Development of a mouse model expressing a bifunctional glutathione-synthesizing enzyme to study glutathione limitation in vivo

Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology an...

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Published in:The Journal of biological chemistry 2024-02, Vol.300 (2), p.105645-105645, Article 105645
Main Authors: Timson, Rebecca C., Khan, Artem, Uygur, Beste, Saad, Marwa, Yeh, Hsi-Wen, DelGaudio, Nicole L., Weber, Ross, Alwaseem, Hanan, Gao, Jing, Yang, Chingwen, Birsoy, Kıvanç
Format: Article
Language:English
Subjects:
animal model
antioxidant
cell death
cell metabolism
ferroptosis
glutathione
iron metabolism
iron-sulfur clusters
metabolism
reactive oxygen species (ROS)
transgenic mice
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Summary:Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.
ISSN:0021-9258
1083-351X
DOI:10.1016/j.jbc.2024.105645