Decoding PRMT1: Studies on the catalytic mechanism, regulation, inhibition, and crosstalk of PRMT1-dependent methylation.

摘要

Arginine methylation is catalyzed by the protein arginine methyltransferase (PRMT) family of enzymes, which transfer a methyl group from S-adenosylmethionine (SAM) to the guanidinium group of an arginine residue. This reaction first produces monomethylated arginine (MMA) that can then be further methylated to produce either asymmetrically dimethylated arginine (ADMA) or symmetrically dimethylated arginine (SDMA). There are nine PRMT family members described to date, with PRMT1 as the predominant member, suggested to be responsible for ~85% of asymmetric dimethylation. In addition, PRMT1-dependent methylation likely plays a significant role in a plethora of diseases (e.g., cancer, heart disease, and ALS). These observations render it imperative that the isozyme be more thoroughly characterized and suggests that potent and selective inhibitors may be useful as therapeutics.;Herein we describe our efforts to decode PRMT1-dependent methylation by investigating the catalytic mechanism, the effects of post-translational modifications and protein-protein interactions on activity, the development of potent and selective inhibitors and inactivators, as well as examining crosstalk between arginine methylation and phosphorylation. Using site-directed mutagenesis and unnatural amino acid incorporation, we have identified key active site residues that are critical for catalysis and/or substrate binding, and have determined the effects of phosphorylation, if any, on enzyme activity. In vitro assays with known interacting proteins has increased our knowledge of the regulation of PRMT1 activity by protein-protein interactions. The use of MS/MS analysis aided in the identification of the site of modification for a potent inactivator of the isozyme, C21, and has led to the design of new inhibitors and inactivators that will likely be more potent and selective for not only PRMT1, but PRMT5 as well. Finally, using a peptide based model, we began to investigate crosstalk between arginine methylation and serine/threonine phosphorylation within kinase consensus sequences and hypothesize that it is an important means of regulation in regards to cell signaling. Overall, the results presented in the following chapters have enhanced our understanding of PRMT1-dependent methylation and have opened doors for future studies involving the regulation of the enzyme and inhibitor design.