Semisynthetic, site-specific ubiquitin modification of α-synuclein reveals differential effects on aggregation

Abstract

The process of neurodegeneration in Parkinson's Disease is intimately associated with the aggregation of the protein α-synuclein into toxic oligomers and fibrils. Interestingly, many of these protein aggregates are found to be post-translationally modified by ubiquitin at several different lysine residues. However, the inability to generate homogeneously ubiquitin modified α-synuclein at each site has prevented the understanding of the specific biochemical consequences. We have used protein semisynthesis to generate nine site-specifically ubiquitin modified α-synuclein derivatives and have demonstrated that different ubiquitination sites have differential effects on α-synuclein aggregation.

Figure 1

Disulfide-directed ubiquitination of α-synuclein. A) Ubiquitin was expressed in E. coli as a fusion to the GyrA intein (1). Intein-mediated thiolysis with cysteamine yielded 2 and subsequent reaction with DTNP generated the activated ubiquitin mixed disulfide (3). α-Synuclein cysteine point mutants (4) were also expressed in E. coli and were then reacted with 3 to generate the corresponding disulfide-directed ubiquitinated derivates. B) Primary sequence of α-syn with all sites of ubiquitination noted in red.

Figure 2

Characterization of ubiquitinated α-synuclein. A) Purified α-synuclein (α-Syn), ubiquitin C-terminal thiol (Ub-SH, 2), α-synuclein lysine to cysteine point mutants (K#C), and the corresponding ubiquitinated derivatives (K#C-Ub) were separated by SDS-PAGE and analyzed by Coomassie staining. B) CD spectra of the same proteins.

Figure 3

Aggregation of ubiquitinated α-synuclein. Purified α-synuclein (α-Syn) and the disulfide-directed ubiquitinated derivatives (K#C-Ub) at a concentration of 100 µM were incubated at 37 °C before analysis by ThT fluorescence (450 nm Ex/482 nm Em) at the indicated timepoints. The same protein samples at day 5 were analyzed by TEM; scale bar: 500 nm). The experiments were performed in triplicate and error bars represent standard deviation.