| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2022: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2021: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Outline of Annual Research Achievements |
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.
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