Simultaneous capture of ISG15 conjugating and deconjugating enzymes using a semi-synthetic ISG15-Dha probe
Chuntong Li1,2, Tian Wang1, Lujun Liang1
1Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Center for Synthetic and Systems Biology, State Key Laboratory of Chemical Oncogenomics (Shenzhen), Tsinghua University, Beijing, 100084 China.
Abstract:
ISG15 is a ubiquitin-like (Ubl) protein attached to substrate proteins by ISG15 conjugating enzymes whose dysregulation is implicated in a multitude of disease processes, but the probing of these enzymes remains to be accomplished. Here, we describe the development of a new activity-based probe ISG15-Dha (dehydroalanine) through protein semi-synthesis. In vitro cross-linking and cell lysate proteomic profiling experiments showed that this probe can sequentially capture ISG15 conjugating enzymes including E1 enzyme UBA7, E2 enzyme UBE2L6, E3 enzyme HERC5, the previously known ISG15 deconjugating enzyme (USP18), as well as some other enzymes (USP5 and USP14) which we additionally confirmed to impart deISGylation activity. Collectively, ISG15-Dha provides a new tool that can simultaneously capture ISG15 conjugating and deconjugating enzymes for biochemical or pharmacological studies.
Electronic Supplementary Material:
Supplementary material is available for this article at 10.1007/s11426-022-1455-x and is accessible for authorized users.
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