Cysteine-Based Mimic of Arginylation Reproduces NeuroprotectiveEffects of the Authentic Post-Translational Modification on ?-Synuclein

Arginylation is an understudied post-translational modification(PTM) involving the transfer of arginine to aspartate or glutamate sidechains ina protein. Among the targets of this PTM is alpha-synuclein (alpha S), a neuronal proteininvolved in regulating synaptic vesicles. The aggregation of alpha S is implicated inneurodegenerative diseases, particularly in Parkinson's disease, and arginylation hasbeen found to protect against this pathological process. Arginylated alpha S has beenstudied through semisynthesis involving multipart native chemical ligation (NCL),but this can be very labor-intensive with low yields. Here, we present a facile way tointroduce a mimic of the arginylation modification into a protein of interest, compatible with orthogonal installation of labels such asfluorophores. We synthesize bromoacetyl arginine and react it with recombinant, site-specific cysteine mutants of alpha S. We validate themimic by testing the vesicle binding affinity of mimic-arginylated alpha S, as well as its aggregation kinetics and monomer incorporationintofibrils, and comparing these results to those of authentically arginylated alpha S produced through NCL. In cultured neurons, wecompare thefibril seeding capabilities of preformedfibrils carrying a small percentage of arginylated alpha S. Wefind that, consistent withauthentically arginylated alpha S, mimic-arginylated alpha S does not perturb the protein's native function but alters aggregation kinetics andmonomer incorporation. Both mimic and authentically modified alpha S suppress aggregation in neuronal cells. Our results providefurther insight into the neuroprotective effects of alpha S arginylation, and our alternative strategy to generate arginylated alpha S enables thestudy of this PTM in proteins not accessible through NCL.