The role of specific phosphorylation patterns in the oligomerization of Tau-R4

Specific phosphorylation patterns control the activity of multiphosphorylated proteins. In case of the Tau protein, multiphosphorylation leads to the formation of different disease-related condensates and aggregates. Studying the role of these specific patterns at the protein level is crucial for understanding the molecular mechanisms of Tauopathies such as Alzheimers Disease. However, due to the extreme difficulty in obtaining recombinant proteins with specific phosphorylation patterns using kinase-based methods, it is practically impossible to study the connection between specific phosphorylation patterns and aggregation events at the protein level. Here we addressed this problem by reducing the system to the peptide level and studying the effect of specific phosphorylation patterns on the condensation and aggregation of a specific domain of Tau, R4 (residues 336-358). To achieve this aim, we have applied advanced methods to synthesize a library of multiphosphorylated peptides derived from R4. We showed that specific phosphorylation patterns stringently control the formation of Tau aggregates and condensates. Phosphorylation of Ser341 promoted aggregation of R4 while phosphorylation of Ser352 promoted its condensation. Interestingly, Ser356 phosphorylation inhibited both processes, which can be overridden by double-phosphorylation at Ser341/Ser352. Differences between the microenvironments of the phosphorylated residues lead to their different effects on R4 aggregation upon phosphorylation. Our results show that working at the domain level using advanced peptide synthesis methods is a highly useful and practical way to provide valuable information about the effects of post translational modifications on protein activity.