Transcription
factor NF-E2 p45-related factor 2 (Nrf2) and its
principal negative regulator, Kelch-like ECH-associated protein 1
(Keap1), comprise a molecular effector and sensor system that robustly
responds to perturbations of the cellular redox homeostasis by orchestrating
a comprehensive cytoprotective program. Under homeostatic conditions,
Nrf2 is a short-lived protein, which is targeted for ubiquitination
and proteasomal degradation. Upon encounter of electrophiles, oxidants,
or pro-inflammatory stimuli, the cysteine sensors in Keap1 are chemically
modified, rendering Keap1 unable to target Nrf2 for degradation, and
consequently leading to accumulation of the transcription factor and
enhanced transcription of cytoprotective genes. A detailed understanding
of the protein–protein interactions between Nrf2 and Keap1
has been achieved by use of various in vitro systems, but few assays
are available to assess these interactions in the context of the living
cell. We previously developed an imaging-based FLIM/FRET methodology
to visualize and measure the interaction between Nrf2 and Keap1 in
single cells. Here, our goal was to improve this methodology in order
to increase throughput and precision, and decrease cell-to-cell variability.
To eliminate the possibility of orientation bias, we incorporated
a flexible linker between Keap1 and the FRET acceptor fluorescent
protein tag. To ensure the correct image capture of Nrf2 fused to
the FRET donor fluorescent protein tag, we matched the maturation
time of the fluorescent tag to the half-life of the endogenous Nrf2,
by using sfGFP as the FRET donor. Using a global binning approach
increased the assay throughput, whereas including the measured instrument
response function in the analysis improved precision. The application
of this methodology revealed a strong covariation of the results with
the expression level of the acceptor. Taking the acceptor level into
account circumvented cell-to-cell variability and enhanced sensitivity
of the measurements of the Keap1–Nrf2 interaction in live cells
