Mechanistic investigations of protein arginine N-methyltransferases and elucidation of their implications in the yeast stress response
department or school: Pharmaceutical Sciences, Faculty of
data provider: DSpace
degree campus: UBCV
creator: Brown, Jennifer
issued: 2023-09-30T07:00:00Z
issued: 2021
related degree: Doctor of Philosophy - PhD
degree grantor: University of British Columbia
description: 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 c…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/79789
note: MECHANISTIC INVESTIGATIONS OF PROTEIN ARGININE N-METHYLTRANSFERASES AND ELUCIDATION OF THEIR IMPLICATIONS IN THE YEAST STRESS RESPONSE by Jennifer Brown B.Sc. (Hons), Trent University, 2014 B.Ed., Queen’s University, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmaceutical Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2021 © Jennifer Brown, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: MECHANISTIC INVESTIGATIONS OF PROTEIN ARGININE N-METHYLTRANSFERASES AND ELUCIDATION OF THEIR IMPLICATIONS IN YEAST STRESS RESPONSE submitted by Jennifer Brown in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmaceutical Sciences Examining Committee: Dr. Adam Frankel, Faculty of Pharmaceutical Sciences, University of British Columbia Supervisor Dr. Brent Page, Faculty of Pharmaceutical Sciences, University of British Columbia Supervisory Committee Member Dr. Calvin Yip, Department of Biochemistry and Molecular Biology, University of British Columbia University Examiner Dr. Kirsten Wolthers, Department of Chemistry, University of British Columbia, Okanagan University Examiner Additional Supervisory Committee Members: Dr. LeAnn Howe, Department of Biochemistry and Molecular Biology, University of British Columbia Supervisory Committee Member Dr. Thibault Mayor, Department of Biochemistry and Molecular Biology, University of British Columbia Supervisory Committee Member iii Abstract 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 be…
has type: Thesis/Dissertation
date issued: 2021-11
is shown at: 10.14288/1.0402332
language: eng
degree discipline: Pharmaceutical Sciences
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
contributor: Frankel, Adam
type: Text
identifier URI: http://hdl.handle.net/2429/79789