Systematic characterization of the enzyme-substrate network in protein Lysine methylation

* 尽管赖氨酸甲基化在正常生理学和复杂疾病（如癌症和神经系统疾病）的发病机制中很重要，但我们对这种翻译后修饰（PTM）的了解很少，除了它在组蛋白修饰中的作用。我们在这里提出定义赖氨酸甲基转移酶（KMTs）和甲基化赖氨酸脱甲基酶（KDMs）的酶-底物网络，以了解赖氨酸甲基化的功能。作为实现这一雄心勃勃的目标的重要一步，我们将专注于表征修饰H3 K4甲基化的KMT和KDM，H3 K4甲基化是一种与活跃转录基因相关的组蛋白标记。一方面，H3 K4甲基化的重要性通过能够修饰标记的大量KMT（>12）和KDM（>6），以及另一方面，通过其中相应基因被消除的小鼠中的致死性或严重发育缺陷来强调。我们假设H3 K4特异性KMT/KDM实际上是非特异性的;相反，它们具有对另外的组蛋白和非组蛋白蛋白的活性。对于这项发现资助，我们将确定底物特异性并鉴定H3 K4修饰的KMT和KDM的甲基化网络。我们的目标是：**（1）我们将通过使用肽微阵列和互补蛋白质组学和生物信息学方法的体外甲基化/去甲基化测定来确定KMT和KDM的底物特异性。所得的特定KMT/KDM的特异性谱将使我们能够预测该酶的底物。我们还将使用肽阵列方法来鉴定可用作功能探针的抑制剂。** (2)我们将阐明KTM/KDM底物网络在细胞中的先进质谱。为此，我们将标记KTM/KDM，在细胞中表达，并进行亲和纯化（AP）-串联质谱（MS/MS）和AP-MRM（多反应监测）分析，以鉴定（通过MS/MS）新底物或验证（通过MRM）上述预测的KTM/KDM-底物网络。**（3）我们将表征H3 K4修饰的KMT/KDM在干细胞分化中的功能。具体而言，我们将敲除或敲除小鼠胚胎干细胞中的单个KMT/KDM，并表征所产生的克隆在神经分化中的缺陷。** 我们的工作不仅揭示了许多新的Lys甲基化位点，并为会聚在H3 K4上的修饰酶的特异性和功能提供了前所未有的见解，本文开发的方法可用于在全球范围内对Lys甲基化进行系统鉴定和功能表征。反过来，这将使针对赖氨酸甲基化的疾病干预的创新策略的发展成为可能。

***Despite the importance of Lys methylation in normal physiology and the pathogenesis of complex diseases such as cancer and neurological disorder, we know little about this post-translational modification (PTM) beyond its role on histone modification. We propose here to define the enzyme-substrate network of lysine methyltransferases (KMTs) and methyllyinse demethylases (KDMs) in order to understand the function of Lys methylation. As an important step towards this ambitious goal, we will focus on characterizing the KMTs and KDMs that modify H3K4 methylation, a histone mark that is associated with actively transcribed genes. The importance of H3K4 methylation is underscored, on the one hand, by the large number of KMTs (>12) and KDMs (>6) that are capable of modifying the mark, and, on the other, by the lethality or severe developmental defects in mice in which the corresponding genes are ablated. We hypothesize that the H3K4-specific KMTs/KDMs are in fact non-specific; but rather, they possess activities towards additional histone and non-histone proteins. For this Discovery Grant, we will determine the substrate specificity and identify the methylation network for the H3K4-modifying KMTs and KDMs. Our Objectives are:******(1) We will determine the substrate specificity of the KMTs and KDMs by in vitro methylation/demethylation assays using peptide microarrays and complementary proteomics and bioinformatics approaches. The resulting specificity profile for a given KMT/KDM will allow us to predict the substrates for that enzyme. We will also use the peptide array approach to identify inhibitors that may be exploited as functional probes.******(2) We will elucidate the KTM/KDM-substrate network in cells by advanced mass spectrometry. To this end, we will tag a KTM/KDM, express it in cells and carry out affinity purification (AP)-tandem mass spectrometry (MS/MS) and AP-MRM (Multiple Reaction Monitoring) analysis to identify (by MS/MS) novel substrates or validate (by MRM) the above predicted KTM/KDM-substrate network. ******(3) We will characterize the function of H3K4-modifying KMTs/KDMs in stem cell differentiation. Specifically, we will knock down or knock out individual KMTs/KDMs from mouse embryonic stem cells and characterize defects of the resulting clones in neural differentiation. ******Our work would not only uncover numerous novel Lys methylation sites and provide unprecedented insights into the specificity and function of modifying enzymes that converges on H3K4, the approach developed herein could be used for systematic identification and functional characterization of Lys methylation on a global scale. This, in turn, would enable the development of innovative strategies for disease intervention targeting Lys methylation.