Identification of the Interactome of Methylated Histones from Human Proteome

描述（由申请人提供）：由组蛋白赖氨酸甲基化介导的分子网络错综复杂。目前，组蛋白赖氨酸甲基化介导的信号通路的作用机制仍不清楚。假设组蛋白在不同赖氨酸残基和不同状态（例如，二-和三甲基化）被相应的结合效应物识别，所述结合效应物随后开启和关闭相关的下游信号传导途径。仍然需要解决的一个主要问题是如何定义每个甲基化组蛋白尾部的甲基化依赖性相互作用组。该项目旨在开发转化技术，以识别与赖氨酸残基处的每个甲基化组蛋白状态结合的效应蛋白的天然库。强大的技术在化学和生物学的接口集成来解决这个问题。具体来说，我们将使用一种新的蛋白质组学技术，称为mRNA展示，其中每个蛋白质都与其自身的mRNA共价连接。将从mRNA展示的人蛋白质组文库中分离与不同甲基化状态结合的蛋白质序列。选择将在非常具体的条件下进行。在捕获甲基化组蛋白尾部/结合配偶体复合物后，甲基化非依赖性结合剂将通过使用未修饰的组蛋白肽去除，而依赖于所需甲基化状态的那些结合配偶体将使用相应的二甲基化或三甲基化组蛋白肽特异性洗脱。由于基因型和表型之间的共轭，所选择的序列可以很容易地扩增迭代轮的选择，使我们能够解决的困难，在确定相对较弱的甲基化依赖的蛋白质-蛋白质相互作用的蛋白质组广泛的规模。作为备用计划，我们将使用含有可交联光亮氨酸部分的甲基化组蛋白尾部肽，以促进捕获与甲基化组蛋白尾部相互作用相对较弱的低丰度蛋白质。虽然我们在这个项目中使用人类蛋白质组解决了这个问题，但该方法可以很容易地应用于模式生物，如C。线虫、斑马鱼和果蝇。 公共卫生相关性：甲基化依赖性相互作用组的可用性将使我们能够破译每个组蛋白甲基化状态的结合效应物，从而大大促进我们对组蛋白赖氨酸甲基化介导的信号通路的理解。它还应该有助于开发治疗和成像剂，允许操纵包括癌症在内的许多人类疾病中涉及的表观遗传过程。

DESCRIPTION (provided by applicant): The molecular networks mediated by histone lysine methylation are intricate and extremely complex. Currently, the action mechanisms of the signaling pathways mediated by histone lysine methylation are still very elusive. It is hypothesized that the histone methylation at different lysine residues and at different states (e.g., di- and trimethylation) are recognized by the corresponding binding effectors that subsequently turn on and off the related downstream signaling pathways. One major problem that remains to be addressed is how to define the methylation-dependent interactome for each methylated histone tail. This project is aimed at developing transforming technologies that allow the identification of the natural repertoire of the effector proteins that bind to each of the methylated histone states at lysine residues. Powerful techniques at the interface of chemistry and biology are integrated to address the problem. Specifically, we will use a novel proteomic technique called mRNA-display in which each protein is covalently linked to its own mRNA. The protein sequences that bind to distinct methylation states will be isolated from an mRNA-displayed human proteome library. The selections will be performed under very specific conditions. After capturing the methylated histone tail/binding partner complexes, the methylation-independent binders will be removed by using the unmodified histone peptide, while those binding partners that are dependent on the desired methylation state will be specifically eluted using the corresponding di- or trimethylated histone peptide. Due to the conjugation between the genotype and the phenotype, the selected sequences can be readily amplified for iterative rounds of selection, allowing us to address the difficulty in identifying the relatively weak methylation-dependent protein-protein interactions on a proteome wide scale. As a backup plan, we will use the methylated histone tail peptides that contain a crosslinkable photo-leucine moiety to facilitate the capture of low abundant proteins whose interactions with methylated histone tails are relatively weak. Although we address the problem using the human proteome in this project, the method can be readily applied to model organisms such as C. elegans, zebrafish and Drosophila. PUBLIC HEALTH RELEVANCE: The availability of the methylation-dependent interactome will allow us to decipher the binding effectors of each histone methylation state and therefore greatly facilitate our understanding of the signaling pathways mediated by histone lysine methylation. It should also aid in the development of therapeutic and imaging agents that allow the manipulation of epigenetic processes involved in a number of human diseases including cancer.