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Thesis Format

Integrated Article

Degree

Master of Science

Program

Biochemistry

Supervisor

Choy, Wing-Yiu

2nd Supervisor

Karttunen, Mikko

Co-Supervisor

Abstract

The nuclear factor erythroid 2-related factor 2 (Nrf2) protein is a critical transcription factor for activating the antioxidant response pathway, a primary defense mechanism against disproportionate levels of oxidants in the cell, via the upregulation of cytoprotective genes. Notably, aberrant activation of Nrf2 in cancer cells increases their resistance to chemotherapy, rendering the treatment ineffective. The focus was to uncover the conformational landscape of Nrf2’s Neh4 and Neh5 domains, which participate in crucial interactions for complete transcriptional activation. Since Nrf2 is an intrinsically disordered protein (IDP), molecular dynamics simulations were employed to capture its dynamic nature and conformational heterogeneity. The Neh4 and Neh5 domains differed in their structural propensities, where the phosphorylation of Neh5’s S193 residue had little effect. This work points to the domains having potentially different binding mechanisms. In addition, performance discrepancies between force fields CHARMM36m and Amber99SB*-ILDNP were identified, potentially contributing to future force field development.

Summary for Lay Audience

The human body is continually exposed to reactive oxygen species (ROS). An excess of ROS results in oxidative stress, leading to the development of highly prevalent diseases through the damage of lipids, proteins, and DNA. However, the human body has a defense mechanism, the antioxidant response pathway, that combats the excessive ROS levels. Activation of the pathway results in the nuclear factor erythroid 2-related factor 2 (Nrf2) protein translocating to the nucleus, binding co-activators and additional transcriptional machinery. Together as a complex, they activate the transcription of cytoprotective genes (i.e., genes that code for detoxifying proteins) via an antioxidant response element (ARE), a promoter region that mediates the binding of elements to aid in transcription. When Nrf2 is inactivated or impaired, the cell cannot protect itself from excessive oxidant levels and thus is open to disease. However, when Nrf2 is activated inappropriately in cancer cells, it can protect them, increasing their resistance to chemotherapy. The appropriate modulation of Nrf2’s activity is critical for maintaining homeostasis. To understand how proteins operate in our system and develop tools to modulate their activity, uncovering their structural properties provides valuable information.

This thesis focused on elucidating the structural and dynamical properties of Nrf2’s transactivation domains, Neh4 and Neh5. The Neh4 and Neh5 domains participate in a crucial interaction for complete transcriptional activation. Notably, Nrf2 is an intrinsically disordered protein (IDP), which means it lacks a stable three-dimensional structure. Disordered proteins are dynamic and capable of folding into many conformations, which makes studying IDPs more challenging to work with using traditional experimental techniques. Therefore, molecular dynamics (MD) simulations, a computational modelling approach, were used to study the dynamic nature of the Neh4 and Neh5 domains.

It was observed that the structural propensities of the Neh4 and Neh5 domains differ, where the phosphorylation of Neh5’s serine residue had no significant effect. This work provides insight into how Neh4 and Neh5 may interact differently with their binding partners. In addition, discrepancies between popular MD simulation setups were identified, which may prove useful for future developments to more accurately characterize IDPs.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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