Substrate Specificity of the HEMK2 Protein Glutamine Methyltransferase and Identification of Novel Substrates

Abstract

Bacterial HEMK2 homologs initially had been proposed to be involved in heme biogenesis or to function as adenine DNA methyltransferase. Later it was shown that this family of enzymes has protein glutamine methyltransferase activity, and they methylate the glutamine residue in the GGQ motif of ribosomal translation termination factors. The murine HEMK2 enzyme methylates Gln 185 of the eukaryotic translation termination factor eRF1. We have employed peptide array libraries to investigate the peptide sequence recognition specificity of murine HEMK2. Our data show that HEMK2 requires a GQX 3 R motif for methylation activity. In addition, amino acid preferences were observed between the ؊3 and ؉7 positions of the peptide substrate (considering the target glutamine as 0), including a preference for Ser, Arg, and Gly at the ؉1 and a preference for Arg at the ؉7 position. Based on our specificity profile, we identified several human proteins that contain putative HEMK2 methylation sites and show that HEMK2 methylates 58 novel peptide substrates. After cloning, expression, and purification of the corresponding protein domains, we confirmed methylation for 11 of them at the protein level. Transfected CHD5 (chromodomain helicase DNA-binding protein 5) and NUT (nuclear protein in testis) were also demonstrated to be methylated by HEMK2 in human HEK293 cells. Our data expand the range of proteins potentially subjected to glutamine methylation significantly, but further investigation will be required to understand the function of HEMK2-mediated methylation in proteins other than eRF1.

Systematic Functional Characterization of Human 21st Chromosome Orthologs inCaenorhabditis elegans

“…To our knowledge, MTQ-2 has not been implicated in nervous system function in any animal model. The mammalian ortholog of mtq-2 , N6AMT1 , was originally named on the basis of the presence of an amino acid motif (D/N/S)PP(Y/FW) which is characteristic of adenine methyltransferases ( Bujnicki and Radlinska 1999 ; Kusevic et al 2016 ). However, no evidence of adenine methylation by MTQ-2 protein has been found in either yeast or mouse, suggesting that N6AMT1 may be a misnomer ( Liu et al 2010 ; Ratel et al 2006 ).…”

Mmp10 is required for post-translational methylation of arginine at the active site of methyl-coenzyme M reductase

“…Because Mmp10 is probably highly expressed in this strain, it is possible that other proteins might have been post-translationally modified. SAM-dependent protein methyltransferases are known to specifically recognize the amino acid sequences flanking the amino acid to be methylated (9, 36, 37). Thus, the M. maripaludis proteome was searched in silico for the potential methylation consensus sequence PxRR 275 (A/S)R(G/A).…”

Trm112, a Protein Activator of Methyltransferases Modifying Actors of the Eukaryotic Translational Apparatus

“…This is further supported by the growth defect phenotype of yeast cells lacking the MTQ2 gene [13,14,63], the cell proliferation defect with arrest in the G1 phase of murine embryonic stem cells depleted of the PRED28α isoform [65], the early mouse embryonic lethality upon disruption of PRED28α isoform [66] and the two-fold reduction in HEK293 human cells growth rate resulting from stable knock-down of the HEMK2 gene [66]. Finally, murine PRED28α and human HEMK2 proteins appear to have a broad substrate specificity [67]. Hence, future studies aimed at clarifying the role of the eukaryotic Mtq2-Trm112 complexes and of the methylation they are catalyzing are needed.…”