ユビキチン化によるアクアポリン２の輸送制御機構の研究

泛素化水通道蛋白2转运调控机制研究

• 批准号: 21F20800
• 负责人: 藤吉 好則
• 金额: $ 1.47万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2021
• 资助国家: 日本
• 起止时间: 2021-07-28 至 2023-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-21F20800/
• 关键词: Aquaporin-2; Ubiquitin; E3 ligase; FSEC; CryoEM; AQP-2

E3 ligases CHIP and NEDD4 family members have been cloned, expressed and purified, however in the later stages of screening the interest focused on NEDD4L protein.Co-elution of AQP2 was examined with several E3 ligases including CHIP, NEDD4, NEDD4L, ITCH, Arih2, however without any binding observed.This suggested involvement of scaffold proteins including 14-3-3 and HSP70 or phosphorylation of NEDD4L. These additional proteins and phospho-variants of NEDD4L were prepared, however as before no binding was observed.Furthermore, fusion constructs of N-terminal (intra-cellular) region of NDFIP1/2, reported to bind NEDD4L, and the AQP2 were prepared. So far, NDFIP2-AQP2 fusion was successfully solubilised and purified with initial binding experiments following soon.Lastly, substrate adaptors SOCS2 and FBX6 of Cul2/5 have been reported to recruit AQP2. Constructs of FBX6 were not soluble. Similar problems were experienced with SOCS2, however when co-expressed with EloB/C, a ternary complex was successfully purified. This formed a stable complex with Cul5R1 and initial SPA analysis was performed. More particles will be collected to enable 3D reconstruction.Due to no binding observed thus far, ubiquitin cascade was reconstituted to validate whether identified E3 ligases modify AQP2 in an isolated system. Although this difficult project had not been successfully finalized and the research period was finished, we will continuously challenge on this project because this is interesting and important for understanding homeostasis of human body through ubiquitination of AQP2.

E3连接酶CHIP和NEDD 4家族成员已被克隆、表达和纯化，但在筛选的后期阶段，兴趣集中在NEDD 4L蛋白上。检测了AQP 2与几种E3连接酶（包括CHIP、NEDD 4、NEDD 4L、ITCH、Arih 2）的共洗脱，但未观察到任何结合，这表明包括14-3-3和HSP 70的支架蛋白或NEDD 4L的磷酸化参与。此外，制备了NDFIP 1/2的N-末端（细胞内）区域的融合构建体，据报道其结合NEDD 4L，和AQP 2。最后，Cul 2/5的底物衔接子SOCS 2和FBX 6被报道可以募集AQP 2。FBX 6的构建体不可溶。S 0 CS2经历了类似的问题，然而当与EloB/C共表达时，成功纯化了三元复合物。 这与Cul 5 R1形成稳定的复合物，并进行初始SPA分析。由于迄今为止没有观察到结合，因此重构泛素级联以验证鉴定的E3连接酶是否在分离的系统中修饰AQP 2。虽然这个困难的项目还没有成功完成，研究期已经结束，但我们将继续挑战这个项目，因为这对于通过AQP 2的泛素化来理解人体内稳态是有趣和重要的。

项目成果

Science Dialogue

科学对话

• 发表时间: 2022
• 作者: P. Andriotis;M. Kirby;A. Takasu;Andrej Paluda
• 通讯作者: Andrej Paluda

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