2023/10/12 by Lennart Brewitz, H. T. Henry Chan, Petra Lukacik +4 · 1 voice · 2 citations
Medicine · Biochemistry, Genetics and Molecular Biology · Immunology and Microbiology · #Peptidase Inhibition and Analysis #Ubiquitin and proteasome pathways #interferon and immune responses
paper · doi:10.1016/j.bmc.2023.117498
openalex publication_date 2023/10/12 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/28
The SARS-CoV-2 papain-like protease (PLpro) and main protease (Mpro) are nucleophilic cysteine enzymes that catalyze the hydrolysis of the viral polyproteins pp1a/1ab. By contrast with Mpro, PLpro is also a deubiquitinase (DUB) that accepts post-translationally modified human proteins as substrates. Here we report studies on the DUB activity of PLpro using synthetic Nε-lysine-branched oligopeptides as substrates that mimic post-translational protein modifications by ubiquitin (Ub) or Ub-like modifiers (UBLs), such as interferon stimulated gene 15 (ISG15). Mass spectrometry (MS)-based assays confirm the DUB activity of isolated recombinant PLpro. They reveal that the sequence of both the peptide fragment derived from the post-translationally modified protein and that derived from the UBL affects PLpro catalysis; the nature of substrate binding in the S sites appears to be more important for catalytic efficiency than binding in the S′ sites. Importantly, the results reflect the reported cellular substrate selectivity of PLpro, i.e. human proteins conjugated to ISG15 are better substrates than those conjugated to Ub or other UBLs. The combined experimental and modelling results imply that PLpro catalysis is affected not only by the identity of the substrate residues binding in the S and S′ sites, but also by the substrate fold and the conformational dynamics of the blocking loop 2 of the PLpro:substrate complex. Nε-Lysine-branched oligopeptides thus have potential to help the identification of PLpro substrates. More generally, the results imply that MS-based assays with Nε-lysine-branched oligopeptides have potential to monitor catalysis by human DUBs and hence to inform on their substrate preferences.