Dissecting the Molecular Basis of NRF2 Activity Modulation in the Oxidative Stress Response
Abstract
Nuclear factor erythroid 2 - related factor 2 (Nrf2) is a major transcription factor coordinating cellular responses to oxidative stress. Through the interactions with other proteins, such as Kelch-like ECH-associated protein 1 (Keap1), p21, CREB-binding protein (CBP), and Retinoid X receptor alpha (RXRα), Nrf2 mediates the transcription of cytoprotective genes in response to environmental and endogenous stresses, a process that is critical for removing toxicants and preventing DNA damage. Therefore, Nrf2 plays a significant role in chemoprevention. However, aberrant activation of Nrf2 favors cancer cells by protecting them from apoptosis and gaining chemoresistance. Therefore, to gain a detailed understanding of the regulation of Nrf2, dissecting the molecular basis of its interactions with targets is paramount.
This thesis investigated the structural basis of Nrf2 and its interactions with CBP/p300, p21, and RXRa. Biophysical techniques, including nuclear magnetic resonance (NMR), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and circular dichroism (CD) spectropolarimetry, were used to characterize the structure of Nrf2 extensively. Our NMR data show that Nrf2 is partially disordered. Interestingly, HDX-MS experiments revealed that even though the protein is overall highly exposed to solvent, there are transient structural elements present in Nrf2. The HDX-MS results agree with the CD result showing that over 50% of the protein is not structured.
Next, a series of biophysical experiments were used to gain a detailed understanding of the Nrf2-CBP/p300 interaction, which plays a vital role in Nrf2 transactivation. The structural properties of Neh4 and Neh5 and their binding to the TAZ1 and TAZ2 domains of CBP/p300 were characterized. It was revealed that both Neh4 and Neh5 domains of Nrf2 are intrinsically disordered, and binding affinities with TAZ1 and TAZ2 are in the micro-molar range. Finally, I confirmed that the C-terminal 24 residues of p21 can interact with both DLG and ETGE motifs of Nrf2 and both Neh6 and Neh7 domains are intrinsically disordered. However, in vitro studies showed no interaction between the Neh7 domain with the RXRα-DBD. In conclusion, the findings, along with the details on Nrf2 interactions, will provide a described picture of the Nrf2 regulation pathway, which could be vital in designing therapeutics against Nrf2-mediated diseases.