Developing Novel Peptide Arrays for Epigenetic Research

开发用于表观遗传学研究的新型肽阵列

• 批准号: 8715130
• 负责人: Zu-Wen Sun
• 金额: $ 22.02万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-07 至 2015-07-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8715130
• 关键词: Acetylation; Affect; Amino Acids; Arginine; Binding; Binding Proteins; Biological; Biology; Biotin; Chromatin; Code; Communities; Consensus; Consensus Sequence; Cysteine; Disease; Enzymes; Epigenetic Process; Event; Glass; Goals; Histone Code; Histones; Human; Immunologic Deficiency Syndromes; Incubated; Libraries; Link; Lysine; Malignant Neoplasms; Methionine; Methods; Methylation; Methyltransferase; Modification; Mono-S; Nuclear; Orphan; Outcome; Pathogenesis; Pattern; Peptide Library; Peptides; Phase; Physiology; Positioning Attribute; Post-Translational Protein Processing; Printing; Process; Protein Array; Proteins; Publishing; Radiolabeled; Regulation; Research; Resources; Slide; Specificity; Substrate Specificity; Technology; Time; chromatin modification; design; enzyme activity; epigenome; histone modification; human disease; innovation; insight; methylome; new technology; novel; public health relevance; radiotracer; research study; screening; success; tool

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.

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（例如癌症和免疫缺陷性疾病）相关。迄今为止，已描述了 100 多种组蛋白翻译后修饰 (PTM)。然而，催化修饰的酶和识别特定 PTM 并将其转导为生物学结果的蛋白质的表征在很大程度上仍不发达。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高通量覆盖人类表观基因组的新技术。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，我们将集中精力开发随机固定甲基赖氨酸定向肽阵列库（OPAL），该库将允许快速筛选所有甲基赖氨酸结合蛋白（效应子）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子和酶，我们将合成四个定向的KGXXXKXXXGK-生物素肽库[X代表随机氨基酸（除半胱氨酸之外的任何氨基酸）]，其中（1）中央-K是未甲基化的； (2)中心-K被单甲基化； (3)中心-K被二甲基化； (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，我们将使用该平台来确认已建立的甲基转移酶 G9a 和 SET7/9 的共有底物识别序列。如果我们观察到效应器（目标 1）和酶（目标 2）的共识目标与已发表的结果一致，则将取得成功并证明进入第 2 阶段是合理的。鉴于这些蛋白质将针对高复杂性阵列进行筛选，OPAL 筛选的结果可能会揭示这些效应子和酶的新靶标，这可能会揭示对这些效应子和酶功能的新见解。 由于所有赖氨酸甲基化状态都可以在单个实验中进行筛选，因此可以考虑这些因素。从长远来看，Epicypher 将创建一套 OPAL 平台，其中包括赖氨酸甲基化以外的 PTM，例如赖氨酸乙酰化和精氨酸甲基化，这将使该公司能够成为研究界的宝贵资源。更一般地说，越来越清楚的是，赖氨酸乙酰化和甲基化不仅存在于组蛋白上，而且也存在于大量非组蛋白上。因此，拟议的 OPAL 平台将首次提供定义“孤儿”效应子和酶的功能所需的资源，并帮助研究界预测哪些非组蛋白可能是酶和效应子的底物和相互作用伙伴传统上与组蛋白密码相关，是理解人类赖氨酸甲基化组的重要一步。

项目成果

A functional proteomics platform to reveal the sequence determinants of lysine methyltransferase substrate selectivity.

功能蛋白质组学平台，揭示赖氨酸甲基转移酶底物选择性的序列决定因素。

• 发表时间: 2018
• 影响因子: 13.6
• 作者: Cornett, Evan M;Dickson, Bradley M;Krajewski, Krzysztof;Spellmon, Nicholas;Umstead, Andrew;Vaughan, Robert M;Shaw, Kevin M;Versluis, Philip P;Cowles, Martis W;Brunzelle, Joseph;Yang, Zhe;Vega, Irving E;Sun, Zu;Rothbart, Scott B
• 通讯作者: Rothbart, Scott B