Deciphering the role of phosphorylation in tau fibril formation using genetic code expansion

Aggregation of the protein tau is a hallmark of a group of neurodegenerative diseases collectively termed tauopathies. These aggregates are found to be hyperphosphorylated, suggesting a connection between phosphorylation and aggregation, but the relationship between the two remains unclear.

We hypothesize that phosphorylation of the intrinsically disordered fuzzy coat limits conformational flexibility, increasing the probability of transitioning into otherwise low probability states that lead to fibrillization. This is evinced by previous findings showing that tau’s propensity for aggregation increases with increasing truncation of the fuzzy coat as long as the fibril core is preserved, and that the introduction of one or two pseudophosphorylated residues (aspartic acid or glutamic acid) is sufficient to induce self-assembly of full-length tau in the absence of non-ionic cofactors.

Pseudophosphorylation fails to recapitulate the full -2 charge and steric bulk of true phosphorylation. Instead of pseudophosphorylation, we utilize genetic code expansion (GCE) to encode phosphoserine at specific phosphosites; we aim to produce tau with disease-relevant phosphorylation patterns to better understand pathological fibril formation.

Herein, we produce tau with phosphoserine substituted tau at disease-relevant phosphosites and screen for fibrillization using a high-throughput plate reader assay.