Fibril-forming motif of non-expanded ataxin-3 revealed by scanning proline mutagenesis

Aims: The misfolding of ataxin-3 in neurons is the hallmark of a neurodegenerative disease, spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD). Ataxin-3 consists of a N-terminal Josephin domain and a C-terminal polyglutamine (polyQ) tract. The length of the polyQ tract is positively correlated with the disease. The aggregation of ataxin-3 in vitro is a two-step process, with the first step involving the aggregation of the Josephin domain and the second step involving an expanded polyQ tract. However, the fibril-forming motif of the Josephin domain is not well understood. Methods: In this study, we employed 3D profile algorithm and scanning proline mutagenesis to identify the fibril-forming motif of non-expanded ataxin-3. Results: By using thioflavin T fluorescence kinetics, sarkosyl-insoluble SDS-PAGE, transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR), we identified the fibril-forming motif of the Josephin domain of non-expanded ataxin-3 as 79VISNAL84. Conclusions: We demonstrated that the proline mutation in the fibril-forming motif of the Josephin domain could inhibit the aggregation of expanded ataxin-3, which shows some therapeutic promise.