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NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells
Published on: January 12, 2020
Nuclear factor p65 interacts with Keap1 to repress the Nrf2-ARE pathway
Miao Yu1, Hui Li, Qiongming Liu
1State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 100850, China.
Cellular Signalling
|January 26, 2011
Summary
NF-κB signaling inhibits the Nrf2-ARE pathway. The p65 subunit of NF-κB interacts with Keap1, leading to decreased Nrf2 activity and promoting its ubiquitination.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Keap1 regulates the Nrf2-dependent antioxidant response.
- Mechanisms of Keap1's regulation of the Nrf2-ARE pathway are not fully understood.
Purpose of the Study:
- To elucidate the molecular mechanisms by which Keap1 influences the Nrf2-ARE signaling pathway.
- To identify potential protein interactions involved in this regulation.
Main Methods:
- Yeast two-hybrid screening to identify Keap1-interacting proteins.
- In vivo and in vitro co-immunoprecipitation assays to confirm physical association.
- RNA interference (RNAi) to knock down Keap1 expression.
- Reporter gene assays to measure Nrf2-dependent transcriptional activity.
- Western blotting to assess protein ubiquitination and nuclear translocation.
Main Results:
- The p65 subunit of NF-κB was identified as a Keap1-binding partner.
- Keap1 and p65 physically associate in cells.
- Overexpression of p65 inhibited Nrf2-dependent transcription.
- Knockdown of Keap1 partially reversed p65-mediated repression of Nrf2.
- p65 reduced Nrf2 binding to DNA and enhanced Nrf2 ubiquitination.
- The N-terminal region of p65 mediated Keap1 interaction and transcriptional suppression.
- p65 enhanced the nuclear translocation of Keap1.
Conclusions:
- NF-κB signaling, via the p65 subunit, inhibits the Nrf2-ARE pathway.
- This inhibition occurs through the physical interaction between p65 and Keap1.
- The p65-Keap1 complex modulates Nrf2 activity by affecting its DNA binding and promoting ubiquitination.
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