Target protein identification in live cells and organisms with a non-diffusive proximity tagging system
Identifying target proteins for bioactive molecules is essential for understanding their mechanisms, developing improved derivatives, and minimizing off-target effects. Despite advances in target identification (target-ID) technologies, significant challenges remain, impeding drug development. Most...
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eLife Sciences Publications Ltd
2024-12-01
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| Online Access: | https://elifesciences.org/articles/102667 |
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| author | Yingjie Sun Changheng Li Xiaofei Deng Wenjie Li Xiaoyi Deng Weiqi Ge Miaoyuan Shi Ying Guo Yanxun V Yu Hai-bing Zhou Youngnam N Jin |
| author_facet | Yingjie Sun Changheng Li Xiaofei Deng Wenjie Li Xiaoyi Deng Weiqi Ge Miaoyuan Shi Ying Guo Yanxun V Yu Hai-bing Zhou Youngnam N Jin |
| author_sort | Yingjie Sun |
| collection | DOAJ |
| description | Identifying target proteins for bioactive molecules is essential for understanding their mechanisms, developing improved derivatives, and minimizing off-target effects. Despite advances in target identification (target-ID) technologies, significant challenges remain, impeding drug development. Most target-ID methods use cell lysates, but maintaining an intact cellular context is vital for capturing specific drug–protein interactions, such as those with transient protein complexes and membrane-associated proteins. To address these limitations, we developed POST-IT (Pup-On-target for Small molecule Target Identification Technology), a non-diffusive proximity tagging system for live cells, orthogonal to the eukaryotic system. POST-IT utilizes an engineered fusion of proteasomal accessory factor A and HaloTag to transfer Pup to proximal proteins upon directly binding to the small molecule. After significant optimization to eliminate self-pupylation and polypupylation, minimize depupylation, and optimize chemical linkers, POST-IT successfully identified known targets and discovered a new binder, SEPHS2, for dasatinib, and VPS37C as a new target for hydroxychloroquine, enhancing our understanding these drugs’ mechanisms of action. Furthermore, we demonstrated the application of POST-IT in live zebrafish embryos, highlighting its potential for broad biological research and drug development. |
| format | Article |
| id | doaj-art-e3f6f4fa770347f5a71bc78b3411499a |
| institution | DOAJ |
| issn | 2050-084X |
| language | English |
| publishDate | 2024-12-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj-art-e3f6f4fa770347f5a71bc78b3411499a2025-08-20T02:39:40ZengeLife Sciences Publications LtdeLife2050-084X2024-12-011310.7554/eLife.102667Target protein identification in live cells and organisms with a non-diffusive proximity tagging systemYingjie Sun0Changheng Li1Xiaofei Deng2Wenjie Li3Xiaoyi Deng4Weiqi Ge5Miaoyuan Shi6Ying Guo7Yanxun V Yu8https://orcid.org/0000-0001-6617-0166Hai-bing Zhou9Youngnam N Jin10https://orcid.org/0009-0007-2632-3237Department of Neurology, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, ChinaDepartment of Neurology, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, ChinaDepartment of Hematology, Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University, Wuhan, ChinaDepartment of Neurology, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, ChinaDepartment of Neurology, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, ChinaDepartment of Neurology, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, ChinaDepartment of Neurology, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, ChinaDepartment of Neurology, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, ChinaDepartment of Neurology, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, ChinaDepartment of Hematology, Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University, Wuhan, China; State Key Laboratory of Virology, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, ChinaDepartment of Neurology, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, ChinaIdentifying target proteins for bioactive molecules is essential for understanding their mechanisms, developing improved derivatives, and minimizing off-target effects. Despite advances in target identification (target-ID) technologies, significant challenges remain, impeding drug development. Most target-ID methods use cell lysates, but maintaining an intact cellular context is vital for capturing specific drug–protein interactions, such as those with transient protein complexes and membrane-associated proteins. To address these limitations, we developed POST-IT (Pup-On-target for Small molecule Target Identification Technology), a non-diffusive proximity tagging system for live cells, orthogonal to the eukaryotic system. POST-IT utilizes an engineered fusion of proteasomal accessory factor A and HaloTag to transfer Pup to proximal proteins upon directly binding to the small molecule. After significant optimization to eliminate self-pupylation and polypupylation, minimize depupylation, and optimize chemical linkers, POST-IT successfully identified known targets and discovered a new binder, SEPHS2, for dasatinib, and VPS37C as a new target for hydroxychloroquine, enhancing our understanding these drugs’ mechanisms of action. Furthermore, we demonstrated the application of POST-IT in live zebrafish embryos, highlighting its potential for broad biological research and drug development.https://elifesciences.org/articles/102667proximity labelingPafAdasatinibchloroquinezebrafishtarget identification |
| spellingShingle | Yingjie Sun Changheng Li Xiaofei Deng Wenjie Li Xiaoyi Deng Weiqi Ge Miaoyuan Shi Ying Guo Yanxun V Yu Hai-bing Zhou Youngnam N Jin Target protein identification in live cells and organisms with a non-diffusive proximity tagging system eLife proximity labeling PafA dasatinib chloroquine zebrafish target identification |
| title | Target protein identification in live cells and organisms with a non-diffusive proximity tagging system |
| title_full | Target protein identification in live cells and organisms with a non-diffusive proximity tagging system |
| title_fullStr | Target protein identification in live cells and organisms with a non-diffusive proximity tagging system |
| title_full_unstemmed | Target protein identification in live cells and organisms with a non-diffusive proximity tagging system |
| title_short | Target protein identification in live cells and organisms with a non-diffusive proximity tagging system |
| title_sort | target protein identification in live cells and organisms with a non diffusive proximity tagging system |
| topic | proximity labeling PafA dasatinib chloroquine zebrafish target identification |
| url | https://elifesciences.org/articles/102667 |
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