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Rowley, Michael James Anthony

University of British Columbia

Arginine methylation is a prevalent post-translational modification in biology present on 0.5% of total arginine proteins in mammalian cells. Protein arginine methylation is a key epigenetic modification on histone proteins, that can promote or inhibit gene expression depending on the type and site of arginine methylation. Protein arginine N-methyltransferases (PRMTs) are the epigenetic writers responsible for arginine methylation and have emerged as therapeutic targets in several health disorders including neurodegenerative and cardiovascular disease, and cancer. PRMT2 was recently added to the list of epigenetic writers associated with oncogenic transcriptional programming, specifically in glioblastoma (GBM), hepatocellular carcinoma (HCC), and renal cell carcinoma (RCC). Unlike the other PRMT enzymes, the function of PRMT2 in cells remains enigmatic due to its lackluster in vitro methylation kinetics, making it difficult to characterize. The purpose of this research is to develop techniques for measuring PRMT2 interactions and to use these techniques to characterize how PRMT2 interacts with other proteins and histones. The development of the filter binding and phosphor screening (FBAPS) assay improved the throughput of the traditional filter binding assay used to characterize kinetic parameters of PRMT enzymes. Characterization of PRMT2 through filter binding assay revealed that exposing the radiolabeled S-adenosyl-L-methionine (SAM) cofactor to elevated temperatures for long periods of time create degradation products that confound analysis. We used differential scanning fluorimetry (DSF) to characterize PRMT2 thermal stability to identify conditions that may improve PRMT2 catalytic activity and to measure PRMT2 binding interactions. We identified several excipients that could thermally stabilize PRMT2 but did not improve catalytic activity. Additionally, we found that PRMT2 can interact with SAM and sinefungin (a SAM analogue) in a thermally stabilizing manner. Using kinetic and biophysical assays, we explored a noncatalytic role for PRMT2 in histone methylation by investigating interactions between PRMT2, histones, and other PRMTs. The result of this study indicated that PRMT2 interacts with PRMT1 in a cofactor- and domain-dependent manner. Further PRMT2 could modulate the histone methylation capacity of PRMT1 in a macromolecular-dependent manner. This work reveals the ability of PRMT2 to serve a noncatalytic role through its SH3 domain in generating site-specific histone methylation marks.

Text

2024-05-09

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88263

Pharmaceutical Sciences

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/