In vivo methylation of OLA1 revealed by activity-based target profiling of NTMT1.

Kaimin Jia,Bingbing Wu,Yulan Xiong,Ping Li,Wei Wu,Ruben Shrestha,Gaochao Huang

doi:10.1039/c9sc02550b

Kaimin Jia, Bingbing Wu + Show 5 more

Open Access

https://doi.org/10.1039/c9sc02550b

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Journal: Chemical Science	Publication Date: Jan 1, 2019
Citations: 12	License type: CC BY 3.0

Affiliation: St Vincent Hospital, Kansas State University

Abstract

N-Terminal methyltransferase 1 (NTMT1) catalyzes the N-terminal methylation of proteins with a specific N-terminal motif after methionine removal. Aberrant N-terminal methylation has been implicated in several cancers and developmental diseases. Together with motif sequence and signal peptide analyses, activity-based substrate profiling of NTMT1 utilizing (E)-hex-2-en-5-ynyl-S-adenosyl-l-methionine (Hey-SAM) revealed 72 potential targets, which include several previously confirmed ones and many unknowns. Target validation using normal and NTMT1 knock-out (KO) HEK293FT cells generated by CRISPR-Cas9 demonstrated that Obg-like ATPase 1 (OLA1), a protein involved in many critical cellular functions, is methylated in vivo by NTMT1. Additionally, Hey-SAM synthesis achieved ≥98% yield for SAH conversion.

Highlights

One of the most common post-translational modi cations catalyzed by protein methyltransferases (PMTs),[1] plays important roles in regulating epigenetics and cell signalling pathways.[2,3] While most studies focus on protein arginine and lysine methyltransferases (PRMT and PKMT, respectively),[4,5,6] the recent discovery of N-Terminal methyltransferase 1 (NTMT1) has attracted much attention due to its wide substrate spectrum toward nonhistone proteins.[7,8,9,10,11,12] Using S-adenosyl-L-methionine (SAM) as a methyl donor, NTMT1 methylates proteins with a speci c Nterminal sequence of XPK a er initial methionine removal.[11,12] Subsequent study employing limited peptide arrays showed that NTMT1 has an expanded substrate speci city,[13] suggesting that N-terminal methylation is a widespread post-translational modi cation.N-terminal methylation has been established as a regulator of protein–DNA and protein–protein interactions for a number of proteins, such as RCC1,11 CENPA/B,7,9,14 DDB2,8 PARP3,10 and MYL9,15 playing important roles in cell mitotic progression, DNA damage repair, and regulation of protein function.[16]
Target validation using normal and N-Terminal methyltransferase 1 (NTMT1) knock-out (KO) HEK293FT cells generated by CRISPR-Cas[9] demonstrated that Obg-like ATPase 1 (OLA1), a protein involved in many critical cellular functions, is methylated in vivo by NTMT1
To identify a SAM analogue that can be accepted by wt NTMT1, we examined all SAM analogues reported so far.[27]

Summary

Introduction

One of the most common post-translational modi cations catalyzed by protein methyltransferases (PMTs),[1] plays important roles in regulating epigenetics and cell signalling pathways.[2,3] While most studies focus on protein arginine and lysine methyltransferases (PRMT and PKMT, respectively),[4,5,6] the recent discovery of NTMT1 has attracted much attention due to its wide substrate spectrum toward nonhistone proteins.[7,8,9,10,11,12] Using S-adenosyl-L-methionine (SAM) as a methyl donor, NTMT1 methylates proteins with a speci c Nterminal sequence of XPK a er initial methionine removal.[11,12] Subsequent study employing limited peptide arrays showed that NTMT1 has an expanded substrate speci city,[13] suggesting that N-terminal methylation is a widespread post-translational modi cation.N-terminal methylation has been established as a regulator of protein–DNA and protein–protein interactions for a number of proteins, such as RCC1,11 CENPA/B,7,9,14 DDB2,8 PARP3,10 and MYL9,15 playing important roles in cell mitotic progression, DNA damage repair, and regulation of protein function.[16]. In vivo methylation of OLA1 revealed by activity-based target profiling of NTMT1 Target validation using normal and NTMT1 knock-out HEK293FT cells edited by CRISPR-Cas[9] demonstrated that Obg-like ATPase 1 (OLA1), a protein involved in many critical cellular functions, is methylated in vivo by NTMT1.

Results

Conclusion