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

蛋白质赖氨酸甲基转移酶（PKMT）是在2000年发现的，当时显示SUV 39 H1在赖氨酸9处甲基化组蛋白H3。然而，最初被鉴定为组蛋白修饰酶的几种PKMT后来被发现也甲基化非组蛋白底物，并且蛋白质赖氨酸甲基化已被认为是参与各种过程的重要翻译后修饰。今天，在人类蛋白质组中报告了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，可能还有更多的赖氨酸甲基化事件有待发现。此外，对于大多数描述的赖氨酸甲基化事件，负责的PKMT尚未被鉴定，并且对于其中许多，甲基化的生物学作用尚不清楚。此外，对于大多数PKMT，迄今为止仅鉴定了少数底物。因此，鉴定负责特定蛋白质甲基化事件的酶是生物化学研究的关键挑战。此外，解决特定赖氨酸甲基化事件的细胞作用的复杂实验是迫切需要的。我们开发并成功地采用了一种新的方法来识别PKMT的非组蛋白靶点。它从用肽阵列测定PKMT的底物特异性开始。然后可以使用特异性谱来搜索人类蛋白质组中的候选底物。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，我们计划开发细胞测定来揭示甲基化事件的生物学作用。本申请的目的是继续这项工作并进一步推进它，如以下工作包（WP）中所述：WP 1：克隆、表达和纯化另外的人PKMT WP 2：测定PKMT的特异性谱并鉴定新的肽底物WP 3：体外和细胞中新蛋白底物的鉴定WP 4：甲基化事件的细胞作用的研究WP 5：开发一个Web服务器，可以识别PKMT和赖氨酸甲基化事件对，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.