- Protocol
- Published:
- Juan Luo1na1,
- Yao Yu1na1,
- Ke Wang1,
- Sizhe He ORCID: orcid.org/0009-0002-1077-26481,
- Longjie Wang ORCID: orcid.org/0009-0000-7722-62841,
- Fangfang Liang2,
- Jason W. Chin ORCID: orcid.org/0000-0003-1219-47573 &
- …
- Shan Tang ORCID: orcid.org/0000-0003-4483-11081
Nature Protocols (2024)Cite this article
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Subjects
- Biological techniques
- Chemical biology
Abstract
Catalytic mechanism-based, light-activated traps have recently been developed to identify the substrates of cysteine or serine hydrolases. These traps are hydrolase mutants whose catalytic cysteine or serine are replaced with genetically encoded 2,3-diaminopropionic acid (DAP). DAP-containing hydrolases specifically capture the transient thioester- or ester-linked acyl–enzyme intermediates resulting from the first step of the proteolytic reaction as their stable amide analogs. The trapped substrate fragments allow the downstream identification of hydrolase substrates by mass spectrometry and immunoblotting. In this protocol, we provide a detailed step-by-step guide for substrate capture and identification of the peptidase domain of the large tegument protein deneddylase (UL36USP) from human herpesvirus 1, both in mammalian cell lysate and live mammalian cells. Four procedures are included: Procedure 1, DAP-mediated substrate trapping in mammalian cell lysate (~8 d); Procedure 2, DAP-mediated substrate trapping in adherent mammalian cells (~6 d); Procedure 3, DAP-mediated substrate trapping in suspension mammalian cells (~5 d); and Procedure 4, substrate identification and validation (~12–13 d). Basic skills to perform protein expression in bacteria or mammalian cells, affinity enrichment and proteomic analysis are required to implement the protocol. This protocol will be a practical guide for identifying substrates of serine or cysteine hydrolases either in a complex mixture, where genetic manipulation is challenging, or in live cells such as bacteria, yeasts and mammalian cells.
Key points
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The method facilitates the discovery of hydrolase substrates in live mammalian cells. In response to a light pulse, a genetically encoded, 2,3-diaminopropionic acid-containing protease stably traps otherwise transient acyl–enzyme intermediates, allowing their unequivocal identification by mass spectrometry.
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The 2,3-diaminopropionic acid-containing enzymes preserve substrate specificity, allowing accurate substrate identification from multiple subcellular compartments. Furthermore, the acyl–enzyme conjugates can be isolated to obtain crystal structures at high resolution.
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Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the accession number PXD046516. All other datasets and materials generated or analyzed in this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank L. Liu (Tsinghua University) and J.-S. Zheng (University of Science and Technology of China) for their helpful discussion and kind support. We thank the Experiment Center for Life Sciences of University of Science and Technology of China for LC–MS/MS characterization. This work was partially carried out at the Instruments Center for Physical Science, University of Science and Technology of China. This work is supported by the National Key Research and Development Program of China (2023YFA0916000), USTC Research Funds of the Double First-Class Initiative (YD9100002019) and Center for Advanced Interdisciplinary Science and Biomedicine of Institute of Health and Medicine (IHM) (QYPY20220013).
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Author notes
These authors contributed equally: Juan Luo, Yao Yu.
Authors and Affiliations
Department of Oncology, The First Affiliated Hospital of USTC, Key Laboratory of Immune Response and Immunotherapy, Centre for Advanced Interdisciplinary Science and Biomedicine of IHM, MOE Key Laboratory for Membraneless Organelles & Cellular Dynamics, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China
Juan Luo,Yao Yu,Ke Wang,Sizhe He,Longjie Wang&Shan Tang
Department of Medical Oncology, Guangxi Medical University First Affiliated Hospital, Nanning, Guangxi Zhuang Autonomous Region, China
Fangfang Liang
Medical Research Council Laboratory of Molecular Biology, Cambridge, UK
Jason W. Chin
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Contributions
S.T. and J.W.C. set the overall direction of the research. J.L., Y.Y. and S.T. wrote the manuscript. J.L. designed and conducted all experiments of substrate trapping and identification from HEK293T and Expi293 cells. Y.Y. carried out the recombinant expression and characterization of UL36USP variants and performed the experiments in mammalian cell lysate. K.W. set up the equipment for photoactivation. L.W. and Y.Y. analyzed the mass data. S.H. and F.L. helped analyze data and shape the manuscript.
Corresponding author
Correspondence to Shan Tang.
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Nature Protocols thanks Yoheir Katsuyama and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Related links
Key references using this protocol
Tang, S. et al. Nature 602, 701–707 (2022): https://doi.org/10.1038/s41586-022-04414-9
Eiamthong, B. et al. Angew. Chem. Int. Ed. 61, e202203061 (2022): https://doi.org/10.1002/anie.202203061
Huguenin-Dezot, N. et al. Nature 565, 112–117 (2019): https://doi.org/10.1038/s41586-018-0781-z
Supplementary information
Supplementary Information
Supplementary Figs. 1–4 and Table 1.
Supplementary Data
Uncropped SDS–PAGE for Supplementary Fig. 1 and uncropped western blot gels for Supplementary Figs. 2 and 3.
Source data
Source Data Fig. 1
Statistical source data.
Source Data Figs. 2 and 4–6
Uncropped western blot gels for Figs. 2, 4 and 6 and uncropped SDS–PAGE for Fig. 6.
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Luo, J., Yu, Y., Wang, K. et al. Capturing acyl–enzyme intermediates with genetically encoded 2,3-diaminopropionic acid for hydrolase substrate identification. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01006-x
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DOI: https://doi.org/10.1038/s41596-024-01006-x
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Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acid
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