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A bifunctional molecule-assisted synthesis of mimics for use in probing the ubiquitination system

Nature Protocols volume 18, pages 530–554 (2023)Cite this article

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Abstract

Ubiquitination regulates almost every life process of eukaryotes. The study of the ubiquitin (Ub) coupling or decoupling process and the interaction study of Ub–Ub binding protein have always been the central focus. However, such studies are challenging, owing to the transient nature of Ub-coupling enzymes and deubiquitinases in the reactions, as well as the difficulty in preparing large quantities of polyubiquitinated samples. Here we describe a recently developed strategy for the efficient preparation of analogs of Ub chains and analogs for Ub coupling and uncoupling intermediates, which facilitate the study of the ubiquitination process. The strategy includes mainly the following steps: (i) the bifunctional molecule conjugation on the only cysteine (Cys) residue of a target protein (usually a Ub or Ub-conjugating enzyme), exposing an orthogonal reactive site for native chemical ligation; (ii) covalent ligation with a Ub-derived thioester, exposing a free sulfhydryl; and (iii) (optional) a disulfide bond formation with a substrate protein (mainly with only one Cys protein) through nonactivity-based cross-linking or with a deubiquitinase (mainly with several Cys residues) through activity-based cross-linking. When the bifunctional molecule and target proteins are obtained, the final products can be prepared in milligram quantities within 2 weeks.

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Fig. 1: Schematic overview of the CAET-assisted strategy.
Fig. 2: Synthetic route of the biofunctional molecule (CAET-Acm).
Fig. 3: Synthesis and characterization of K29-Ub chain mimics.
Fig. 4: Synthesis and characterization of Ubc2-Ub-K48Ub-Y/degron.
Fig. 5: Synthesis and characterization of Otud2-Ub-K27Ub.
Fig. 6: Identification and HDX-MS application of Otud2-Ub-K27Ub.
Fig. 7: Ub sequences and modifications.

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Data availability

The data associated with these results are included in the Supplementary Information and Source Data files.

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Acknowledgements

We thank H. Deng, X. Meng and X. Tian in the Protein Chemistry and Proteomics Facility, Tsinghua University Technology Center for Protein Research, for protein MS analysis. This work was supported by Shanghai Rising-Star Program (no. 22QA1404900), the National Key R&D Program of China (no. 2021YFC2100201) and the National Natural Science Foundation of China (nos. 22277073, 22207070, 91849129 and 22077078). Q.Z. thanks the funding by the National Facility for Translational Medicine (Shanghai).

Author information

Author notes

  1. These authors contributed equally: Qingyun Zheng, Tian Wang, Junxiong Mao.

Authors and Affiliations

  1. Institute of Translational Medicine, National Center for Translational Medicine (Shanghai), School of Chemistry and Chemical Engineering, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China

    Qingyun Zheng, Tian Wang, Lujun Liang, Chong Zuo & Man Pan

  2. Institute of Translational Medicine, School of Medicine, Shanghai University, Shanghai, China

    Qingyun Zheng, Lujun Liang, Yuanyuan Yu & Honggang Hu

  3. Tsinghua-Peking Center for Life Sciences, Department of Chemistry, Tsinghua University, Beijing, China

    Tian Wang, Junxiong Mao, Guochao Chu, Lujun Liang, Yangwode Jing & Chong Zuo

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Contributions

M.P. and H.H. led the project and supervised the project. Q.Z. and M.P. designed all experiments and prepared the manuscript. G.C., Y.Y. and H.H. synthesized the bifunctional molecule. T.W., J.M. and C.Z. synthesized the free Ub chain mimics. J.M. and L.L. synthesized the Ub coupling intermediate mimics. T.W. and Y.J. synthesized the Ub decoupling intermediate mimics. Q.Z. and T.W. performed the experiments on HDX-MS and cross-linking site identification. All authors contributed to the writing of the manuscript and approved the final version.

Corresponding authors

Correspondence to Honggang Hu or Man Pan.

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Key references using this protocol

Pan, M. et. al. Nature 600, 334–338 (2021): https://doi.org/10.1038/s41586-021-04097-8.

Zheng, Q. et al. Angew. Chem. In. Ed. 59, 13496–13501 (2020): https://doi.org/10.1002/anie.202002974.

Extended data

Extended Data Fig. 1 Intermediate products of the Acm-deprotection reaction and CAET-assisted NCL reaction for preparation of Ubc2-Ub-K48Ub-Y/degron.

a, RP-HPLC trace (214 nm) and ESI-MS of purified product 6. b, RP-HPLC trace (214 nm) and ESI-MS of purified product 8.

Extended Data Fig. 2 LC–MS characterization of Ub-K27C-CAET module and monitoring the reaction efficiency of the DUB intermediate mimic.

LC–MS characterization of Ub-K27C-CAET module and monitoring the reaction efficiency of the DUB intermediate mimic. a, RP-HPLC trace (214 nm) and ESI-MS of purified product 12. b, SDS–PAGE analysis of the activity-based cross-linking between the enzyme Otud2 and K27-CAET-AT2 diUb.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–5 and Methods 1 and 2.

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Source data

Source Data Fig. 3

Unprocessed gels for Fig. 3.

Source Data Fig. 4

Unprocessed gels for Fig. 4c.

Source Data Fig. 5

Unprocessed gels for Fig. 5e.

Source Data Extended Data Fig. 2

Unprocessed gels for Extended Data Fig. 2b.

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Zheng, Q., Wang, T., Mao, J. et al. A bifunctional molecule-assisted synthesis of mimics for use in probing the ubiquitination system. Nat Protoc 18, 530–554 (2023). https://doi.org/10.1038/s41596-022-00761-z

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