| Budget Amount *help |
¥14,600,000 (Direct Cost: ¥14,600,000)
Fiscal Year 2006: ¥5,600,000 (Direct Cost: ¥5,600,000)
Fiscal Year 2005: ¥9,000,000 (Direct Cost: ¥9,000,000)
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| Research Abstract |
In this study, we achieved following results :
1. Identification of interacting proteins with transcription factor Nrf2
To identify cooperative factors of Nrf2, we purified proteins that specifically interact with recombinant Nrf2 protein and analyzed by mass spectrometry. As a result, TRRAP, TIF1β, TIF1γ, BAF53a, BRG1 were identified as novel interacting proteins with Nrf2. It was demonstrated that BRG1 interacts with Nrf2 and selectively mediates HO-1 gene induction in response to the oxidative stress.
2. Analysis of functional domains of Nrf2 in living cell.
To analyze the effect of interacting proteins, stable knockdown cell lines of BRG1 and TRRAP were established and recruitment of Nrf2 and BRG1 was investigated. In BRG1 knockdown cell, Nrf2 binding to ARE was not changed, but the recruitment of BRG1 was abolished by the Nrf2 knockdown. By TRRAP knockdown, Nrf2-ARE binding was enhanced. We are now examining the recruitment of Nrf2 and other Nrf2-interacting proteins to ARE using Tetracycline-inducible Nrf2 expression cells.
3. Construction of reporter gene responsive to electrophile, diethyl maleate (DEM).
To screen molecules involved in the activation of Nrf2 using a siRNA library, reporter gene with human NQO1 or HO-1 promoter fused with green fluorescence protein or thymidine kinase gene was constructed. We are almost the step to transfect the reporter construct into culture cells and start to screening.
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