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

基本信息

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

项目摘要

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 NEDD4 家族成员已被克隆、表达和纯化，但在筛选的后期阶段，人们的兴趣集中在 NEDD4L 蛋白上。使用多种 E3 连接酶（包括 CHIP、NEDD4、NEDD4L、ITCH、Arih2）检查了 AQP2 的共洗脱，然而没有观察到任何结合。这表明支架蛋白（包括 14-3-3 和 HSP70）或磷酸化的参与NEDD4L。制备了这些额外的蛋白质和 NEDD4L 的磷酸变体，但与以前一样没有观察到结合。此外，制备了 NDFIP1/2 N 末端（细胞内）区域的融合构建体，据报道可结合 NEDD4L 和 AQP2。到目前为止，NDFIP2-AQP2 融合蛋白已成功溶解并纯化，并进行了初步结合实验。最后，Cul2/5 的底物接头 SOCS2 和 FBX6 据报道可招募 AQP2。 FBX6 的构建体不可溶。 SOCS2 也遇到了类似的问题，但是当与 EloB/C 共表达时，三元复合物被成功纯化。这与 Cul5R1 形成稳定的复合物，并进行了初始 SPA 分析。将收集更多颗粒以实现 3D 重建。由于迄今为止尚未观察到结合，因此重建泛素级联以验证已识别的 E3 连接酶是否会在隔离系统中修饰 AQP2。虽然这个困难的项目还没有成功完成并且研究期也结束了，但我们将继续挑战这个项目，因为这对于通过 AQP2 泛素化来理解人体的稳态很有趣并且很重要。