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

***尽管Lys甲基化在正常生理和复杂疾病（如癌症和神经系统疾病）的发病机制中具有重要意义，但除了对组蛋白修饰的作用外，我们对这种翻译后修饰（PTM）知之甚少。我们建议定义赖氨酸甲基转移酶（KMTs）和赖氨酸去甲基化酶（KDMs）的酶-底物网络，以了解赖氨酸甲基化的功能。作为实现这一宏伟目标的重要一步，我们将重点研究修饰H3K4甲基化的kmt和kdm， H3K4甲基化是一种与活性转录基因相关的组蛋白标记。一方面，大量的kmt（>2）和kdm （bbb6）能够修饰标记，另一方面，在相应基因被切除的小鼠中，H3K4甲基化的重要性得到了强调。我们假设h3k4特异性kmt / kdm实际上是非特异性的；相反，它们具有针对额外组蛋白和非组蛋白的活性。在这项研究中，我们将确定底物特异性，并确定修饰h3k4的kmt和kdm的甲基化网络。我们的目标是：******(1)我们将使用肽微阵列和互补蛋白质组学和生物信息学方法，通过体外甲基化/去甲基化分析来确定kmt和kdm的底物特异性。所得到的给定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能被用作功能探针的抑制剂。******(2)我们将利用先进的质谱技术阐明细胞中的KTM/ kdm -底物网络。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（多重反应监测）分析，以（通过MS/MS）鉴定新的底物或（通过MRM）验证上述预测的KTM/KDM-底物网络。******(3)我们将描述h3k4修饰的kmt / kdm在干细胞分化中的功能。具体来说，我们将从小鼠胚胎干细胞中敲除或敲除单个kmt / kdm，并表征由此产生的克隆在神经分化中的缺陷。******我们的工作不仅将揭示许多新的赖氨酸甲基化位点，并提供前所未有的见解，以修饰酶的特异性和功能，集中在H3K4上，本文开发的方法可用于全球范围内赖氨酸甲基化的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得以发展。

***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.