title: Protocol for a Steady-State FRET Assay in Cancer Chemoprevention.
abstract: Cancer chemoprevention is an important strategy to prevent, reverse, or suppress the development of cancer. One of the target pathways that has emerged in recent years is the Keap1-Nrf2-ARE system that regulates the protection of cells against various carcinogens and their metabolites. Increased concentrations of the redox transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) induces the activation of antioxidant and phase 2 detoxifying genes. Nrf2 is regulated by substrate adaptor protein Kelch-like ECH-associated protein 1 (Keap1) that can target Nrf2 for ubiquitination and degradation by the proteasome. The interaction between Nrf2 and Keap1 can be disrupted at the protein-protein interface in order to increase Nrf2 activity for potential therapeutic purposes. This chapter describes a protocol for a steady-state fluorescence or Förster resonance energy transfer (FRET) assay to examine the Keap1-Nrf2 protein-protein interaction (PPI), to investigate the effects of Nrf2 mutations on Keap1 binding and finally to identify potential inhibitors of this PPI. In the assay system Keap1 is conjugated to an YFP protein at the N-terminus whereas an Nrf2-derived 16-mer peptide containing a high-affinity "ETGE" motif is conjugated to a CFP protein at the N-terminus.
authors.affiliation: UCL School of Pharmacy, University College London, 29/39 Brunswick Square, London, WC1N 1AX, UK.
authors.forename: Marjolein C A
authors.initials: MC
authors.lastname: Schaap
authors.affiliation: UCL School of Pharmacy, University College London, 29/39 Brunswick Square, London, WC1N 1AX, UK.
authors.forename: Andreia M R
authors.initials: AM
authors.lastname: Guimarães
authors.affiliation: UCL School of Pharmacy, University College London, 29/39 Brunswick Square, London, WC1N 1AX, UK.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Wilderspin
authors.affiliation: UCL School of Pharmacy, University College London, 29/39 Brunswick Square, London, WC1N 1AX, UK. g.wells@ucl.ac.uk.
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Wells
mesh_terms: D020816:Amino Acid Motifs; D018890:Chemoprevention; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000378:metabolism / Q000517:prevention & control*; D014170:Transformation, Genetic
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D049452:Green Fluorescent Proteins
keywords: FRET; Keap1; Nrf2; Peptide inhibitors; Protein–protein interactions; Small-molecule inhibitors