| 引用本文： |
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董思琪,许 可.CtBP1 SUMO化修饰的分子机制研究[J].同济大学学报（医学版）,2024,45(5):656-664. [点击复制]
- DONG Siqi,XU Ke.Diagnosis of anterior cruciate ligament injuries in MRI images based on semi-supervised residual network deep learning[J].Journal of Tongji University(Medical Science),2024,45(5):656-664. [点击复制]
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| 摘要: |
| 目的 在体外重组表达、纯化CtBP1发生SUMO化修饰时参与反应的各个蛋白: CtBP1（底物）、SUMO1（SUMO蛋白）、Ubc9（E2结合酶）和PC2（E3连接酶），体外组装CtBP1 SUMO化修饰复合体CtBP1-SUMO1-Ubc9-PC2，通过结构解析捕捉CtBP1发生SUMO化修饰的中间状态，阐明CtBP1 SUMO化修饰的分子机制。方法 通过pRSFDuet-1载体构建人源CtBP1、SUMO1、Ubc9、PC2表达质粒，系统表达纯化各蛋白组分，并通过分子筛检测并优化各个蛋白的状态，以及CtBP1-SUMO1-Ubc9-PC2复合体的形成，通过单颗粒冷冻电镜解析复合体结构。结果 通过原核表达系统成功表达并纯化获得了参与CtBP1 SUMO化反应复合体中的各个蛋白CtBP1、SUMO1、Ubc9与PC2，获得了CtBP1 SUMO化修饰复合体，通过负染和冷冻电镜的样品优化初步获得了CtBP1 SUMO化修饰复合体的冷冻电镜结构。结论 成功获得了CtBP1 SUMO化反应中间状态CtBP1-SUMO1-Ubc9-PC2复合体蛋白以及初步的冷冻电镜结构，为阐明CtBP1发生SUMO化修饰的分子机制奠定了重要基础。 |
| 关键词: CtBP1 SUMO化 单颗粒冷冻电镜 |
| DOI：10.12289/j.issn.2097-434523379 |
| 通信作者: |
| 投稿时间：2024-02-26 |
| 录用日期：2024-04-01 |
| 基金项目:国家重点研发计划(2021YFA1302200)；国家自然科学基金(32301018) |
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| Diagnosis of anterior cruciate ligament injuries in MRI images based on semi-supervised residual network deep learning |
| DONG Siqi,XU Ke |
| ((Shanghai Key Laboratory of Anesthesiology and Brain Functional Modulation, Clinical Research Center for Anesthesiology and Perioperative Medicine, Translational Research Institute of Brain and Brain-Like Intelligence, Shanghai Fourth People’s Hospital, School of Medicine, Tongji University, Shanghai 200434, China)) |
| Abstract: |
| Objective To purify the proteins involved in the SUMOylation of CtBP1 in vitro and to elucidate molecular mechanisms of SUMOylation of CtBP1. Methods The expression plasmids of human CtBP1(substrate), SUMO protein SUMO1, E2-binding enzyme Ubc9 and E3 ligase PC2 were constructed by the pRSFDuet-1 vector, and the protein components were expressed and purified in the prokaryotic system. The state of each protein and the formation of the CtBP1-SUMO1-Ubc9-PC2 complex were detected and optimized by molecular sieve, and the structure of the complex was analyzed by single-particle cryo-electron microscopy(cryo-EM). Results The proteins CtBP1, SUMO1, Ubc9 and PC2 involved in the CtBP1 SUMOylation modification complex were successfully expressed and purified in the prokaryotic expression system, and the CtBP1 SUMOylation modification complex was obtained. The cryo-EM structure of the CtBP1 SUMOylation modification complex was observed by negative staining and cryo-EM sample optimization. Conclusion The intermediate state of CtBP1 SUMOylation, the CtBP1-SUMO1-Ubc9-PC2 complex protein and the cryo-EM structure observation have been obtained in the study, which provides the basis for elucidating the molecular mechanism of SUMOylation of CtBP1. |
| Key words: CtBP1 SUMOylation single-particle cryo-EM |
