Mass Spectrometric Studies of KEAP1-NRF2 Binding Interactions

Chemoprevention is an active cancer preventive strategy to inhibit, delay or reverse human carcinogenesis using naturally occurring or synthetic chemical agents. In recent years, it has emerged as one of the major approaches for reducing cancer. Nrf2 is a member of the NF-E2 family of nuclear basic leucine zipper transcription factors. In the cell nucleus, Nrf2 binds to the 5’ upstream regulatory antioxidant response element (ARE) regions of phase II genes and accelerates their transcription. Under basal conditions, Nrf2 concentration in the cell is low because of continuous ubiquitination by the Culln3-based (Cul3) E3 ligase ubiquitination complex and degradation (Zhang 2006). Therefore, phase II gene expression is suppressed under basal conditions. Keap1 serves as a bridge between Nrf2 and Cul3 and binds both proteins. When cells are under oxidative stress, signals are transmitted to the Keap1-Nrf2 complex, which interrupts ubiquitination of Nrf2 and increases its concentration in cells, including the nucleus. Consequently, protective phase II enzymes are up-regulated by the increasing level Nrf2 in the nucleus (Yamamoto, Itoh et al. 2003). The Keap1-Nrf2 system has been shown to play an important role in the chemoprevention process. However, how the Keap1 protein interacts with transcription factor Nrf2 is still unclear. In this thesis, an ultrafiltration mass spectrometry screening assay was developed for characterizing protein-protein interactions. To minimize the interference from trypsin acting on the protein target during the binding assay, a trypsin deactivation step was included in the assay. This step significantly reduced the activity of trypsin remaining in the peptide mixture used in the binding incubation. This ultrafiltration mass spectrometry method should be suitable for use as an alternative to H/D exchange or protein footprinting to study protein-protein interactions. In this investigation, the Keap1 tryptic peptide 337QSLSYLEAYNPSDGTWLR354 was identified as having the highest affinity for the Neh2 domain of Nrf2. These results support the view that the human Kelch domain (amino acid 327-amino acid 609) is at least in part responsible for association between Keap1 and Nrf2.