-domain interaction of Zn(7)MT3 to determine the molecular mechanism for protection against the neuronal cytotoxicity of A beta(1-42) with copper ions. With this in mind, the alpha and beta single-domain proteins, heterozygous beta(MT3)-I +/-(MT1), and a linker-truncated mutant a dagger 31-34 were prepared and characterized. In the presence/absence of various Zn(7)MT3 proteins, the A beta(1-42)-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 beta domain cannot abolish A beta(1-42)-Cu2+-induced aggregation, and neither the beta domain nor the alpha domain can quench the production of ROS because of the redox cycling of A beta-Cu2+. Similarly to wild-type Zn(7)MT3, the heterozygous beta(MT3)-I +/-(MT1) possesses the characteristic of alleviating A beta(1-42) 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 alpha domain plays an important role in modulating the stability of the metal-thiolate cluster, and the alpha-beta domain-domain interaction through the linker is critical for its protective role in the brain.