As we age, proteasome function decreases, resulting in the accumulation of intrinsically disordered proteins (IDPs), which has been directly linked to many neurodegenerative diseases. This highlights the need to develop methods to prevent or halt the dysregulation or accumulation of disordered proteins. Enhancement of the proteolytic activity of the proteasome offers a promising method to prevent IDP accumulation. However, methods to evaluate and probe this activity remain limited. Furthermore, the available activity assays are not easily modified to meet high through-put screening demands. To meet the demands of a high-throughput screen, we describe the development of a cell-based AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay) designed to measure the rate of proteolytic degradation of a well-known IDP, ornithine decarboxylase (ODC), that is tagged with a green fluorescent protein (GFPSpark) on its N-terminal. GFPspark folds much quicker than other GFP counterparts while maintaining the stable characteristic β-barrels. Its high degree of stability proves to be a challenge for even the 19S cap, of the 26S proteasome complex, to unfold. ODC, on the other hand, is a homodimeric enzyme that plays a key role in the biosynthesis of polyamines. In its monomeric form, ODC is inactive, intrinsically disordered, and can be degraded by the 20S proteasome. The opposing structural characteristics of GFPspark and ODC, make their conjugated protein combination, an ideal substrate for the evaluation of 20S proteasome activation by small molecules. The use of the cell-based AlphaLISA enabled us to screen the NIH Clinical Library and the Prestwick library, and we identified Erlotinib as a new 20S proteasome enhancer.

Development of a cell-based AlphaLISA assay and utilization in a high-throughput screen for small molecule proteasome enhancers

Fall 2022 abstracts, posters, and presentations will launch in mid-September.

ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.