The utility of FRET/FLIM-based bioassays for monitoring Nrf2 activation in single live cells

The Kelch-like ECH-associated protein 1 - Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) partnership is vital for protection against oxidative stress, and its dysregulation has been linked to the pathogenesis of numerous chronic diseases. Under homeostatic conditions, Keap1 targets Nrf2 for ubiquitination and proteasomal degradation. When Keap1 is inactivated by electrophiles or Keap1-Nrf2 protein-protein interaction (PPI) inhibitors (termed inducers), Nrf2 accumulates and, in association with a small musculoaponeurotic fibrosarcoma (sMaf) protein, induces the transcription of networks of genes encoding cytoprotective proteins. To understand the PPIs between Nrf2 and Keap1 and the effect of inducers in the cellular context, we expressed GFP-Nrf2 and Keap1-mCherry fusion proteins and employed Förster resonance energy transfer (FRET)-based multiphoton fluorescence lifetime imaging microscopy (FLIM). PPI inhibitors, but not electrophiles, caused dissociation of GFP-Nrf2 from Keap1-mCherry. To monitor the protein stability of Nrf2, we developed a genetically encoded FLIM-timer-tagged Nrf2, which combines sfGFP and mCherry connected by a linker positioning the fluorophores in a way that promotes FRET. In cells co-expressing stoichiometric amounts of FLIM-timer-Nrf2 with Keap1 from a single promoter, the addition of sulforaphane, the classical Keap1-targeting electrophile, decreased the fluorescence lifetime of the Nrf2-FLIM-timer, consistent with Nrf2 stabilization. Such decrease was also observed upon depletion of either the N-terminal Keap1-binding motif of Nrf2, or a degron within its Neh6 domain that confers β-TrCP-dependent degradation. These findings show the utility of FRET/FLIM-based bioassays for monitoring Nrf2 activation and illustrate their potential for exploring various aspects of the oxidative stress response.