AbstractThe transcription factor Nrf2 is a master regulator of cytoprotective gene expression. Nrf2 is negatively controlled by Keap1, a sensor protein which allows Nrf2 to respond to changing cellular conditions. In the basal state, Nrf2 binds to two sites of a Keap1 dimer allowing its ubiquitination in a Cullin-3/Rbx1-dependent manner. In response to electrophiles and oxidants (termed inducers, which bind directly to Keap1) ubiquitination of Nrf2 is inhibited; consequently, Nrf2 accumulates and activates transcription.
We have developed aFLIM-based assay to study the dynamic interaction between Keap1 and Nrf2 in single live cells. Combinations of wild type and mutant proteins revealed that under basal conditions the Keap1-Nrf2 complex exists in two conformations, one in which Nrf2 is bound to both members or the Keap1 dimer (‘closed’ conformation), and a second in which Nrf2 interacts with a single Keap1 monomer (‘open’ conformation). We found that following exposure to a range of inducers the Cul3-Keap1-Nrf2 complex does not dissociate, but remains intact. Furthermore, we found that inducers lead to the accumulation of the Keap1-Nrf2 complex in the ‘closed’ conformation. Interestingly, blockage of the proteasome also leads to the accumulation of the complex in the closed conformation, suggesting that the binding of Nrf2 and its subsequent Keap1-dependent ubiquitination follows a cyclical pattern. We believe that the existence of a Keap1-Nrf2 binding cycle benefits the cell, as it allows other signaling pathways, such as those mediated by p21 and p62, to regulate Nrf2 activity in the absence of inducers.
Together our results show that the interaction between Keap1 and Nrf2 is more dynamic than previously anticipated and that inducers function to modulate this dynamism, leading to Nrf2 stabilisation and cytoprotective gene expression.
|Date of Award||2013|
|Supervisor||Albena Dinkova-Kostova (Supervisor)|