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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：
10006583
负责人：
Or P. Gozani
金额：
$ 19.59万
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 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10006583
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是生物学中重要的翻译后修饰。此修改发生在许多蛋白质中，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、伴侣蛋白、剪接体因子和细胞骨架蛋白。赖氨酸甲基化经常调节蛋白质：蛋白质相互作用，通常通过招募甲基赖氨酸结合因子，并且涉及调节多种多样的生物现象，如转录、翻译、DNA损伤反应、信号转导和蛋白伴侣功能。这些修饰是由S-腺苷甲硫氨酸（SNMet）依赖性赖氨酸甲基转移酶催化的（KMT），其中大多数属于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是非常重要的，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，已经开发了促进KMT的底物鉴定的方法，包括基于候选物的方法，肽和蛋白质阵列，以及利用HATMet类似物的化学亲和质谱技术用生物正交反应基团衍生化。虽然这些方法有助于表征由于某些KMT的底物选择性，底物鉴定仍然是一个持续的挑战。戈扎尼（斯坦福大学）、Trievel（密歇根大学）、Mehl（俄勒冈州州立大学）和Larsen（密歇根大学）密歇根州）的实验室已经建立了跨学科的合作，设计和实施一种新的发现SET结构域KMT的蛋白质底物的方法。这种新方法是对现有方法的补充技术，并基于在活性位点中引入亲电非天然氨基酸（UAA KMT的。这种UAA将促进与赖氨酸氨基的邻近诱导的化学交联，蛋白质底物，随后通过质谱法鉴定交联的底物。我们设想这种方法将能够系统地表征SET的衬底选择性结构域KMT，产生关键的见解，他们的生物学功能，以及如何失调，这些酶可能导致异常的蛋白质甲基化和疾病。