Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Master of Science

Program

Biochemistry

Supervisor

Choy, James W.Y.

2nd Supervisor

Prado, Marco

Joint Supervisor

Abstract

Oxidative and proteotoxic stress are common hallmarks of Neurodegenerative diseases (NDs). Cellular proteostasis is maintained through Heat shock protein (Hsp) 90 and Stress-inducible protein 1 (STIP1) modulating the stability of their substrates (clients). Hsp90/heat shock factor (HSF)1 pathway activation attenuates proteotoxicity. Meanwhile, activating the Kelch-like ECH associated protein 1 (Keap1)/ nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway combats oxidative stress. Numerous studies attempted to individually manipulate the expression of Hsp90 or Nrf2 to treat NDs.

Novel interactions of Hsp90 with Nrf2 and Keap1 were discovered via yeast-2-hybrid screening (unpublished data). We analyzed their interactions through NMR spectroscopy, ITC, protein-binding assay, Western blotting, and RT-qPCR. We demonstrated that Hsp90 and STIP1 are modulators of both Nrf2 and Keap1’s protein stability. Keap1 directly binds with STIP1 and Hsp90. Keap1 interacts Hsp90 via the Kelch domain. Our study revealed potential crosstalk between Keap1/Nrf2 and Hsp90/HSF1 cytoprotective pathways and the possibility of co-modulating them.

Summary for Lay Audience

This work addresses the biophysical and biological analysis of Nrf2 and Keap1 as client proteins of Hsp90 chaperone machinery, which can provide valuable insights into treatment designs towards neurodegenerative diseases (ND). Heat shock protein (Hsp) 90, one of the most abundant and evolutionary conserved molecular chaperones, modulates protein folding, proteostasis maintenance, and proteotoxicity clearance. Substrate proteins that interact with m Hsp90 are called clients. The proper folding and functionality of Hsp90 clients are maintained through the chaperone networks and co-chaperones, such as Stress-inducible protein 1 (STIP1). This network and the clients of Hsp90 are found to play important roles in neurodegenerative diseases. The inhibition of Hsp90 activates the heat shock factor (HSF)1- mediated heat shock response (HSR), which attenuates the harmful aggregated protein Amyloid-b (Ab) and tau-related toxicity in Alzheimer’s disease. Meanwhile, Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2), a master regulator of antioxidative response, combats oxidative stress and the proteotoxicity, such as the neuronal Aβ oligomer toxicity. Numerous studies are devoted to separately manipulating the level of Hsp90 or Nrf2 in treating ND. Here we provide insights into the crosstalk of the Nrf2- and Hsp90-mediated cytoprotective pathways implying the potential in co-modulating their levels.

Novel interactions of Hsp90 with Nrf2 and Kelch-like ECH associated protein 1 (Keap1), the negative regulator of Nrf2, were discovered via yeast-two-hybrid screening by our collaborator, Dr. Duennawald. Following this result, we analyzed their interactions through NMR spectroscopy, ITC, protein-binding assay, Western blotting, and RT-qPCR. Our findings showed that both Hsp90 and STIP1 interacts with Nrf2 and Keap1. Specifically, Keap1 interacted with Hsp90 via the Kelch domain. In addition, by using the genetically modified mouse models recently developed by Dr. Prado’s lab, we investigated the functional consequences of these interactions in vivo. Our data indicated that STIP1 plays a critical role in Hsp90’s ability to modulate the protein stability of Keap1 and Nrf2. The interactions of Hsp90 with Keap1 and Nrf2 reveal exciting underlying crosstalk in Keap1/Nrf2 and Hsp90/HSF1 cytoprotective pathways and the potential in co-modulating these pathways. Further characterizing their interacting mechanisms and cellular functions may provide valuable insights in treating neurodegenerative diseases.

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