Unnatural Amino Acid Chemistry for Lysine Methyltransferase Substrate Discovery

赖氨酸甲基转移酶底物发现的非天然氨基酸化学

基本信息

批准号：
9808782
负责人：
Or P. Gozani
金额：
$ 25.88万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-02 至 2021-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9808782
关键词：
Active Sites Affinity Alanine Amber Amino Acyl-tRNA Synthetases Benchmarking Binding Binding Proteins Biochemical Biological Assay Biological Phenomena Biological Process Biology Cancer Etiology Cell Cycle Progression Cell physiology Cells Chemicals Chemistry Chromatin Codon Nucleotides Cytoskeletal Proteins DNA Damage Disease Enzymes Epigenetic Process Exhibits Family Gene Expression Generations Genetic Genetic Transcription Goals Heat shock proteins Histone-Lysine N-Methyltransferase Histones Human Human Genome In Vitro Knowledge Laboratories Lysine Malignant Neoplasms Mass Spectrum Analysis Methodology Methods Methylation Methyltransferase Michigan Modeling Modification Molecular Chaperones Mutation Oregon Orphan Pathologic Peptides Phenylalanine Phosphotransferases Physiological Post-Translational Protein Processing Production Protein Array Protein Methylation Proteins Research Ribosomal Proteins Ribosomes S-Adenosylmethionine SET Domain SMYD3 gene Series Signal Pathway Signal Transduction Site Substrate Specificity Techniques Tertiary Protein Structure Testing Translations Tyrosine Universities amino group analog base covalent bond crosslink design enzyme model enzyme substrate experimental study in vitro activity insight interdisciplinary collaboration mutant novel novel strategies novel therapeutic intervention protein protein interaction recruit response screening transcription factor unnatural amino acids

项目摘要

Project Abstract Protein lysine methylation represents a prominent post-translational modification in biology. This modification occurs in a multitude of proteins, including histones, transcription factors, chromatin modifying enzymes, ribosomal proteins, cytoplasmic signaling enzymes, chaperones, spliceosomal factors, and cytoskeletal proteins. Lysine methylation frequently modulates protein:protein interactions, often through the recruitment of methyllysine binding factors, and has been implicated in regulating a diverse array of biological phenomena, such as transcription, translation, DNA damage response, signal transduction, and protein chaperone function. These modifications are catalyzed by S-adenosylmethionine (AdoMet)-dependent lysine methyltransferases (KMTs), the majority of which belong to the SET domain family. The human genome encodes over 50 predicted SET domain KMTs. It is fundamentally important to elucidate the substrate selectivities of these enzymes, as methylation of their substrates defines their respective biological functions. Toward this goal, several techniques have been developed to facilitate substrate identification of KMTs, including candidate-based approaches, peptide and protein arrays, and a chemical affinity-mass spectrometry technique that utilizes AdoMet analogs derivatized with bio-orthogonally reactive groups. Although these methods have aided in characterizing the substrate selectivities of certain KMTs, substrate identification remains a persistent challenge. The Gozani (Stanford University), Trievel (University of Michigan), Mehl (Oregon State University), and Larsen (University of Michigan) laboratories have established an interdisciplinary collaboration to devise and implement a novel method for discovering protein substrates of SET domain KMTs. This new approach is complementary to current techniques and is based upon the introduction of an electrophilic unnatural amino acid (UAA) in the active sites of KMTs. This UAA will facilitate proximity-induced chemical crosslinking with the lysine epsilon amino group in protein substrates, with subsequent identification of the crosslinked substrates by mass spectrometry. We envision that this methodology will enable systematic characterization of the substrate selectivities of SET domain KMTs, yielding key insights into their biological functions and how dysregulation of these enzymes may contribute to aberrant protein methylation and disease.

项目摘要蛋白质赖氨酸甲基化代表了生物学中一种重要的翻译后修饰。本次修改存在于多种蛋白质中，包括组蛋白、转录因子、染色质修饰酶、核糖体蛋白、细胞质信号酶、分子伴侣、剪接体因子和细胞骨架蛋白。赖氨酸甲基化经常通过招募赖氨酸来调节蛋白质：蛋白质相互作用甲基赖氨酸结合因子，并与调节多种生物现象有关，例如转录、翻译、DNA损伤反应、信号转导和蛋白质伴侣功能。这些修饰由 S-腺苷甲硫氨酸 (AdoMet) 依赖性赖氨酸甲基转移酶催化（国民党），其中大部分属于 SET 域家族。人类基因组编码超过 50 个预测的 SET 域 KMT。阐明这些酶的底物选择性至关重要，因为它们底物的甲基化决定了它们各自的生物学功能。为了实现这一目标，需要采取多种技术已开发用于促进 KMT 的底物识别，包括基于候选的方法，肽和蛋白质阵列，以及利用 AdoMet 类似物的化学亲和质谱技术用生物正交反应基团衍生。尽管这些方法有助于表征由于某些 KMT 的底物选择性，底物识别仍然是一个持续的挑战。戈扎尼人（斯坦福大学）、Trievel（密歇根大学）、Mehl（俄勒冈州立大学）和 Larsen（密歇根大学）密歇根州）实验室建立了跨学科合作来设计和实施一种新颖的发现SET结构域KMT的蛋白质底物的方法。这种新方法是对当前方法的补充技术，基于在活性位点引入亲电子非天然氨基酸（UAA）国民党的。该UAA将促进与赖氨酸ε氨基的邻近诱导化学交联蛋白质底物，随后通过质谱法鉴定交联底物。我们预计该方法将能够系统地表征 SET 的底物选择性域 KMT，从而深入了解其生物学功能以及这些酶的失调如何可能导致异常蛋白质甲基化和疾病。