Examining the Role of Threonine Phosphorylation in Ubiquitin’s Function Using Chemical Protein Synthesis
The phosphorylation of ubiquitin significantly enhances the complexity of the ubiquitin code. However, the molecular consequences of ubiquitin phosphorylation at threonine residues remain largely uncharacterized. In this study, we present an effective method for the total chemical synthesis of threonine-phosphorylated ubiquitin, producing tens of milligrams of all six in vivo-identified threonine-phosphorylated ubiquitin analogues: pUbT7, pUbT12, pUbT14, pUbT22, pUbT55, and pUbT66. The biochemical activities of phosphorylated ubiquitin analogues were examined in vitro. Our results show that threonine phosphorylation has a differential impact on E2 charging, with phosphorylation at residue Thr7 exhibiting significant inhibition. In addition, threonine phosphorylation significantly affects the E1-E2-E3-mediated assembly and deubiquitinase-mediated disassembly of polyubiquitin chains in a site-specific manner. Collectively, this work provides new insights into the effect of phosphorylation on the ubiquitin code.
Authors
- Jing Shi (ORCID: https://orcid.org/0000-0003-0180-4265)
- Chuntong Li (ORCID: https://orcid.org/0000-0002-9360-3489)
- Lujun Liang (ORCID: https://orcid.org/0000-0001-7199-8777)
- Ligong Yuan (ORCID: https://orcid.org/0000-0002-8903-1967)
- Maoshen Sun (ORCID: https://orcid.org/0000-0002-8602-4817)
- L. Y. Shi (ORCID: https://orcid.org/0000-0003-1331-5477)
- Yingyue Zhang (ORCID: https://orcid.org/0000-0002-2056-2946)
- Feng Zhao (ORCID: https://orcid.org/0000-0001-6767-8105)
- Yijie Liu (ORCID: https://orcid.org/0000-0002-8149-2080)
- Shuangqing Sun (ORCID: https://orcid.org/0000-0002-4227-4857)
- Li Xu (ORCID: https://orcid.org/0000-0001-6750-9539)
- B J Wang
Institutions
- University of Science and Technology of China (CN)
- Howard Hughes Medical Institute (US)
- Harvard University (US)
- Bioanalytica (Switzerland) (CH)
- Institute of Bioorganic Chemistry (BY)
- Tsinghua University (CN)
Publication Details
- Journal
- JACS Au
- Published
- 2025-04-22
- DOI
- https://doi.org/10.1021/jacsau.5c00067
- Citations
- 3
- Primary Topic
- Ubiquitin and proteasome pathways
- Type
- article
- Field-Weighted Citation Impact
- 2.42
Funders
- National Natural Science Foundation of China
- Scientific Research Foundation of Education Department of Anhui Province of China