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Published on: March 14, 2019
Diffusion dynamics of the Keap1-Cullin3 interaction in single live cells
Liam Baird1, Albena T Dinkova-Kostova
1Jacqui Wood Cancer Centre, Division of Cancer Research, Medical Research Institute, University of Dundee, Dundee, Scotland, UK.
Biochemical and Biophysical Research Communications
|March 5, 2013
Summary
Small molecules activate the Nrf2 pathway by altering Keap1 protein conformation, not by dissociating it from Cul3. This finding clarifies how Nrf2 is stabilized, impacting cellular defense mechanisms.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Biochemistry
Background:
- Transcription factor Nrf2 (NF-E2 p45-related factor 2) controls cytoprotective genes.
- Keap1 (Kelch-like ECH associated protein 1) normally targets Nrf2 for degradation via Cul3.
- Small molecule inducers activate Nrf2, but the mechanism of Keap1 inactivation is debated.
Purpose of the Study:
- To investigate the diffusion dynamics of the Keap1-Cul3 interaction in live cells.
- To determine if small molecule inducers dissociate the Keap1-Cul3 complex.
- To elucidate the mechanism by which inducers stabilize Nrf2.
Main Methods:
- Developed a quantitative fluorescence recovery after photobleaching (FRAP) system.
- Utilized Keap1-EGFP and mCherry-Cul3 fusion proteins in single live cells.
- Applied four distinct types of small molecule inducers (CDDO, sulforaphane, STCA, H2O2).
Main Results:
- Confirmed Keap1 and Cul3 interact in single live cells.
- Observed no dissociation of the Keap1-Cul3 complex upon treatment with various inducers.
- Inducers target distinct cysteine residues in Keap1 with varying potencies.
Conclusions:
- Small molecule inducers do not dissociate the Keap1-Cul3 complex.
- Inducer-mediated inactivation of Keap1's repressor function results from conformational changes, not complex dissociation.
- Conformational changes in Keap1 lead to Nrf2 stabilization and subsequent upregulation of cytoprotective genes.
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