Molecular mechanism of metal-ion-induced protofibril formation of Ataxin-3

Abstract Ataxin-3, the causative protein of Machado-Joseph disease, comprises an enzymatic domain, Josephin domain (JD), and a polyglutamine repeat (polyQ). The polyQ expansion promotes the fibrillogenesis of ataxin-3, which is responsible for the disease onset. The fibrillogenesis proceeds through two distinct steps: protofibril formation, in which JD undergoes self-assembly, and subsequent fibrillation. Both polyQ elongation and metal ions accelerate fibrillation; however, the effects of these factors on each stage remain unclear. This study comprehensively analyzed the effects of metal ions on ataxin-3 protofibril formation using spectroscopic analyses. The protofibril formation of JD and that with a 28-residue polyQ tract (Q28) did not complete within 14 d under metal-free conditions. In contrast, the introduction of Cu²⁺ or Zn²⁺ markedly accelerated the process: JD denatured within 1 d, and protofibrils were formed within 4 d. The acceleration was similar between JD and Q28, indicating that metal binding primarily affects JD. Thiol (-SH) assays and mass spectrometry showed that Cu²⁺ binds to thiols and induces intra- and intermolecular disulfide bonds, whereas Zn²⁺ binds to thiols but does not promote disulfide formation. These observations suggest that Cu²⁺ and Zn²⁺ enhance the protofibril formation through interactions with specific JD sites, but the underlying mechanisms differ between the two metal ions.