A Simple, Quick, and Scalable Route to Fluorogenic Ubiquitin and Ubiquitin-Like Protein Substrates for Assessing Activities of Deubiquitinases and Ubiquitin-Like Protein-Specific Proteases

Abstract

Ubiquitin (Ub) and ubiquitin-like proteins (UBLs) regulate essential cellular processes as protein modifiers. While Ub signaling is well studied, many UBL pathways remain poorly defined, partly due to the limited availability of suitable UBL substrates. Here, we report the synthesis of fluorogenic Ub-ACA and UBL-ACA probes using the activated cysteine-based protein ligation (ACPL) technique to conjugate recombinant Ub and UBLs containing a C-terminal Gly to Cys mutation with glycyl-2-(7-amino-2-oxo-2H-chromen-4-yl)acetic acid (Gly-ACA), a water-soluble fluorophore. This one-step strategy that allows replacing Cys with Gly-ACA enables simple, quick, and scalable synthesis of Ub-ACA and 11 UBL-ACAs. Five UBL-ACAs represent the first reported fluorogenic substrates for their respective UBLs. Afforded Ub-ACA and 10 UBL-ACAs were demonstrated to be active toward a panel of DUBs or UBL-specific proteases. Notably, SUMO4-ACA was cleaved by SENP1 with efficiency comparable to the other three SUMO-ACA probes despite SUMO4’s distinct structure compared to the other three SUMOs. In human cell lysates, all 12 probes are efficiently cleaved. URM1 has no known proteases. Our results indicate that URM1-specific protease(s) exist in human cells and are yet to be identified. Given their simple and scalable synthesis, these new fluorogenic Ub-ACA and UBL-ACA substrates are highly versatile tools for studying Ub and UBL pathways and drug discovery research.