Measuring the Interaction of Transcription Factor Nrf2 with Its Negative Regulator Keap1 in Single Live Cells by an Improved FRET/FLIM Analysis

Chemical Research in Toxicology
Dina DikovskayaAlbena T Dinkova-Kostova

Abstract

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 varia...Continue Reading

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Citations

Aug 14, 2020·Current Protocols in Toxicology·Dina Dikovskaya, Albena T Dinkova-Kostova
Jan 13, 2021·Disease Models & Mechanisms·Julia M T AuerDimitrios K Papadopoulos

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