Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Pathology and Laboratory Medicine

Supervisor

Duennwald, Martin L.

Abstract

To cope with the dynamic range of stressful stimuli that a cell experiences within its lifetime, a host of adaptive cell survival and cell stress response pathways have evolved. The antioxidant and heat shock responses are two key cell stress response pathways primarily involved in the detoxification and elimination of oxidative stress and the maintenance of protein integrity, respectively. Traditionally, these responses are regarded and studied as two independent pathways. In this exploratory work, we hypothesize that oxidative damage to Nrf2 and Keap1 and their interactions with Hsp90 alter their function within the cellular antioxidant stress response. By establishing and characterizing a novel yeast model for human Nrf2, the transcriptional master regulator of the antioxidant response, a previously unexplored interaction was found between Nrf2 and the major heat shock response protein, Hsp90. Further investigation into this interaction using mammalian and breast cancer cells reveals the co-involvement of these proteins in key aspects of protein oxidation, protein misfolding, and cellular responses to cancer therapy. Additionally, Nrf2 and its regulating protein Keap1 were observed to misfold and form protein inclusions upon exposure to oxidative stress, which might implicate a previously unknown mechanism of Nrf2 regulation by inclusion formation. These findings suggest that investigating the antioxidant and heat shock responses in parallel may provide an additional layer of knowledge that is relevant to both basic science and clinical research.

Summary for Lay Audience

All living things experience stress from the environment that can be harmful to cells within the body. As a means of protection, cells have evolved numerous cell stress response pathways to eliminate these insults. This includes the antioxidant response, which protects against harmful free radicals, and the heat shock response, which protects cells from protein-damaging stress. Traditionally, these responses are regarded and studied as two independent and separate pathways. In this exploratory work, we hypothesize that oxidative damage to Nrf2 and Keap1 and their interactions with Hsp90 alter their function within the cellular antioxidant stress response. Using laboratory yeast, a new binding interaction was discovered between the key cell stress protein, Nrf2, which regulates the antioxidant response, and Hsp90, which is a key player in the heat shock response. Further investigation into these interactions using human cells (including cancer cells) shows their co-involvement in important aspects of protein folding and cellular responses to cancer therapy. Additionally, Nrf2 and its regulating protein Keap1 were observed to misfold and form clusters known as inclusions inside the cell under certain stress conditions which could function as a previously unknown “on/off switch” for their cellular activity. Since these two cell stress response pathways overlap, these findings suggest that studying the antioxidant and heat shock responses in parallel may provide important information that is relevant to both basic science and clinical research.

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