2021 Fiscal Year Annual Research Report
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
| Project/Area Number |
21F20800
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
藤吉 好則 東京医科歯科大学, 高等研究院, 特別栄誉教授 (80142298)
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| Co-Investigator(Kenkyū-buntansha) |
PALUDA ANDREJ 東京医科歯科大学, 高等研究院, 外国人特別研究員
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| Project Period (FY) |
2021-07-28 – 2023-03-31
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| Keywords | AQP-2 / Ubiquitin / E3 ligase |
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| Outline of Annual Research Achievements |
The initial aim of this project was to establish purification of AQP-2 in Sf9 insect cells and to prepare constructs of E3 ligases CHIP, NEDD4, and NEDD4L. Full length AQP-2 in fusion with eGFP were prepared by solubilisation using two different detergents (n-Dodecyl β-D-maltoside and n-nonyl-β-D-glucoside) and purified using affinity beads and size-exclusion chromatography. Subsequently, constructs of E3 ligases CHIP full-length, NEDD4L ΔN185, and NEDD4L ΔN593 were cloned, expressed using bacterial system, and purified by affinity beads and size-exclusion chromatography.
Following this, AQP-2 and respective E3 ligases were mixed and analysed by FSEC. Fluorescent signal of eGFP was followed to evaluate shift in the elution volume suggesting complex formation. All construct exhibited minor shift towards higher molecular weight species. To follow up, GST fusions with C-terminal fragments of AQP-2 of various lengths were prepared. All constructs comprised K270, a residue considered to be modified by ubiquitin and thus responsible for internalisation of AQP-2. These were subject of GST-pulldown assay and also showed weak binding to the NEDD4L ΔN593. This construct was then subject of preliminary negative staining experiment.
In addition to the current targets, a list of further AQP-2 modifying E3 ligases was compiled based on the data currently available in the public databases and literature.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
AQP-2 solubilised in two different detergents has been successfully prepared as a platform from majority of planned experiments.
Two out of three originally proposed target E3 ligases have been successfully cloned, expressed and purified.
Co-elution and pulldown experiments suggested week interaction among AQP-2 and NEDD4L.
Material for the initial EM experiments has been prepared.
Additional E3 ligase targets have been identified and their constructs have been ordered and designed.
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| Strategy for Future Research Activity |
The main goal for the future period will be to prepare suitable samples of AQP-2 and E3 ligases CHIP and NEDD4L for single-particle analysis by CryoEM. As these have only showed week binding, crosslinking or fusion of these proteins might be required to enhance binding. Alternatively, additional approaches to stabilise these interactions might be needed. HSP70, NDFIP1, and 14-3-3e have been shown to regulate activity of these E3 ligases and may potentially stabilise weak interactions between AQP-2 and E3 ligases CHIP and NEDD4L.
Furthermore, additional candidates that modify AQP-2 have been identified. Depending on the progress with original candidates, E3 ligases Arih2, ITCH, MIB2, TRIM27, RNF139, HERC4 and Cullin-related substrate adaptors SOCS2 and FBX6 will be considered. These comprise a variety of E3 ligase families and potentially use different modes of binding to their substrates and AQP-2.
Apart from structural approaches a set of proteins that form ubiquitylation pathway will be obtained from a collaborator to recreate this system in vitro and will enable evaluation of the modification of AQP-2 by the purified E3 ligases.
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