PRMT3 is a distinct member of the protein arginine N-methyltransferase family. Conferral of substrate specificity by a zinc-finger domain

S-Adenosyl-l-methionine-dependent protein arginine N-methyltransferases (PRMTs) catalyze the methylation of arginine residues within a variety of proteins. At least four distinct mammalian family members have now been described, including PRMT1, PRMT3, CARM1/PRMT4, and JBP1/PRMT5. To more fully define the physiological role of PRMT3, we characterized its unique putative zinc-finger domain and how it can affect its enzymatic activity. Here we show that PRMT3 does contain a single zinc-finger domain in its amino terminus. Although the zinc-liganded form of this domain is not required for methylation of an artificial substrate such as the glutathione S-transferase-fibrillarin amino-terminal fusion protein (GST-GAR), it is required for the enzyme to recognize RNA-associated substrates in RAT1 cell extracts. The recombinant form of PRMT3 is inhibited by high concentrations of ZnCl(2) as well as N-ethylmaleimide, reagents that can modify cysteine sulfhydryl groups. We found that we could distinguish PRMT family members by their sensitivity to these reagents; JBP1/PRMT5 and Hsl7 methyltransferases were inhibited in a similar manner as PRMT3, whereas Rmt1, PRMT1, and CARM1/PRMT4 were not affected. We were also able to define differences in these enzymes by their sensitivity to inhibition by Tris and free arginine. Finally, we found that the treatment of RAT1 cell extracts with N-ethylmaleimide leads to a loss of the major PRMT1-associated activity that was immune to inhibition under the same conditions as a GST fusion protein. These results suggest that native forms of PRMTs can have different properties than their GST-catalytic chain fusion protein counterparts, which may lack associated noncatalytic subunits.     

Design and synthesis of novel PRMT1 inhibitors and investigation of their binding preferences using molecular modelling

Protein arginine methyltransferase 1 (PRMT1) catalyses the methylation of substrate arginine by transferring the methyl group from SAM (S-adenosyl-l-methionine), which leads to the formation of S-adenosyl homocysteine (SAH) and methylated arginine. We have shown previously that the Asp84 on PRMT1 could be a potential inhibitor binding site. In the current study, 28 compounds were designed and synthesized that were predicted to bind the Asp84 and substrate arginine sites together. Among them, 6 compounds were identified as potential PRMT1 inhibitors, and showed strong inhibitory effects on cancer cell lines, especially HepG2. The most potent PRMT1 inhibitor, compound 13d, was selected for molecular dynamic simulations to investigate binding poses. Based on the free energy calculations and structural analysis, we predicted that the ethylenediamine group would tightly bind to Asp84, and the trifluoromethyl group should occupy part of substrate arginine binding site, which is consistent with our original goal. Our results show for the first time that PRMT1 inhibitors can target the Asp84 binding site, which will be helpful for future drug discovery studies.