INVESTIGATING THE PROTEIN ALPHA-N-TERMINAL METHYLTRANSFERASE 1 (NTMT1)-MEDIATED PATHWAYS

α-N-methylation (Nα-methylation), a crucial post-translational modification catalyzed by protein N-terminal methyltransferases (NTMTs), involves the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to the Na amino group of substrate proteins. NTMT1/2 are known to methylate canonical Nα sequences starting with X-P-K/R, but recent findings indicate that noncanonical sequences like SSKR and ASKR are also substrates, suggesting a broader substrate scope than previously recognized. Despite the prediction of over 300 potential human protein substrates, only a small fraction has been validated and the functions of Nα-methylation remain largely unexplored. In addition, the specific Nα-methylation readers that recognize and translate this modification into downstream effects have not yet been identified, leaving a significant gap in our understanding of Nα-methylation.

To elucidate NTMT1-mediated pathways, we designed and synthesized Nα-methylated PPKT-photoaffinity probes to identify the readers of Nα-methylation, along with unmethylated analog as the control. Using photoaffinity labeling, we identified developmentally regulated GTP-binding protein 1 (DRG1) and peptidyl-prolyl cis-trans isomerase A (PPIA) as top candidates after validation with biochemical and cellular assays. Additionally, we characterized protein arginine deiminase 1 (PAD1) and zinc finger CCCH-type containing 15 (ZC3H15) as NTMT1 substrates. Our results demonstrated that NTMT1-mediated methylation increases the half-life of PAD1, whereas it promotes degradation and reduces protein half-life of ZC3H15, indicating that the impact of Nα-methylation on protein stability is substrate-dependent. Our proteomic analyses revealed different protein interactions between unmethylated and Nα-methylated PAD1/ZC3H15, suggesting a regulatory role for Nα-methylation in modulating protein-protein interactions. Specifically, proteasome subunit and ubiquitin-related proteins were identified as potential Nα-methylation readers of ZC3H15, implicating them in its degradation pathway. Finally, we assessed the cellular selectivity of NTMT1 bisubstrate inhibitor NAH-C3-GPKK. Chemoproteomic studies confirmed NTMT1 as the top enriched target for NAH-C3-GPKK, demonstrating the inhibitor’s high selectivity for NTMT1. Similarly, we also evaluated the selectivity of two Nicotinamide N-Methyltransferase bisubstrate inhibitors LL320 and II399, with II399 incorporating the unconventional SAM mimic, displaying improved selectivity over LL320. These findings confirm that using the unconventional SAM mimic is an effective approach to create highly selective and cell-active inhibitors for SAM-dependent methyltransferases.