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

赖氨酸甲基化(KME)是一种广泛存在的蛋白质翻译后修饰，由蛋白质赖氨酸甲基转移酶(PKMT)引入。它调节许多生物过程，包括蛋白质稳定性、信号通路、免疫反应、基因表达和细胞周期。细胞内病原体的蛋白质可以通过PTM引入宿主细胞的酶来修饰，但人PKMT对SARS-CoV-2蛋白的赖氨酸甲基化位点还没有研究过。SARS-CoV-2蛋白质组包含562个赖氨酸残基，本项目的目的是系统地研究24个人PKMT的潜在甲基化。过去已经证明，大多数被选择的PKMT对非组蛋白蛋白都有活性。PKMT小组包括人类细胞中大多数经过充分研究和高活性的这类酶。因此，该项目将提供系统地覆盖一个生物体的所有蛋白质的生化KME分析的第一个例子。SARS-CoV-2蛋白的甲基化分析将包括三个步骤：首先，利用呈现SARS-CoV-2蛋白质组所有赖氨酸残基的多肽阵列，鉴定和确认SARS-CoV-2蛋白多肽中的KME位点(目标1)。然后用体外甲基化的纯化蛋白检测SARS-CoV-2蛋白中的KME位点(Aim 2)，最后检测人细胞中SARS-CoV-2蛋白中的KME事件(Aim 3)。该项目将提供基于不同序列环境中约590个赖氨酸残基的24个PKMT的无偏活性数据(562个SARS-CoV-2赖氨酸残基和每个PKMT的1-2个参考底物)。利用大量的甲基化数据，计划在不同于相应PKMT的已知特异性图谱的序列环境中识别新的底物。然后对这些新发现的具有不同序列的底物进行系统的特异性分析，测试该序列的所有单一突变的甲基化，以获得相应PKMT的全面和公正的特异性数据(AIM 4)。还将对选定的新基质进行结构调查(目标5)。这里计划的项目将在SARS-CoV-2和PKMT研究领域产生重要影响。SARS-CoV-2蛋白质甲基化数据将代表第一次完整的KME分析，包括生物体的所有蛋白质。它们将使关于KME事件的生物效应及其在SARS-CoV-2致病性中的作用的后续研究成为可能。此外，我们的数据将允许外推蛋白质中赖氨酸甲基化的全球发生，并显著提高我们对PKMT作为一类重要酶的底物识别的理解。

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.