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 res

SUBMITTER: Chanda S 

PROVIDER: S-EPMC12455564 | biostudies-literature | 2025 Sep

REPOSITORIES: biostudies-literature