Synthesis of a Bis-thio-acetone (BTA) Analogue of the Lysine Isopeptide Bond and its Application to Investigate the Effects of Ubiquitination and SUMOylation on α-Synuclein Aggregation and Toxicity

The reversible modification of protein by the small protein ubiquitin and other ubiquitin-like modifiers plays important roles in virtually every key biological process in eukaryotic cells. The establishment of a range of chemical methods for the preparation of ubiquitinated proteins has enabled the site-specific interrogation of the consequences of these modifications. However, many of these techniques require significant levels of synthetic expertise, somewhat limiting their widespread application by the biological community. To overcome this issue, the creation of structural analogues of the ubiquitin-protein linkage that can be readily prepared with commercially available reagents and buffers is an important goal. Here we present the development of conditions for the facile synthesis of bis-thio-acetone (BTA) linkages of ubiquitinated proteins in high yields. Additionally, we apply this technique to the preparation of the aggregation prone protein α-synuclein bearing either ubiquitin or the small ubiquitin-like modifier (SUMO). With these proteins, we demonstrate that the BTA linkage recapitulates the previously published effects of either of these proteins on α-synuclein, suggesting that it is a good structural mimic. Notably, the BTA linkage is chemically and enzymatically stable, enabling us to study the consequences of site-specific ubiquitination and SUMOylation on the toxicity of α-synuclein in cell culture, which revealed modification and site-specific differences.