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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Anatomy and Cell Biology

Supervisor

Lajoie, Patrick

Abstract

The budding yeast Saccharomyces cerevisiae has been used extensively to uncover the genetic mechanisms that control basic cellular processes, including survival, maintenance, and response to stressors. One metric of yeast survival is chronological lifespan (CLS), which is the amount of time non-dividing yeast cells can survive at stationary phase. Variations in CLS following genetic alteration are used to understand the function of specific genes and pathways in cellular aging. Many factors contribute to aging, including accumulation of toxic misfolded secretory proteins in the endoplasmic reticulum (ER stress), to which the cell responds through activation of ER stress signaling pathways, such as the Unfolded Protein Response (UPR).

In this thesis, we first developed new fluorescent assays and a corresponding software program to measure yeast CLS and investigate how ER stress responses impact yeast CLS. Using these assays, we found that inositol was important for CLS, especially in cells with compromised UPR. We also found that UPR-dependent upregulation of the ER chaperone Kar2 is critical for CLS, and we demonstrate that deletion of ER-associated degradation (ERAD) components accelerates chronological aging. We argue that the capacity of the ER quality control machinery to degrade misfolded secretory proteins is an important determinant of ER stress sensitivity and, by extension, yeast CLS. Finally, we aimed to identify ways to modulate ER stress and the UPR; we worked to decipher the mechanism of a compound previously hypothesized to act as a “chemical chaperone” to alleviate ER stress by directly improving protein folding, trafficking, and degradation – tauroursodeoxycholic acid, or TUDCA. We found that, while TUDCA alleviates ER stress, this process can be uncoupled from UPR signaling. We also found little evidence that TUDCA works as a true chemical chaperone. By contrast, it alleviates ER stress indirectly through activation of another stress response, the Cell Wall Integrity (CWI) pathway. We thus discovered a novel mechanism of modulating ER stress. Overall, this thesis has identified new factors influencing ER stress regulation, including chronological aging, protein quality control mechanisms, the CWI pathway, and small molecules such as TUDCA.

Summary for Lay Audience

In order to survive, all living things must be able to respond to insults, or stressors, in their environments. Some common stressors include starvation, toxins, and aging. During aging, cells can also become more sensitive to all other stressors. This can result in age-related diseases, such as Huntington’s disease, Alzheimer’s disease, and type 2 diabetes. In this thesis, the first goal was to determine why this occurs. Using a budding yeast model, we first developed new methods to study yeast aging with existing equipment and a new software program. From there, we were able to identify cellular processes and genes that determine a cell’s lifespan. These included the cell’s natural stress responses (such as the Unfolded Protein Response, or UPR), nutrient levels and pH, and its built-in quality control processes (such as the breakdown of damaged or misfolded proteins). The loss or alteration of any of these, especially when combined with a specific form of stress known as Endoplasmic Reticulum Stress (ER stress), resulted in a short lifespan. Finally, using this information, we aimed to find ways to change cellular sensitivity to ER stress, which could be used to treat age-related diseases. One way to do so utilized a traditional Chinese medicine called TUDCA (a chemical originally derived from bear bile) which is currently in clinical trials for several diseases worsened by aging and ER stress. Despite success in treating these diseases, TUDCA’s mechanism is still a mystery. We hypothesized that it may work by altering the cell’s stress responses to make the cell less sensitive to additional stress. We found that it reduced stress response activation via the UPR, but that it could still reduce stress sensitivity in cells lacking the UPR. It seemed to work from another, unexpected angle: a quality control mechanism that usually maintains yeast cell wall integrity (CWI). This indirect way of altering stress sensitivity could offer a new target for developing stress-reducing drugs. Overall, this thesis discovered new information about what occurs during aging and cellular stress exposure and identifies new ways in which these processes can be chemically altered.

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