UBC Theses and Dissertations
Mechanistic investigations of protein arginine N-methyltransferases and elucidation of their implications in the yeast stress response Brown, Jennifer
Protein arginine N-methyltransferases (PRMTs) catalyze the transfer of methyl groups from the methyl donor S-adenosyl-L-methionine (SAM) to polypeptide substrates. PRMTs are conserved among eukaryotes and many of their biological roles, including transcriptional regulation, DNA repair, and RNA processing, have been well elucidated. Another emerging research area is their role in stress pathways. Because of their significance in biological processes, PRMTs are promising drug targets for many human diseases. Knowledge of an enzyme’s kinetic mechanism and roles in cellular pathways can contribute to effective design of PRMT inhibitors. The purpose of this project was to examine how PRMTs bind to and methylate their substrates, and explore their roles in the stress response using Saccharomyces cerevisiae as a model organism. Using a combination of steady-state enzyme kinetics and biophysical assays, including differential scanning fluorimetry (DSF), we show that the predominant mammalian PRMT binds its substrates in a sequential manner where target substrate binding follows cofactor binding. By using knockout strains, we demonstrate that methylarginines are expelled from cells undergoing stress and that methylarginine expulsion is driven by autophagy. Further, we demonstrate that yeast do not take up methylarginines from their environment because methylarginines can inhibit nitric oxide production which is important for long-term cell survival under stressful growth conditions. Last, through studying in vitro methylation of yeast lysates using recombinantly expressed enzymes, we identify putative yeast methyltransferase Ynl092wp as a protein histidine N-methyltransferase that binds its substrates using a sequential mechanism and predict that Ykl162cp is an RNA methyltransferase. Here, we use a novel approach to examine the PRMT binding mechanism and we demonstrate that this novel technique is applicable to other enzyme families. Further, we demonstrate how yeast cells can evade cell toxicity by controlling methylarginine flux. Therefore, the research described herein encompasses the full importance and impact of PRMTs and their substrates in eukaryotic biological pathways.
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