Activity-based substrate profiling of N-terminal methyltransferase 1 (NTMT1)

Abstract

N-terminal methyltransferase 1 (NTMT1) is a new protein methyltransferase that catalyzes the N-terminal methylation of proteins with a specific N-terminal sequence motif after methionine removal. Protein N-terminal methylation plays multiple cellular roles in DNA-protein interaction, chromatin segregation, and DNA damage repair. Dysregulation of NTMT1 has been implicated in several cancers and developmental disease. Thus, it is vital to identify NTMT1 targets to reveal its involvement in various disease pathology. Here, an activity-based substrate profiling strategy was developed to uncover the potential substrates of NTMT1. For proof of concept, Hey-SAM, a SAM surrogate containing a terminal alkynyl group, was able to alkylate the previously confirmed NTMT1 substrate, regulator of chromosome condensation (RCC1), with a high efficiency in the presence of wild type NTMT1. The alkylated RCC1 was subsequently labelled with a biotin group via Cu(I) catalyzed click chemistry, which allowed its purification by streptavidin beads. This strategy was applied to profile NTMT1 substrates from human cell lysates. Human embryonic kidney 293FT (HEK293FT) cell line was edited to generate an NTMT1 knockout (KO) variant using clustered regularly interspaced short palindromic repeats (CRISPR)-cas9 system, which could produce hypomethylated cell lysates specifically for NTMT1. Potential NTMT1 substrates were treated with Hey-SAM, labeled with a biotin group, enriched by streptavidin beads, separated on SDS-PAGE, digested by trypsin, and identified by LC-MS. Statistical and motif analysis revealed 70 putative NTMT1 targets. Among them, three proteins, RCC1, protein SET, and 60S ribosomal protein L23a (RL23A) were reported to be NTMT1 substrates previously. Validation of selected targets confirmed that polybromo-1 (PB1, peptide analog), SH3 and PX domain-containing protein 2B (SPD2B, peptide analog), 40S ribosomal protein S14 (RS14), and Obg-like ATPase 1 (OLA1) were NTMT1 substrates in vitro. Methylation of OLA1 was further investigated in vivo using normal and NTMT1 KO HEK293FT cells. OLA1 was reported to play multiple roles in several cellular processes including centrosome amplification, antioxidant response, heat shock response and cell growth. Overexpressed OLA1 in normal cells is dimethylated and this modification is abolished in KO cells. These results confirm that NTMT1 is responsible for catalyzing the N-terminal methylation of OLA1 in vivo. Moreover, our preliminary study revealed that methylation of OLA1 does not alter its subcellular location and catalytic activity as an ATPase in vitro. Further study is in need to elucidate the cellular function of this important modification on OLA1.