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.

项目概要/摘要 作为一种天然存在的琥珀抑制系统，吡咯赖氨酸 (Pyl) 掺入机制已转变为 成为进行基于琥珀抑制的非规范氨基酸 (ncAA) 诱变的巨大工具 在原核和真核细胞中。通过异位表达 tRNAPyl 和吡咯赖氨酰-tRNA 合成酶 (PylRS) 或用 ncAA 装载 tRNAPyl 的 PylRS 突变体，约 200 个 ncAA 已被基因编码 由各种细胞中的琥珀密码子。作为 Pyl 工程研究领域的最早的先驱之一 NCAA基因整合系统中，PI团队贡献了三分之一以上 编码的 ncAA。这些 ncAA 包含多种功能，允许在这两个领域进行无数的应用 学术界和工业界可能。经过十多年为 ncAA 设计 Pyl 系统 合并后，该领域现在能够使用基于 Pyl 系统的 ncAA 诱变技术来进行重大研究 探索解决根本性重要的生物学问题。过去，PI 的实验室设计了一种 多种基于 n​​cAA 诱变的方法用于合成具有翻译后赖氨酸的蛋白质 修饰（赖氨酸 PTM）。一种允许泛素和泛素样直接功能化的新方法 PI 实验室还开发了与其他蛋白质结合的蛋白质。通过所有这些方法 随着成果的实现，PI 的实验室正在将其研究重点转向使用他们开发的技术来研究基础和 根本上重要的生物学问题。将推进五个具体方向。第一个方向是使用 NCAA 诱变技术可生产设计核小体（具有明确赖氨酸 PTM 的核小体） 探测表观遗传擦除器（包括 SIRT6、SIRT7、HDAC1 和）靶向的组蛋白赖氨酸位点和 PTM 类型 LSD1（天然复合物中的 HDAC1 和 LSD1）。第二个方向是使用重构设计器 核小体作为探针，从细胞中富集其结合伙伴，其身份可以使用 基于质谱的蛋白质组分析。第三个方向是对设计者进行冷冻电镜分析 核小体与 SIRT6、SIRT7、HDAC1 和 LSD1（天然复合物中的 HDAC1 和 LSD1）结合， 阐明四种酶识别目标赖氨酸位点和 PTM 的结构基础 染色质。第四个方向是合成不同的三泛素亚型并用它们作为探针来富集 来自细胞的结合伙伴，其身份将通过基于质谱的蛋白质组分析得到确认。 最后但并非最不重要的方向是合成环 GMP-AMP 合酶 (cGAS)，这是一种前沿传感器 人类细胞利用赖氨酸检测病原体的双链 DNA 并触发先天免疫反应 PTM 并研究赖氨酸 PTM 在调节活性、细胞定位和细胞半衰期中的功能作用 cGAS 的寿命。 PI研究的长期目标是推动基于Pyl系统的ncAA的应用 诱变技术与其他化学生物学技术相结合，增强基础生物学能力 研究。