Probing the effects of N-terminal acetylation on -synuclein structure, aggregation and cytotoxicity

Parkinson's disease is associated with the aberrant aggregation of alpha-synuclein within brain cells. Although the causes of this process are still unclear, post-translational modifications of alpha-synuclein are likely to play a modulatory role. Since alpha-synuclein is constitutively N-terminally acetylated, we previously investigated how this protein modification affects the aggregation behavior of the protein using a variety of methods in vitro and in cell systems. This chapter describes the production of N-terminally acetylated (NTA) alpha-synuclein, the preparation of different seeds of NTA alpha-synuclein for aggregation assays and the experimental methods for the kinetic analysis of the aggregation process of NTA alpha-synuclein. We also detail our protocol to evaluate the effects of preformed protofibrils of NTA alpha-synuclein in cell-based assays. These methods can be applied to study other post-translational modifications of alpha-synuclein, or adapted for the study of N-acetylation of other aggregation-prone proteins.