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

Abstract

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.

Modulation of the Interactions Between α-Synuclein and Lipid Membranes by Post-translational Modifications

“…In a recent study, mono-ubiquitin was synthetically added to α-synuclein via a BTA linkage, to allow ubiquitination to remain in reducing environments (156). This approach revealed that ubiquitination at K6, K23, K43, and K96 had no effect on monomeric α-synuclein change to secondary structure via CD, but all reduced amyloid aggregation.…”

Development and application of ubiquitin-based chemical probes

“…Matthew et al proposed the use of 1,3-dibromoacetone in a stepwise crosslinking, which generates a single crosslinked product with a high yield. 90 3.3.3 Azide-alkyne cycloaddition. Cu-catalysed azidealkyne cycloaddition (CuAAC) reactions can be used to construct isopeptide bond mimics.…”

Chemical Protein Ubiquitylation with Preservation of the Native Cysteine Residues

“…Different methods for generating free ubiquitin chains have been developed as previously reviewed . Enzymatic and chemical methods have also been reported to produce monoubiquitylated and polyubiquitylated proteins. Among them, disulfide‐bond‐mediated ubiquitin ligation has been successfully used to produce monoubiquitylated H2B, PCNA, α‐synuclein, and Ras, as well as polyubiquitylated α‐globin and PCNA .…”