Crosslinking-Assisted Substrate Identification for Lysine Demethylases

赖氨酸脱甲基酶的交联辅助底物鉴定

• 批准号: 10388729
• 负责人: Kabirul Islam
• 金额: $ 3.44万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10388729
• 关键词: Active Sites; Affect; Amino Acids; Autoimmune Diseases; Biochemical; Biological Process; Cells; Chromatin; Complex; DNA Repair; Development; Drug Targeting; Engineering; Enzymes; Excision; Family; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Histones; Human; Lead; Light; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methods; Methylation; Neoplasm Metastasis; Pathologic; Photosensitivity; Point Mutation; Process; Proteins; Proteome; Regulator Genes; Work; alpha ketoglutarate; chemoproteomics; crosslink; demethylation; drug development; histone demethylase; human disease; improved; member; methyl group; non-histone protein; novel; overexpression; protein protein interaction; public health relevance; tumor

Crosslinking-Assisted Substrate Identification for Lysine Demethylases Abstract. Reversible lysine methylation on histone proteins constitutes a primary mechanism for gene regulation. Of particular importance is the oxidative removal of the methyl groups by a conserved family of Fe2+- and 2-ketoglutarate-dependent lysine demethylases (KDMs) that significantly affect transcriptional potential of a gene. However, gene regulatory activity of KDMs is inherently complex due to their ability to demethylate a wide range of non-histone proteins. This raises an important question: Are the biological functions of a given KDM manifested through its histone or non-histone substrates or both? Currently, no method exists to characterize KDM substrates in proteome-wide manner. To circumvent the challenge, we propose to develop a novel chemoproteomic approach termed ‘crosslinking-assisted substrate identification (CASI)’. The CASI platform involves engineering of a KDM active site with a photosensitive amino acid for light-mediated crosslinking with the bound substrates followed by identification of the crosslinked species using quantitative mass spectrometry. Using development- and cancer-relevant lysine demethylase 4A (KDM4A) as paradigm, we show that such engineering approach is feasible. We plan to extend this approach to all the members of the KDM4 family and to identify their distinct substrates from human cells. Subsequent biochemical studies of the newly identified non- histone substrates would lead to improved understanding of how KDM4-mediated demethylation of critical cellular proteins controls protein-protein interactions, reprograms gene expression, repairs DNA damage and promotes tumor metastasis. We anticipate the CASI approach to be highly general and applicable to any chromatin-modifying enzyme to elucidate their functions in a manner not attainable by existing methods.

赖氨酸脱甲基酶的交联辅助底物鉴定 抽象。组蛋白上可逆的赖氨酸甲基化是基因突变的主要机制。 调控特别重要的是通过Fe 2 +-的保守家族氧化除去甲基。 和2-酮戊二酸依赖性赖氨酸脱甲基酶（KDM），这些酶显著影响转录潜能。 基因然而，KDM的基因调控活性是固有的复杂性，这是由于它们能够使广泛的DNA去甲基化。 一系列非组蛋白。这就提出了一个重要的问题：给定的KDM的生物学功能 通过组蛋白或非组蛋白底物或两者来表现？目前，没有方法可以表征 KDM底物在蛋白质组范围内的方式。为了规避挑战，我们建议开发一种新的 化学蛋白质组学方法称为“交联辅助底物鉴定（CASI）”。CASI平台 涉及用光敏氨基酸改造KDM活性位点，用于光介导的交联， 结合的底物，然后使用定量质谱法鉴定交联物质。 使用发育和癌症相关的赖氨酸脱甲基酶4A（KDM 4A）作为范例，我们表明， 工程方法可行。我们计划将这种方法扩展到KDM 4家族的所有成员， 来识别它们与人类细胞不同的底物。随后对新发现的非- 组蛋白底物将有助于更好地理解KDM 4介导的关键细胞的去甲基化， 细胞蛋白控制蛋白质-蛋白质相互作用，重新编程基因表达，修复DNA损伤， 促进肿瘤转移。我们预计CASI方法是高度通用的，适用于任何 染色质修饰酶，以阐明其功能的方式不能达到现有的方法。