Specificity and novel substrates of human protein glutamine methyltransferases
Final Report Abstract
The murine HEMK2 enzyme is a protein glutamine methyltransferase that methylates Q185 of the eukaryotic translation termination factor eRF1. We have employed SPOT peptide array methylation experiments to investigate the peptide sequence specificity of HEMK2 showing that it requires a G-Q-X3-K motif for methylation activity. In addition, amino acid preferences were observed between the -3 and +7 position of the peptide substrate (considering the target glutamine as 0), including a preference for S, R and G at the +1 and a preference for R at the +7 position. Based on our specificity profile, we identified several human proteins, which 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 Chromodomain helicase DNA binding protein-5 (CHD5) and Nuclear protein in Testis (NUT) 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. Unfortunately, it was not possible to demonstrate the biological role of these additional glutamine methylation events. In the course of the project a specific antibody against methylated Q185 on eRF1 was generated and made available to the research community. Moreover, specificity and activity of other Protein lysine methyltransferases including NSD1, NSD2, Clr4, SET8 and SETD2 were investigated.
Publications
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Activity and specificity of the human SUV39H2 protein lysine methyltransferase. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Vol. 1849. 2015, Issue 1, pp. 55-63.
Schuhmacher MK, Kudithipudi S, Kusevic D, Weirich S, Jeltsch A
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Investigation of the methylation of Numb by the SET8 protein lysine methyltransferase. Scientific Reports, Vol. 5. 2015, Article number: 13813.
Weirich S., Kusevic D., Kudithipudi S., Jeltsch A.
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Substrate Specificity of the HEMK2 Protein Glutamine Methyltransferase and Identification of Novel Substrates. Journal of Biological Chemistry, Vol. 291. 2016, Issue 12, pp. 6124-6133.
Kusevic D, Kudithipudi S, Jeltsch A
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Clr4 specificity and catalytic activity beyond H3K9 methylation. Biochimie, Vol. 135. 2017, pp. 83-88.
Kusevic D., Kudithipudi S., Iglesias N., Moazed D., Jeltsch A.
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Kinetic Analysis of the Inhibition of the NSD1, NSD2 and SETD2 Protein Lysine Methyltransferases by a K36M Oncohistone Peptide. ChemistrySelect Vol. 2. 2017, Issue 29, pp. 9532-9536.
Schuhmacher M.K., Kusevic D., Kudithipudi S.,Jeltsch A.
