Molecular Mechanism of Metal-Ion-Induced Protofibril Formation of Ataxin-3

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 a 28-residue polyQ tract (Q28) did not complete within 14 days under metal-free conditions. In contrast, the introduction of Cu²⁺ or Zn²⁺ markedly accelerated the process: JD denatured within one day, and protofibrils formed within four days. The acceleration was similar between JD and Q28, indicating that metal binding primarily affects JD. 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.