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）：越来越多的人认识到，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，已经描述了100多种组蛋白翻译后修饰（PTM）。然而，催化修饰的酶和识别并将特异性PTM转化为生物学结果的蛋白质的表征在很大程度上仍然不发达。鉴于异常的PTM与人类疾病的关联，迫切需要开发创新的工具来表征参与表观遗传调控的蛋白质。我们建议开发一种新的技术，利用蛋白质阵列的高通量覆盖的人类表观基因组。总体目标是建立将蛋白质阵列技术应用于表观遗传学研究的方法，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。在这项提案中，我们将集中精力开发一个随机固定的甲基赖氨酸定向肽阵列库（OPAL），这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。为了询问所有效应子和酶，我们将合成四个定向KGXXXKXXXGK-生物素肽文库[X代表随机氨基酸（除半胱氨酸外的任何氨基酸）]，其中（1）中心- K是未甲基化的;（2）中心- K是单甲基化的;（3）中心- K是二甲基化的;和（4）中心- K是三甲基化的。OPAL平台将通过确定已知甲基赖氨酸效应蛋白（HP 1、RAG 2、ORC 1和PHF 19）的结合特异性进行验证。此外，我们将使用该平台来确认甲基转移酶G9 a和SET 7/9的已建立的共有底物识别序列。如果我们观察到效应物（目标1）和酶（目标2）的共识靶点与已发表的结果一致，则将取得成功并证明进展至II期是合理的。鉴于这些蛋白质将针对高复杂性阵列进行筛选，OPAL筛选的结果可能揭示这些效应物和酶的新靶标，这将可能揭示对蛋白质的功能的新见解。 这些因素，因为所有赖氨酸甲基化状态可以在单一实验中筛选。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，如赖氨酸乙酰化和精氨酸甲基化，这将使该公司成为研究界的宝贵资源。更普遍地，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，而且存在于大量非组蛋白蛋白上。因此，拟议的OPAL平台将首次提供必要的资源来定义“孤儿”效应子和酶的功能，并帮助研究界预测哪些非组蛋白可能是传统上与组蛋白密码相关的酶和效应子的底物和相互作用伴侣，并作为理解人类赖氨酸甲基化的重要步骤。

项目成果

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

• DOI: 10.1126/sciadv.aav2623
• 发表时间: 2018-11
• 期刊: Science advances
• 影响因子: 13.6
• 作者: Cornett EM;Dickson BM;Krajewski K;Spellmon N;Umstead A;Vaughan RM;Shaw KM;Versluis PP;Cowles MW;Brunzelle J;Yang Z;Vega IE;Sun ZW;Rothbart SB
• 通讯作者: Rothbart SB