SpyTag/SpyCatcher-mediated protein ubiquitination to investigate 20S and 26S proteasomal degradation

Abstract

Ubiquitination significantly influences human health and disease because it plays an essential role in many cellular signaling pathways. To investigate the effects of different types of ubiquitination, various strategies based on synthesis, semisynthesis, or expression have been developed for protein ubiquitination. Here, we introduce a new method for protein ubiquitination using the SpyTag/SpyCatcher system. By combining protein expression with chemical synthesis, we created enhanced green fluorescent protein (eGFP) with ubiquitin chains consisting of 1-4 units linked through Lys48. This allowed us to study how different ubiquitin chains affect proteasomal degradation by the 26S and 20S proteasomes. While the 26S proteasome only trimmed the ubiquitin chain, the 20S proteasome degraded the different ubiquitin variants, highlighting the flexibility of the 20S proteasome in degrading complex ubiquitinated proteins.