Crosslinking-Assisted Substrate Identification for Lysine Demethylases

赖氨酸脱甲基酶的交联辅助底物鉴定 摘要。组蛋白上的可逆赖氨酸甲基化构成了基因的主要机制 监管。特别重要的是通过 Fe2 保守家族氧化去除甲基基团 - 和 2-酮戊二酸依赖性赖氨酸脱甲基酶 (KDM) 显着影响 a 的转录潜力 基因。然而，KDM 的基因调控活性本质上是复杂的，因为它们具有广泛的去甲基化能力。 一系列非组蛋白。这就提出了一个重要的问题：特定 KDM 的生物学功能是否 通过其组蛋白或非组蛋白底物或两者表现出来？目前还没有方法来表征 以全蛋白质组方式进行 KDM 底物。为了规避这一挑战，我们建议开发一部小说 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，以实现光介导的交联 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 (KDM4A) 作为范例，我们表明，这样的 工程方法可行。我们计划将这种方法扩展到 KDM4 家族的所有成员， 鉴定其与人类细胞的独特底物。随后对新发现的非- 组蛋白底物将有助于更好地了解 KDM4 介导的关键去甲基化 细胞蛋白质控制蛋白质-蛋白质相互作用、重新编程基因表达、修复 DNA 损伤和 促进肿瘤转移。我们预计 CASI 方法具有高度通用性，适用于任何 染色质修饰酶以现有方法无法实现的方式阐明其功能。

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.