Measuring Changes in Keap1-Nrf2 Protein Complex Conformation in Individual Cells by FLIM-FRET.

Measuring Changes in Keap1-Nrf2 Protein Complex Conformation in Individual Cells by FLIM-FRET. Curr Protoc Toxicol. 2020 Sep;85(1):e96 Authors: Dikovskaya D, Dinkova-Kostova AT Abstract The nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)-mediated stress response is a major cellular defense mechanism against endogenous and exogenous oxidants, electrophiles, and pro-inflammatory agents. A number of Nrf2 inducers are being developed to therapeutically stimulate this pathway. Inducers are typically sensed by Kelch-like ECH-associated protein 1 (Keap1), a negative regulator and a binding partner of Nrf2. Modifications of Keap1 by oxidants or electrophiles, or its targeting by compounds that disrupt its interaction with Nrf2, alter the conformation of the Keap1-Nrf2 protein complex, which initiates the accumulation of Nrf2 required for mounting a stress response. To detect conformational changes in the Keap1-Nrf2 complex in live cells, we have developed a procedure based on Fluorescence Lifetime Imaging-Förster Resonance Energy Transfer (FLIM-FRET). The procedure includes a FLIM time course in cells expressing fluorescently-tagged Nrf2 and Keap1, followed by an extended analysis pipeline that quantifies changes in fluorescence lifetime of labeled Nrf2. The analysis visualizes and removes intensity-dependent bias in fluorescence lifetime measured with the Time-Correlated Single Photon Counting (TCSPC) approach, thereby improving the...
Source: Current Protocols in Toxicology - Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research