Reagents to study lysine methylation and proteomic survey of the human methyllysi

描述（申请人提供）：赖氨酸甲基化研究是一个新兴领域；对组蛋白翻译后修饰及其在表观遗传调控中的作用的研究取得了巨大进展。近年来已经发现了许多甲基化或去甲基化组蛋白的酶，其中一些酶的失调与肿瘤发生有关。从组蛋白的ptm研究中出现的一个趋势是，许多修饰组蛋白的酶也可以修饰和调节非组蛋白的功能。我们对非组蛋白上赖氨酸甲基化的了解几乎不存在，因为到目前为止已经确定了三种真核蛋白上的甲基化位点。本提案的目标是开发必要的工具，使赖氨酸甲基化的研究和进行人类甲基赖氨酸蛋白质组的第一次调查。我们提出了生产针对单、二、三甲基赖氨酸的泛细胞特异性抗体的策略。针对三种形式的甲基赖氨酸的pan特异性抗体的产生将使科学家能够轻松地研究任何蛋白质上的赖氨酸甲基化。我们从HeLa细胞中鉴定含甲基赖氨酸蛋白的策略是新颖的，因为它结合了简单的细胞提取物的预分离，以减少样品的复杂性，因此增加了我们用pan特异性抗体免疫亲和纯化甲基赖氨酸肽的可能性。本提案中概述的实验将极大地促进我们对信号转导途径中赖氨酸甲基化的生物学理解。我们设想，pan特异性抗体和人类蛋白质组中甲基赖氨酸蛋白的目录将为这一尚未开发的研究领域的重要突破奠定基础。鉴于对赖氨酸甲基化酶的放松管制与包括癌症在内的各种人类疾病有关，我们相信本提案中描述的工具和发现将导致未来的科学突破，对人类健康产生直接影响。

DESCRIPTION (provided by applicant): The study of lysine methylation is an emerging field; tremendous progress has been made in understanding this post-translational modification (PTM) on histones and its roles in epigenetic regulation. Many of the enzymes that methylate or demethylate histones have been identified in the recent years, and deregulation of several of these enzymes has been linked to tumorgenesis. One trend that emerges from the study of PTMs on histones is that many of the enzymes that modify histones can also modify and regulate the function of nonhistone proteins. Our understanding of lysine methylation on nonhistone proteins is almost non-existent since methylation sites on three eukaryotic proteins have been identified so far. The goals of this proposal are to develop the necessary tools that will enable the study of lysine methylation and to conduct the first survey of the human methyllysine proteome. We propose strategies to produce pan specific antibodies to the mono-, di-, and trimethyllysine. The generation of pan specific antibodies to the three forms of methyllysine will enable scientists to easily study lysine methylation on any protein. Our strategy to identify methyllysine-containing proteins from HeLa cells is novel in that it incorporates a simple prefractionation of the cell extracts to reduce the complexity of the sample and therefore increase our likelihood to immunoaffinity purify the methyllysine peptides with the pan specific antibodies. The experiments outlined in this proposal will greatly advance our understanding of the biology of lysine methylation in signal transduction pathways. We envision that pan specific antibodies and the catalog of methyllysine proteins in the human proteome will be foundation for important breakthroughs in this untapped area of research. Given that deregulation of lysine methylation enzymes have been linked to various human diseases, including cancer, we believe that the tools and discoveries describe in this proposal will lead to future scientific breakthroughs that will have a direct impact on human health. PUBLIC HEALTH RELEVANCE: Deregulation of lysine methylation has been linked to various human diseases, including cancer. This proposal outlines the development of much-needed reagents and methods for the study of lysine methylation. The successful completion of this proposed project will lead the development of important tools for the study of lysine methylation and build the foundation for future scientific discoveries that will have a direct impact on public health.