PROJECT SUMMARY Protein arginine methylation catalyzed by Protein arginine methyltransferase 1 (PRMT1) is of intense current interest as an anti-cancer therapeutic target, as well as a mediator of lung, kidney, neurodegenerative and cardiovascular pathologies. To date, the majority of studies characterizing the consequences of PRMT1-mediated protein methylation have not considered the biochemical mechanisms by which PRMT1 activity is altered, which is pivotal in understanding how PRMT1 participates in cellular homeostasis, the progression of the aforementioned disease states, and in developing treatment protocols to control PRMT1-dependent cellular events. The two hypotheses being tested in this project are: 1) changes in oligomeric state and site-specific cysteine oxidation of the PRMT1 protein affect substrate targeting and activity, and 2) that a cysteine residue in PRMT1 can be harnessed to create/upgrade isoform-specific PRMT1 inhibitors. In Aim 1, novel fluorescence-based biophysical methods will be used to characterize the dynamics of PRMT1 oligomer formation in intact cells. Strategically designed variants of PRMT1 which present as tetramers, dimers, or monomers will be used to identify binding and catalytic differences in the targeting by each oligomer, aiding in the ongoing effort to understand molecular recognition rules of PRMT1 for its substrates. The effect of sulfenylation at a cysteine residue near the active site of PRMT1 will also be characterized, allowing for the development of stable oxidized and reduced mimics of PRMT1 to be used as research tools in areas of human health affected by oxidative stress. The objectives in Aim 1 are built upon initial in vitro findings by the PI and are expected to apply to the long-term goal of characterizing mechanisms of regulating PRMT1 activity in vivo. In Aim 2, the strategy of using a nucleophilic cysteine near the active site of PRMT1 to enable covalent inhibition by adenosine derivatives will be explored. This strategy would be a valuable way to salvage inhibitors that show a lack of isoform-specificity, but otherwise perform well to inhibit PRMT activity. Covalent inhibitors represent a novel, rationale avenue for developing PRMT1 drugs and research tools. Undergraduate and graduate researchers with interests in future careers in biomedical research or medicine will be involved in the project and receive training in experimental design and analysis, notebook keeping, scientific writing and presentation.

Project Narrative PRMT1 is a human protein that plays a critical role in the ability of cells to communicate and organize chemical reactions in the cell. In this project we will investigate how grouping PRMT1 molecules together and how stress damage of PRMT1 affects the ability of the protein to function. Additionally, we will test a new way of controlling the activity of PRMT1 using designed small molecules.