Analysis of the protein lysine methylome of SARS-CoV-2

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Lysine methylation (Kme) is a widespread post-translational protein modification, which is introduced by protein lysine methyltransferases (PKMTs). It regulates many biological processes, including protein stability, signaling pathways, immune response, gene expression and cell cycle. It is well established that proteins of intracellular pathogens can be modified by PTM introducing enzymes from the host cell, but lysine methylation sites of SARS-CoV-2 proteins by human PKMTs has not yet been studied. The SARS-CoV-2 proteome contains 562 lysine residues and it is the aim of this project to investigate their potential methylation by 24 human PKMTs in a systematic manner. Most of the selected PKMTs have already been shown to be active on non-histone proteins in the past. The panel of PKMTs include most of the well-investigated and highly active enzymes of this class in human cells. Therefore, this project will provide the first example of a biochemical Kme analysis systematically covering all proteins of one organism. The methylation analysis of SARS-CoV-2 proteins will comprise three-steps: first to identify and confirm Kme sites in peptides derived from SARS-CoV-2 proteins using peptide arrays presenting all lysine residues of the SARS-CoV-2 proteome (Aim 1). Then to detect Kme sites in SARS-CoV-2 proteins using purified proteins methylated in vitro (Aim 2), and finally to detect Kme events in SARS-CoV-2 proteins in human cells (Aim 3). The project will provide unbiased activity data of 24 PKMTs based on about 590 lysine residues in different sequence contexts (562 SARS-CoV-2 lysine residues and 1-2 reference substrates for each PKMT). Using this large amount of methylation data, it is planned to identify new substrates in sequence contexts differing from the known specificity profiles of the corresponding PKMTs. These newly discovered substrates with diverging sequence will then be subjected to a systematic specificity analysis by testing methylation of all single mutations of this sequence to obtain comprehensive and unbiased specificity data for the corresponding PKMT (Aim 4). Selected new substrates will also be investigated structurally (Aim 5). The project planned here will have important impacts in the SARS-CoV-2 and PKMT research fields. The SARS-CoV-2 protein methylation data will represent the first complete Kme analysis including all proteins of an organism. They will enable follow-up studies regarding the biological effects of the Kme events and their role in SARS-CoV-2 pathogenicity. Moreover, our data will allow extrapolations regarding the global occurrence of lysine methylation in proteins and significantly improve our understanding of the substrate recognition of PKMTs as an important class of enzymes.