Chemoenzymatic Semisynthesis of Phosphorylated ?-Synuclein Enables Identification of a Bidirectional Effect on Fibril Formation.

ABSTRACT: Post-translational modifications (PTMs) impact the pathological aggregation of ?-synuclein (?S), a hallmark of Parkinson's disease (PD). Here, we synthesize ?S phosphorylated at tyrosine 39 (pY₃₉) through a novel route using in vitro enzymatic phosphorylation of a fragment followed by ligation to form the full-length protein. We can execute this synthesis in combination with unnatural amino acid mutagenesis to include two fluorescent labels for Förster resonance energy transfer (FRET) studies. We determine the effect of pY₃₉ on the aggregation of ?S and compare our authentically phosphorylated material to the corresponding glutamate 39 "phosphomimetic." Intriguingly, we find that ?S-pY₃₉ can either accelerate or decelerate aggregation, depending on the fraction of phosphorylated protein. The ?S-E₃₉ mutant can qualitatively reproduce some, but not all, of these effects. FRET measurements and analysis of existing structures of ?S help to provide an explanation for this phenomenon. Our results have important implications for the treatment of PD patients with tyrosine kinase inhibitors and highlight the importance of validating phosphomimetics through studies of authentic PTMs.

SUBMITTER: Pan B 

PROVIDER: S-EPMC7724256 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature