Crosslinking-Assisted Substrate Identification for Lysine Demethylases

赖氨酸脱甲基酶的交联辅助底物鉴定

基本信息

批准号：
10428551
负责人：
Kabirul Islam
金额：
$ 29.35万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
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/10428551
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

项目摘要

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。特别重要的是通过保守的 Fe2+- 家族氧化去除甲基。和 2-酮戊二酸依赖性赖氨酸脱甲基酶 (KDM) 显着影响 a 的转录潜力然而，KDM 的基因调控活性本质上是复杂的，因为它们能够广泛去甲基化。这提出了一个重要的问题：特定 KDM 的生物学功能是什么？通过其组蛋白或非组蛋白底物或两者表现出来？目前，没有方法可以表征？为了规避这一挑战，我们建议开发一种新型的蛋白质组底物。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，这样的工程方法是可行的，我们计划将这种方法扩展到 KDM4 系列的所有成员，并且以确定其与人类细胞的独特底物的后续生化研究。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 损伤和我们预计 CASI 方法具有高度通用性，适用于任何疾病。染色质修饰酶以现有方法无法实现的方式阐明其功能。