The Function Of Fibril-Forming Motifs In Tau Protein And The Location Of Fibril-Forming Motif In Ataxin-3

Protein misfolding and aggregating into amyloid fibrils are the pathological hallmarks of protein misfolding diseases including neurodegenerative diseases. Different amyloidogenic proteins are associated with different protein misfolding diseases. The structure of amyloid includes the fibril core and the outside domain. The sequence in the fibril core is called fibril-forming motif.The aggregation of microtuble associated protein Tau in neurons is the hallmark of Alzheimer disease. Studies have shown that 275VQIINK280(PHF6*) and 306VQIVYK311(PHF6) are the fibril-forming motifs of Tau protein. However, the relationship between the fibril-forming motifs and the fibrillizationof Tau protein is not well understood. In this study, we have constructed the deletion mutation of its fibril-forming motifs and the insertion mutations of fibril-forming motifs from other amyloidogenic proteins. As evidenced by thioflavin T(ThT) fluorescence kinetics, transmission electron microscopy, and circular dichroism, insertion of fibril-forming motifs from other amyloidogenic proteins could replace PHF6*/PHF6, driving Tau to form fibrils with different kinetic parameters and different morphologies. The cross-seeding assays have indicated that the seeding efficiency is the highest when the fibril-forming motif of the seed is the same as that of the monomer. Our results suggest that fibril-forming motifs play a key role in fibrillization kinetics, fibril morphology, and fibril seeding of amyloidogenic proteins.The aggregation of Ataxin-3 in neurons is the hallmark of spinocerebellar ataxia type 3(SCA3), also called Machado-Joseph disease(MJD). Ataxin-3 consists of a folded N-terminal Josephin domain and a C-terminal polyglutamine(polyQ) tract. The length of polyQ tract is positive correlation with the disease. Studies have shown that the aggregation of Ataxin-3 in vitro was a two-step process. The first step is the aggregation of Josephin domain, and the second step is the aggregation of expanded polyQ tract. In this study, we have investigated the length thresholdof the C-terminal polyQ tract and the fibril-forming motif of Josephin domain,respectively. We have constructed Ataxin-3221-361 with different length of polyQ tract, and found that 6Q and 22Q do not aggregate in vitro but 34Q and 46Q do aggregate. We have employed 3D-profile algorithm and scanning proline mutagenesis to identify the fibril-forming motif of non-expanded Ataxin-3. By using ThT fluorescence kinetics, sarkosyl insoluble SDS-PAGE, transmission electron microscopy, circular dichroism, and fourier transform infrared spectroscopy, we have identified the fibril-forming motif of Josephin domain as VISNAL. We have also demonstrated that the proline mutation in fibril-forming motif of Josephin domain can inhibit the aggregation of expanded Ataxin-3.