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: 10220076
• 负责人: Kabirul Islam
• 金额: $ 29.37万
• 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/10220076
• 关键词: Acetylation; Acetyltransferase; Active Sites; Address; Affect; Amber; Amino Acids; Autoimmune Diseases; Binding; Biochemical; Biological Process; Catalysis; Cell physiology; Cells; Chemicals; Chromatin; Complex; DNA; DNA Modification Process; DNA Repair; Deacetylase; Development; Disease model; Drug Targeting; Elongation Factor; Engineering; Environment; Enzymes; Epitopes; Excision; Family; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Glucosyltransferase; Glycoside Hydrolases; Heritability; Histones; Human; Human Genome; Immunoprecipitation; Laboratories; Lead; Length; Light; Link; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methods; Methylation; Methyltransferase; Modification; Mutagenesis; Neoplasm Metastasis; Nucleic Acids; Pathologic; Peptide Initiation Factors; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Photosensitivity; Physiological; Point Mutation; Post-Translational Protein Processing; Process; Protein Biosynthesis; Protein Engineering; Proteins; Proteome; Proteomics; RNA; Reagent; Regulation; Regulator Genes; Structure; Techniques; Testing; Transferase; Validation; Work; acetyl phosphate; alpha ketoglutarate; base; chemoproteomics; covalent bond; crosslink; demethylation; drug development; histone demethylase; human disease; improved; interest; irradiation; mRNA Stability; member; methyl group; non-histone protein; novel; overexpression; packaging material; protein protein interaction; public health relevance; spatiotemporal; tool; tumor; unnatural amino acids

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法底物鉴定 抽象的。组蛋白可逆性赖氨酸甲基化是基因的主要机制 监管。特别重要的是由保守的Fe2+-家族氧化去除甲基。 和2-酮戊二酸依赖的赖氨酸去甲基酶(KDM)，它们显著影响A细胞的转录潜能 吉恩。然而，KDMS的基因调节活性是复杂的，因为它们有能力广泛地去甲基化 一系列非组蛋白蛋白。这提出了一个重要的问题：给定的KDM的生物学功能 通过其组蛋白或非组蛋白底物，或两者兼而有之？目前，还没有方法来表征 KDM底物以蛋白质组的方式。为了规避这一挑战，我们提议开发一部小说 化学蛋白质组学方法被称为“交联辅助底物鉴定(CASI)”。CASI平台 包括设计具有光敏氨基酸的KDM活性部位，用于光介导的交联剂 结合底物，然后用定量质谱仪鉴定交联物。 使用与发育和癌症相关的赖氨酸去甲基酶4A(KDM4A)作为范例，我们证明了 工程方法是可行的。我们计划将这一方法推广到KDM4家族的所有成员，并 以从人类细胞中鉴定出它们的不同底物。新发现的非霍奇金杆菌的后续生化研究 组蛋白底物将有助于更好地理解KDM4介导的关键分子去甲基化 细胞蛋白质控制蛋白质之间的相互作用，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方法将非常通用，并适用于任何 染色质修饰酶，以现有方法无法实现的方式阐明它们的功能。