Developing Novel Peptide Arrays for Epigenetic Research

描述（由申请人提供）：人们越来越认识到，表观遗传过程中的扰动与多种人类疾病（如癌症和免疫缺陷疾病）有关。迄今为止，已有超过100种组蛋白翻译后修饰（ptm）被描述。然而，催化修饰的酶和识别并转导特定ptm的蛋白质的表征仍然很大程度上不发达。鉴于异常ptm与人类疾病的关联，迫切需要开发创新工具来表征参与表观遗传调控的蛋白质。我们建议开发一种利用蛋白质阵列对人类表观基因组进行高通量覆盖的新技术。总体目标是建立蛋白质阵列技术应用于表观遗传研究的方法，并验证蛋白质阵列技术作为发现染色质修饰事件如何调节表观遗传生物学的新工具。在本提案中，我们将重点开发一个随机固定的甲基赖氨酸取向肽阵列库（OPAL），该库将允许快速筛选所有甲基赖氨酸结合蛋白（效应器）和赖氨酸甲基转移酶（酶）的底物或结合特异性。为了询问所有的效应物和酶，我们将合成四个定向的kgxxxkxxxgk -生物素肽文库[X表示随机氨基酸（除半胱氨酸外的任何氨基酸）]，其中(1)中心- K未甲基化；(2)中心- K被单甲基化；(3)中心- K二甲基化；(4)中心- K被三甲基化。OPAL平台将通过确定已知甲基赖氨酸效应蛋白（HP1、RAG2、ORC1和PHF19）的结合特异性进行验证。此外，我们将使用该平台确认甲基转移酶G9a和SET7/9的公认底物识别序列。如果我们观察到效应物（目标1）和酶（目标2）的共识目标与已发表的结果一致，则将取得成功，并有理由进入第二阶段。考虑到这些蛋白质将在高复杂性阵列中筛选，OPAL筛选的结果可能会揭示这些效应物和酶的新靶点，这可能会揭示对蛋白质功能的新见解

DESCRIPTION (provided by applicant): It has been increasingly appreciated that perturbations in epigenetic processes are associated with diverse human diseases such as cancer and immunodeficiency disorders. To date, more than 100 histone protein posttranslational modifications (PTMs) have been described. However, characterization of the enzymes that catalyze the modifications and the proteins that recognize and transduce specific PTMs to biological outcomes remains largely underdeveloped. Given the association of aberrant PTMs with human disease, there is a compelling need to develop innovative tools to characterize proteins involved in epigenetic regulation. We propose to develop a new technology utilizing protein arrays for high-throughput coverage of the human epigenome. The overall goals are to establish methods for protein array technology to be applied for epigenetic research, and validate protein array technology as a new tool in the discovery of how chromatin modification events regulate epigenetic biology. In this proposal, we will focus our efforts on developing a random fixed methyl-lysine Oriented Peptide Array Library (OPAL) which will allow the rapid screening of all methyl-lysine binding proteins (effectors) and lysine methyltransferases (enzymes) for their substrate or binding specificities. In order to interrogate all effectors and enzymes, we will synthesize four oriented KGXXXKXXXGK-biotin peptide libraries [X represents random amino acids (any amino acid except cysteine)] in which the (1) central- K is unmethylated; (2) central- K is mono-methylated; (3) central- K is di-methylated; and (4) central- K is tri-methylated. The OPAL platform will be validated by determining the binding specificities of known methyl-lysine effector proteins (HP1, RAG2, ORC1, and PHF19). In addition, we will use this platform to confirm the established consensus substrates recognition sequences for the methyltransferases G9a and SET7/9. Success will be achieved and progression to Phase 2 justified if we observe that the consensus targets for the effectors (Aim 1) and enzymes (Aim 2) are consistent with published results. Given that these proteins will be screened against high complexity arrays the results of the OPAL screen may reveal novel targets for these effectors and enzymes, which will likely reveal new insights into the function of these factors since all lysine methylation states can be screened in a single experiment. In the long term, Epicypher will create a suite of OPAL platforms that include PTMs other than lysine methylation, such as lysine acetylation and arginine methylation, which will enable the company to serve as a valuable resource to the research community. More generally, it is becoming clear that lysine acetylation and methylation are not only found on histones, but on a large number of non-histone proteins as well. Thus, the proposed OPAL platforms would, for the first time, provide the resources necessary to define the function of "orphan" effectors and enzymes, and help the research community predict which non-histone proteins could be substrates and interacting partners for enzymes and effectors that have been traditionally associated with the histone code and serve as an important step in understanding the human lysine methylome.