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

E3连接酶CHIP和NEDD4家族成员已经被克隆、表达和纯化，但在后期的筛选阶段，人们的兴趣主要集中在NEDD4L蛋白上。AQP2与几种E3连接酶包括CHIP、NEDD4、NEDD4L、ITCH、Arih2进行了共洗脱实验，但没有观察到任何结合。这表明包括14-3-3和HSP70在内的支架蛋白参与了NEDD4L的磷酸化。这些额外的蛋白质和NEDD4L的磷酸变异体被制备出来，但是和以前一样没有观察到结合。此外，还制备了与NEDD4L结合的NDFIP1/2的N端(细胞内)区域的融合载体，以及AQP2。到目前为止，NDFIP2-AQP2融合蛋白已被成功溶解和纯化，并进行了初步的结合实验。最后，报道了Cul2/5的底物适配子SOCS2和FBX6招募了AQP2。FBX6的构建物不是可溶的。SOCS2也经历了类似的问题，但当与EloB/C共表达时，成功地纯化了一个三元复合体。这与Cul5R1形成了稳定的络合物，并进行了初步的SPA分析。由于到目前为止还没有观察到结合，因此重组泛素级联以验证已识别的E3连接酶是否在隔离系统中修饰AQP2。虽然这个困难的项目还没有成功完成，研究阶段已经结束，但我们将继续挑战这个项目，因为这对于通过泛素化AQP2来理解人体的动态平衡是有趣的，也是重要的。

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.