Use of Genetically Encoded Tools to Interrogate Mechanisms of Glutathione Homeostasis from Mitochondria to Mice

Author

Rebecca Timson

Date of Award

2024

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Birsoy Laboratory

Abstract

Glutathione is an evolutionarily ancient molecule that underlies many critical biological processes across all categories of life. It acts as a cofactor in some metabolic reactions, modifies proteins to modulate their activity, and participates in detoxification of xenobiotic compounds. Despite these wide-ranging functions, glutathione is best known as an antioxidant. Some consider it akin to the fountain of youth because it decreases throughout the course of aging while oxidative damage accumulates.In addition to aging, glutathione dysregulation occurs in a plethora of diseases, including neurodegenerative diseases like Alzheimer’s and Parkinson’s Disease, liver disease, and metabolic disease, among others. However, the specific roles of glutathione that contribute to disease progression remain poorly understood. In part, this is because the function of glutathione in cellular metabolism is still not fully characterized. Glutathione is thought to be critical for mitochondrial function, but it is not known how glutathione gets into mitochondria. Because of this, it has been very difficult to specifically deplete mitochondrial glutathione, resulting in circumstantial evidence that maintenance of glutathione abundance is critical for mitochondrial function. We set out to expand the tools available to characterize the function of compartmentalized glutathione pools. In the first half of this work, I engineered a bacterial enzyme to synthesize glutathione in mitochondria, effectively bypassing both endogenous glutathione synthesis and transport into mitochondria. Using this tool, I tried to identify the mitochondrial glutathione transporter by performing CRISPR-Cas9 screens that relied upon the assumption that the mitochondrial glutathione transporter(s) is/are essential for cell proliferation, and that enabling mitochondrial glutathione synthesis would enable identification of these no-longer essential genes. While this limited approach failed to identify the mitochondrial glutathione transporters, we identified new roles for several genes, including a mechanism of resistance to the most prevalently used glutathione synthesis inhibitor. To identify alternative molecules that may inhibit glutathione synthesis, I designed and performed a high-throughput chemical screen. I was able to identify several candidate molecules, although they are not direct inhibitors of glutathione synthesis. Further identification of the targets of these inhibitors may provide additional insight into the function of glutathione in cellular metabolism. Ultimately, in collaboration with several of my colleagues, we used alternative approaches to identify two putative mitochondrial transporters required for mitochondrial glutathione import. We found that, as expected, mitochondrial glutathione is required for cell viability. However, we found that this was due to glutathione’s role as a critical cofactor in iron sulfur biosynthesis, not due to its role as an antioxidant, emphasizing the importance of considering unbiased approaches to determine the function of glutathione in disease progression. In the second half of this work, I developed two mouse models with unregulated glutathione availability. I was surprised to find that that constitutively high glutathione is in compatible with embryonic development, demonstrating that homeostatic mechanisms of maintaining glutathione levels are critical for viability. However, unregulated glutathione synthesis was compatible with life in adult animals and resulted in an increase of GSH up to 5-fold in some tissues. Interestingly, this increase in GSH was compatible with normal tissue metabolism, underscoring that regulation of glutathione synthesis is required for some essential embryonic processes. Future studies with these mouse models with high glutathione will enable interrogation of glutathione sufficiency in disease progression.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Timson, Rebecca, "Use of Genetically Encoded Tools to Interrogate Mechanisms of Glutathione Homeostasis from Mitochondria to Mice" (2024). Student Theses and Dissertations. 785.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/785