Abstract
As the master control to regulate redox homeostasis in cells, Nrf2 (Nuclear factor erythroid 2-related factor 2) is one of the most important transcription factors in the body. It binds to the antioxidant response element at the promoter region on the target DNA, transcribing a series of downstream antioxidant enzymes. Despite much research over the past few decades, many aspects of the functional mechanisms and regulations of the Nrf2 pathway, especially in relation with Keap1 (Kelch-like ECH-associated protein 1), are still poorly understood. By creating fluorescent variants of Nrf2 and Keap1 via genetically encoded fluorescent unnatural amino acid incorporation and other approaches, we enabled the direct monitoring of the localization and trafficking of Nrf2 and Keap1, as well as their interactions through FRET activities in live cells in real time under a confocal fluorescent microscope. By mutation of a cysteine residue at C151 and a few critical lysine residues in the NES (nuclear export signal) region of Keap1, respectively, the imaging data indicated that C151 was critical to maintain Nrf2 degradation but not enough to cause Nrf2 dissociation and NES in Keap1 was related to Nrf2 nuclear export. Similarly, mutations of a few lysine residues in the NLS (nuclear localization signal) segment and the ETGE binding motif in Nrf2 were made to probe their roles in nucleus import recognition and interactions with Keap1, respectively, in live cells. It was demonstrated that the NLS in Nrf2 was needed for the nuclear import of both Nrf2 and Keap1, and the ETGE motif was critical to Nrf2-Keap1 binding. Cranberries are rich in dietary polyphenols including quercetin and they have shown excellent antioxidant effects but the mechanisms behind these effects are not well understood. Interfering with the Nrf2 pathway as well as direct free radical scavenging ability have been implicated. Molecular probe RCY (Hydro-Cy5) was used to monitor reactive oxygen species in live cells and to evaluate the antioxidant effects of quercetin as well as cranberry extracts. Promotion of nuclear translocation of Nrf2 by quercetin was observed in live cells, providing convincing evidence that quercetin could influence the Nrf2 pathway as a mechanism to exhibit its antioxidant effects. Finally, to study and monitor the important redox-active metal ions, methods have been developed for a novel near-infrared ratiometric fluorescent probe CR-Ac in monitoring the dynamic changes of subcellular Cu2+ and quantification of Cu2+ levels in live cells at nanomolar level for the first time.