Multicellular organisms are equipped with elaborate networks of cytoprotective proteins (e.g., glutathione transferases, NAD(P)H: quinone oxidoreductase 1, heme oxygenase 1) that defend against the damaging effects of oxidants and electrophiles, the principal contributors to the pathogenesis of chronic diseases. Under basal conditions, these genes whose transcription is dependent on transcription factor Nrf2, are not expressed at their maximum capacity, but can be upregulated (induced) by a variety of synthetic and natural agents (inducers).
Normally, Keap1 binds and targets transcription factor Nrf2 for ubiquitination and proteasomal degradation via association with the Cullin 3 (Cul3)-based E3 ubiquitin ligase. Inducers react and chemically modify specific cysteine residues of Keap1 which consequently loses its ability to repress Nrf2. This leads to increased stabilization of Nrf2, its nuclear translocation, binding to the ARE (in combination with small Maf), and ultimately transcriptional activation of cytoprotective genes. Voluminous experimental evidence supports the view that induction of cytoprotective proteins is a very effective strategy for protection against a plethora of oxidants and carcinogens. Thus, mechanistic understanding of the intricate regulation of the Keap1/Nrf2 pathway is critical.
This project aims to investigate in detail the consequences of binding of inducers to Keap1 on the protein localization, turnover, and trafficking in vitro and in live cells in real space and time. This aim will be accomplished by using the cell-permeable fluorescent trivalent arsenical derivatives FlAsH and ReAsH, in combinations with recombinant or ectopically expressed chimeric versions of Keap1 that are fused to either red or green fluorescent proteins. FlAsH and ReAsH are fluorescent due to the presence of fluorescein and resorufin, respectively, within their structures, and are able to react with cysteine residues due to the high-affinity interaction of arsenic for sulfhydryl groups. Live cell imaging and fluorescence resonance energy transfer (FRET) approaches will be employed to determine the spatio-temporal dynamics of induction of the Keap1/Nrf2 pathway.