Specificity analysis of human protein lysine methyltransferases and proteomwide identification of novel substrate proteins

蛋白赖氨酸甲基转移酶（PKMT）是在2000年发现的，当时发现SUV39H1在赖氨酸9位点甲基化组蛋白H3。然而，一些最初被确定为组蛋白修饰酶的pkmt后来被发现也可以将非组蛋白底物甲基化，并且蛋白质赖氨酸甲基化已被认为是涉及各种过程的重要翻译后修饰。今天，在人类蛋白质组中报道了8000多个赖氨酸甲基化事件（Phosphosite Plus, 2016），但由于蛋白质组学方法的局限性，可能还有更多的事件有待发现。此外，对于大多数已描述的赖氨酸甲基化事件，负责的PKMT尚未确定，对于其中许多事件，甲基化的生物学作用尚不清楚。此外，大多数pkmt到目前为止只鉴定了少数底物。因此，鉴定负责特定蛋白质甲基化事件的酶是生化研究的关键挑战。此外，迫切需要复杂的实验来解决特定赖氨酸甲基化事件的细胞作用。我们开发并成功地采用了一种新的方法来鉴定pkmt的非组蛋白靶点。首先用肽阵列测定pkmt的底物特异性。特异性谱可以用来搜索候选底物的人类蛋白质组。然后在体外和细胞中研究这些肽和蛋白质底物的甲基化。最后，我们计划开发细胞分析来揭示甲基化事件的生物学作用。本申请的目标是继续这项工作，并进一步推进以下工作包（WP）：WP1：克隆、表达和纯化额外的人类PKMTsWP2：确定pkmtts的特异性特征并鉴定新的肽底物wp3：鉴定体外和细胞中的新蛋白底物swp4：研究甲基化事件的细胞作用swp5；开发一个Web服务器，可以识别pkmt对和赖氨酸甲基化事件，使我们的研究结果可用于该领域的所有研究。

Protein lysine methyltransferases (PKMT) were discovered in 2000 when it was shown that SUV39H1 methylates histone H3 at lysine 9. However, several PKMTs that were initially identified as histone modifying enzymes were later found to methylate non-histone substrates as well and protein lysine methylation has been recognized as an important post-translational modification involved in various processes. Today, more than 8000 lysine methylation events are reported in the human proteome (Phosphosite Plus, 2016), but due to limitations in proteomics approaches, it is likely that many more are still to be discovered. Moreover, for most described lysine methylation events the responsible PKMT has not been identified and for many of them, the biological role of the methylation is not known. Moreover, for most PKMTs only few substrates have been identified up to now. For this reason, the identification of enzymes responsible for specific protein methylation events is a critical challenge for biochemical research. Moreover, sophisticated experiments addressing the cellular role of specific lysine methylation events are urgently needed. We developed and successfully employed a novel approach for the identification of non-histone targets of PKMTs. It starts with the determination of the substrate specificity of PKMTs with peptide arrays. The specificity profile can then be used to search the human proteome for candidate substrates. The methylation of these peptide and protein substrates is then investigated in vitro and in cells. Finally, we plan to develop cellular assays to uncover the biological role of the methylation event. The goals of this application are to continue this work and further advance it as described in the following work packages (WP):WP1: Cloning, expression and purification of additional human PKMTsWP2: Determination of the specificity profile of PKMTs and identify novel peptide substratesWP3: Identification of novel protein substrates in vitro and in cellsWP4: Investigation of the cellular role of the methylation eventsWP5: Development of a Web server allowing to identify pairs of PKMTs and lysine methylation events to make the results of our study available for all researches in the field.