Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Choy, Wing-Yiu

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.

Summary for Lay Audience

Proteins perform essential tasks in the body, such as growth and maintenance, performing biochemical reactions like digestion, acting as messenger molecules (as hormones), and many more. A particular group of proteins called transcription factors: molecules that control a gene's activity by determining whether the gene's DNA is transcribed into RNA are essential in promoting or inhibiting the activities of the genes. My thesis focuses on a transcription factor named nuclear factor erythroid 2 - related factor 2 (Nrf2). Nrf2 is vital in coordinating cellular responses to oxidative stress and consists of seven regions that carry out different functions, also known as domains (Neh1-7). Under stress, Nrf2 activates cytoprotective gene transcription to remove harmful toxicants and avoid DNA damage. Therefore, Nrf2 plays an important role in cancer, diabetes, and neurodegenerative diseases. A group of proteins such as Kelch-like ECH-associated protein 1 (Keap1), p21, CREB-binding protein (CBP), and Retinoid X receptor alpha (RXRα) interacts with Nrf2 and changes its activity. However, the information about how Nrf2 interacts with other proteins (except Kelch) is scarce.

I investigated the structural basis of Nrf2 using different techniques, including nuclear magnetic resonance (NMR), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and circular dichroism (CD) spectropolarimetry. The results revealed that Nrf2 is partially disordered, yet the protein has some structural content (alpha helix and beta sheet). Then, Nrf2's interaction with CBP was characterized, which is vital in cytoprotection. It was found that two disordered Neh4 and Neh5 domains of Nrf2 involve the TAZ1 and TAZ2 domains of CBP. Further, I investigated the Nrf2-p21 and Nrf2- RXRα interactions, which showed p21 was able to interact with the Neh2 domain, but no interaction between Nrf2 and RXRa was detected. The findings of my thesis could help design therapeutics against cancer and neurodegenerative diseases.

Download

Available for download on Tuesday, December 31, 2024

Share

COinS