Characterizing CMT-causing variants in tryptophanyl- tRNA synthetase

Wu, Fanqi

Characterizing CMT-causing variants in tryptophanyl- tRNA synthetase

Files

Wu_Fanqi_MSc_2025_thesis.pdf (14.23 MB)

Date

2025-12-10

Authors

Wu, Fanqi

Abstract

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that link amino acids to their cognate tRNAs. Neurological conditions, such as Charcot-Marie-Tooth (CMT) disease, have been linked to variants identified in these enzymes. I created a humanized yeast model to assess the underlying disease-causing mechanism associated with two CMT variants, H257R and D314G, present in the human tryptophanyl-tRNA synthetase (WARS1). Using proteomic analysis, protein biochemistry, and yeast growth assays, I found that while both variants retain their typical structure, their stability and function differ. D314G exhibited decreased stability and triggers stress- related pathways, whereas H257R showed mild functional impairment that can be ameliorated by tryptophan supplementation. This work presents the first WARS1 humanized yeast model that was subsequently used to better understand the disease- causing molecular mechanism of WARS1 variants and investigate potential therapeutic approaches, such as amino acid supplementation.

Summary for Lay Audience

Our cells rely on a group of enzymes called aminoacyl-tRNA synthetases to build proteins, the molecules that perform nearly all essential functions of life. When these enzymes malfunction due to genetic mutations, they can disrupt protein production, leading to diseases that affect the nervous system. This research focuses on one of these enzymes, tryptophanyl-tRNA synthetase (WARS1), which has been linked to a hereditary nerve disorder called the Charcot-Marie-Tooth disease. To understand how changes in WARS1 cause disease, this project uses yeast cells as a simple and powerful model system. Yeast shares many key biological processes with human cells, allowing scientists to test human genes in a controlled environment. By introducing human WARS1 and its disease-related variants into yeast, we can observe how these changes affect cell growth, protein production, and stress responses. This study found that yeast carrying disease-associated WARS1 variants show slower growth and signs of stress in pathways that sense nutrients and regulate protein synthesis. These stress responses are like those observed in human cells under nutrient or metabolic stress. Understanding how these mutations disturb cellular balance helps reveal why nerve cells are especially vulnerable in patients. Ultimately, this research lays a foundation for investigating how genetic mutations affect protein homeostasis and cell health. It may also help identify cellular pathways that could be targeted in the future to reduce the effects of similar mutations in human disease.