Use of the Noncanonical Amino Acid Mutagenesis Technique in Combination with Other Approaches to Study Functions of Posttranslational Lysine Modifications in Proteins

使用非常规氨基酸诱变技术与其他方法相结合来研究蛋白质翻译后赖氨酸修饰的功能

• 批准号: 10591531
• 负责人: Wenshe Ray Liu
• 金额: $ 51.94万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10591531
• 关键词: Academia; Address; Amber; Amino Acids; Amino Acyl-tRNA Synthetases; Binding; Biological; Biology; Cells; Charge; Chemicals; Chromatin; Codon Nucleotides; Complex; Cryoelectron Microscopy; Cyclic GMP; Disease; Engineering; Enzymes; Epigenetic Process; Eukaryotic Cell; Genetic; Goals; HDAC1 gene; Half-Life; Health Promotion; Histones; Human; Industry; Innate Immune Response; KDM1A gene; Lysine; Mass Spectrum Analysis; Methods; Mission; Modification; Molecular; Mutagenesis; Nucleosomes; Polyubiquitination; Post-Translational Regulation; Prokaryotic Cells; Protein Biosynthesis; Protein Isoforms; Proteins; Proteomics; Public Health; Regulation; Research; Role; Site; System; Techniques; Ubiquitin; Ubiquitin Like Proteins; United States National Institutes of Health; biological research; ds-DNA; mutant; novel; pathogen; pyrrolysine; reconstitution; sensor; therapeutic target; tool

PROJECT SUMMARY/ABSTRACT As a naturally existing amber suppression system, the pyrrolysine (Pyl) incorporation machinery has turned into an enormous tool for undergoing amber suppression-based noncanonical amino acid (ncAA) mutagenesis in both prokaryotic and eukaryotic cells. By ectopically expressing tRNAPyl and pyrrolysyl-tRNA synthetase (PylRS) or a PylRS mutant that charges tRNAPyl with an ncAA, about 200 ncAAs have been genetically encoded by the amber codon in various cells. As one of the original pioneers in the research field of engineering the Pyl system for the genetic incorporation of ncAAs, the PI’s group has contributed more than one third of the total encoded ncAAs. These ncAAs contain a large variety of functionalities, allowing a myriad of applications in both academia and industry possible. After more than a decade of engineering the Pyl system for the ncAA incorporation, the field is now able to use the Pyl system-based ncAA mutagenesis technique to conduct grand explorations to address fundamentally important biological questions. In the past, the PI’s lab has devised a variety of ncAA mutagenesis-based approaches for the synthesis of proteins with posttranslational lysine modifications (lysine PTMs). A novel method that allows direct functionalization of ubiquitin and ubiquitin like proteins for their conjugation with other proteins has also been developed in the PI’s lab. With all these methods coming to fruition, the PI’s lab is shifting its research focus to use their developed techniques to study basic and fundamentally important biological questions. Five specific directions will be pursued. The first direction is to use the ncAA mutagenesis technique to produce designer nucleosomes (nucleosomes with defined lysine PTMs) for probing histone lysine sites and PTM types targeted by epigenetic erasers including SIRT6, SIRT7, HDAC1, and LSD1 (HDAC1 and LSD1 in their native complexes). The second direction is to use reconstituted designer nucleosomes as probes to enrich their binding partners from cells whose identities can be determined using mass spectrometry-based proteomic analysis. The third direction is to conduct cryo-EM analysis of designer nucleosomes bound with SIRT6, SIRT7, HDAC1, and LSD1 (HDAC1 and LSD1 in their native complexes) to elucidate the structural basis of the four enzymes in their recognition of targeted lysine sites and PTMs in chromatin. The fourth direction is to synthesize different triubiquitin isoforms and use them as probes to enrich binding partners from cells whose identities will be confirmed with mass spectrometry-based proteomic analysis. The last but not least direction is to synthesize cyclic GMP-AMP synthase (cGAS), which is a frontline sensor in human cells that detect double-stranded DNA from pathogens and triggers innate immune responses, with lysine PTMs and to study the functional roles of lysine PTMs in regulating activity, cellular localization and cellular half- life of cGAS. A long-term goal of the PI’s research is to push applications of the Pyl system-based ncAA mutagenesis technique in combination with other chemical biology techniques to enhance basic biological research.

项目摘要/摘要 作为一种自然现有的琥珀抑制系统，吡咯氨氨酸（PYL）掺入机器已转动 进入一个巨大的工具，用于接受基于琥珀的非官方氨基酸（NCAA）诱变 在原核生物和真核细胞中。通过生态表达trnapyl和吡咯基-TRNA合成酶 （塔）或向Trnapyl充电NCAA的塔突变体，大约200个NCAA已被编码约200个NCAA 由各种细胞中的琥珀色密码子。作为工程研究领域的原始开拓者之一。 NCAA的遗传掺入系统，PI组贡献了总数的三分之一以上 编码的NCAA。这些NCAA包含多种功能，在两者中都有无数的应用 学术界和行业可能。经过十多年的工程，NCAA的塔系统 合并，该领域现在能够使用基于塔系统的NCAA诱变技术进行宏大 探索以解决根本重要的生物学问题。过去，PI的实验室设计了 基于NCAA诱变的多种方法，用于与翻译后歌词合成蛋白质 修饰（赖氨酸PTMS）。一种新的方法，允许直接功能化泛素和泛素喜欢 Pi的实验室也开发了与其他蛋白质结合的蛋白质。所有这些方法 浮出水面，PI的实验室正在将其研究重点转移到使用其开发技术来研究基本和 将追求五个具体的指示。第一个方向是使用 NCAA诱变技术可生产设计器核小体（具有定义赖氨酸PTM的核小体）的NCAA诱变技术 探测由SIRT6，SIRT7，HDAC1和 LSD1（HDAC1和LSD1在其本地复合物中）。第二个方向是使用重组的设计师 核小体是从细胞中富集其结合伴侣的问题的问题 基于质谱的蛋白质组学分析。第三个方向是进行设计师的冷冻EM分析 与SIRT6，SIRT7，HDAC1和LSD1（其天然复合物中的HDAC1和LSD1）结合的核小体与 在识别目标赖氨酸位点和PTM中的四种酶的结构基础 染色质。第四个方向是合成不同的三泛素同工型，并将其用作富集的问题 通过基于质谱的蛋白质组学分析，将确认其身份的结合伴侣。 最后但并非最不重要的方向是合成环状GMP-AMP合酶（CGA），它是前线传感器 从病原体和触发先天免疫调查的双链DNA的人类细胞，带有歌词 PTMS并研究赖氨酸PTM在调节活性，细胞定位和细胞中的功能作用 CGA的生活。 PI研究的长期目标是推动基于塔的NCAA的应用 诱变技术与其他化学生物学技术相结合，以增强基本生物学 研究。