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.
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Acknowledgements
This work was supported by the National Key Research & Development Program of China (2021YFA1200104, 2022YFC3401500), the National Natural Science Foundation of China (21621003, 22137005, 21971133, 22027807, 22034004, 92253302, 22227810), and the Tsinghua University Spring Breeze Fund (2020Z99CFY043, 2021Z99CFZ002). We thank Xiaolin Tian, Dingfei Yan, and Prof. Haiteng Deng in the MOE Key Laboratory of Bioinformatics of Tsinghua University for sample analysis. We thank Maoshen Sun for his help with the measurement of Michaelis constant.
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Supporting information The supporting information is available online at http://chem.scichina.com and http://link.springer.com/journal/11426. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.
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Li, C., Wang, T., Liang, L. et al. Simultaneous capture of ISG15 conjugating and deconjugating enzymes using a semi-synthetic ISG15-Dha probe. Sci. China Chem. 66, 837–844 (2023). https://doi.org/10.1007/s11426-022-1455-x
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DOI: https://doi.org/10.1007/s11426-022-1455-x