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

项目摘要/摘要 作为一种自然存在的琥珀抑制系统，吡咯赖氨酸(PYL)掺入机械已将 变成了一种巨大的工具，用于进行基于琥珀抑制的非规范氨基酸(NCAA)突变 在原核细胞和真核细胞中。通过异位表达tRNAPyl和吡咯烷基-tRNA合成酶 (PylRS)或将tRNAPyl与NCAA连接的PylRS突变体，大约有200个NCAA被基因编码 不同细胞中的琥珀色密码子。作为工程研究领域的最早开拓者之一 在NCAA的遗传整合系统中，Pi的群体贡献了三分之一以上 编码的NCAA。这些NCAA包含大量功能，允许在这两个应用程序中使用无数个应用程序 学术界和工业界都有可能。经过十多年的设计，NCAA的PYL系统 通过整合，该领域现在能够使用基于PYL系统的NCAA突变技术来进行GRAGE 探索从根本上解决重要的生物学问题。在过去，PI的实验室曾设计出一种 基于NCAA突变的多种蛋白质翻译后赖氨酸合成方法 修饰(赖氨酸PTMS)。一种允许泛素及其类似物直接功能化的新方法 用于与其他蛋白质结合的蛋白质也在PI的实验室中开发出来。用所有这些方法 随着成果的实现，PI的实验室正在将其研究重点转移到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。将追求五个具体方向。第一个方向是使用 NCAA突变技术用于产生设计的核小体(具有特定赖氨酸PTM的核小体) 探查SIRT6、SIRT7、HDAC1等表观遗传擦除器靶向的组蛋白赖氨酸位点和PTM类型 LSD1(HDAC1和LSD1在其天然复合体中)。第二个方向是使用可重组的设计器 核小体作为探针，从细胞中丰富其结合伙伴，其身份可以用 基于质谱学的蛋白质组分析。第三个方向是对设计师进行冷冻-EM分析 与SIRT6、SIRT7、HDAC1和LSD1(其天然复合体中的HDAC1和LSD1)结合的核小体 阐明这四种酶识别靶向赖氨酸位点和PTMS的结构基础 染色质。第四个方向是合成不同的Triubiquitin亚型，并将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学的蛋白质组分析来确认。 第四个但并非最不重要的方向是合成环状GMP-AMP合成酶(CGAS)，这是一种前线传感器 检测病原体的双链DNA并与赖氨酸一起触发先天免疫反应的人类细胞 并研究赖氨酸PTMS在调节活性、细胞定位和细胞半衰期中的功能作用。 CGAS的生活。PI研究的一个长期目标是推动基于PYL系统的NCAA的应用 与其他化学生物技术相结合的诱变技术以增强基础生物 研究。

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.