Dynamics of Arginine Methylation in Cellular Processes

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Background. The methylation of arginine residues within proteins by enzymes called protein arginine N-methyltransferases (PRMTs), is a modification (PTM) found predominantly in chromatin-associated and RNA-binding proteins, and has been shown to play regulatory roles in many important cellular processes. Once proteolytically liberated, free dimethylated arginines can competitively inhibit nitric oxide synthases and prevent their functions in regulating vascular tone, pain, neurotransmission, and the inflammatory response to microorganisms. This PTM has recently emerged as a biomarker for a growing list of diseases including cancer, cardiovascular, and inflammatory lung diseases. Unlike other transient PTMs, however, arginine methylation is a stable mark on proteins. For the past few years my laboratory has established techniques to study arginine methylation so that we may determine how PRMTs contribute to cellular programming. Recently, we were able to see using proteomic techniques large changes in arginine methylation between proteins from cultured human lung epithelial cells treated with tumor necrosis factor-alpha or lipopolysaccharide and proteins from untreated cells. These experiments also revealed protein mediators of the inflammatory response as new PRMT substrates. Research Program Objectives. The long-term objective of our lab is to understand the independent and combined contributions of PRMTs toward cell proliferation, differentiation, and death. One of our short-term goals, for which we are requesting NSERC support, is to establish PRMT-specific arginine methylation networks in different inflammatory response pathways. Scientific Approach. We will subject cultured human cell lines to treatments that cause an NF-kappaB-mediated inflammatory responses. Proteins from treated and untreated control cells will be harvested and either blotted with dimethylarginine-specific antibodies or used for proteomic analysis as part of an ongoing collaboration with Drs. Leonard Foster and Nichollas Scott at the UBC Centre for High-Throughput Biology. Our proteomic approach takes advantage of a recently reported enrichment strategy for methylated peptides using hydrophilic interaction liquid chromatography prior to sample analysis by mass spectrometry. Methylated proteins from control and treatment groups will then be compared for relative quantitation, allowing us to observe changes in PTMs as a way to establish arginine methylation networks in inflammation. The next step will be to determine specific PRMT involvement in inflammatory pathways either by employing cell-based assays in which specific PRMT expression levels will be decreased via siRNA, or by pharmacologically inhibiting specific PRMTs in cultured cells with small molecules. We will also co-immunoprecipitate methylated proteins from cells to determine which PRMTs associate with their substrates by Western blot and mass spectrometry. These substrates will then be tested with in vitro methylation assays to demonstrate PRMT substrate specificity. Impact. Arginine methylation has been demonstrated to perform specific functions in important cellular processes, but this PTM has yet to be studied in the broader context of the cell. By observing arginine methylation changes on the global scale of the proteome, we will identify substrates whose modifications may serve critical roles in inflammatory events, and demonstrate how this PTM participates in various signaling pathways. This work will provide mechanistic insight into how the integration of PRMT activities contributes to cellular programming and ultimately determines cell fate. Our findings should impact the fields of cancer biology, inflammation, and biochemistry.