Simultaneous capture of ISG15 conjugating and deconjugating enzymes using a semi-synthetic ISG15-Dha probe

Chuntong Li; Tian Wang; Lujun Liang; Guochao Chu; Jiachen Zhang; Wei He; Lei Liu; Jinghong Li

doi:10.1007/s11426-022-1455-x

Simultaneous capture of ISG15 conjugating and deconjugating enzymes using a semi-synthetic ISG15-Dha probe

Sci China Chem. 2023;66(3):837-844. doi: 10.1007/s11426-022-1455-x. Epub 2023 Jan 11.

Authors

Chuntong Li^{1

2}, Tian Wang¹, Lujun Liang¹, Guochao Chu¹, Jiachen Zhang^{1

3}, Wei He³, Lei Liu^{1

2}, Jinghong Li^{1

2}

Affiliations

¹ Department 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.
² Center for BioAnalytical Chemistry, Hefei National Laboratory of Physical Science at Microscale, University of Science and Technology of China, Hefei, 230026 China.
³ School of Pharmaceutical Sciences, 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.

Keywords: ISG15-Dha probe; ISGylation; activity based probe; proteomics; semi-synthesis.