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，但在筛选的后期阶段，兴趣集中在NEDD4L蛋白上。研究了几种E3连接酶（CHIP、NEDD4、NEDD4L、ITCH、Arih2）对AQP2的共洗脱作用，但未观察到任何结合。这表明支架蛋白包括14-3-3和HSP70或NEDD4L的磷酸化参与其中。这些额外的蛋白质和NEDD4L的磷酸化变体被制备出来，但是和以前一样没有观察到结合。此外，我们还制备了NDFIP1/2 n端（细胞内）区域的融合构建物，该区域与NEDD4L和AQP2结合。到目前为止，NDFIP2-AQP2融合已成功溶解和纯化，随后将进行初步结合实验。最后，据报道Cul2/5的底物适配器SOCS2和FBX6可以招募AQP2。FBX6的结构体不溶。SOCS2也遇到了类似的问题，然而，当与EloB/C共表达时，成功地纯化了三元配合物。这与Cul5R1形成了稳定的配合物，并进行了初步的SPA分析。更多的粒子将被收集，使3D重建。由于到目前为止没有观察到结合，我们重组了泛素级联，以验证在分离系统中所鉴定的E3连接酶是否修饰了AQP2。虽然这个困难的项目没有顺利完成，研究期也没有结束，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内稳态是有趣和重要的。

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.