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

项目总结/摘要 作为一种天然存在的琥珀抑制系统，吡咯赖氨酸（Pyl）掺入机制已转向 成为一个巨大的工具，进行琥珀抑制为基础的非规范氨基酸（ncAA）诱变 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 例如，在使用pylRS（PylRS）或用ncAA装载tRNAPyl的PylRS突变体的情况下，已经遗传编码了约200个ncAA。 在不同的细胞中被琥珀密码子所控制作为工程学研究领域的先驱之一， 在ncAA基因整合系统中，PI小组贡献了总数的三分之一以上。 编码的ncAA。这些ncAA包含各种各样的功能，允许在两种环境中的无数应用程序 学术界和工业界都有可能。在为ncAA设计Pyl系统十多年后， 并入后，该领域现在能够使用基于Pyl系统的ncAA诱变技术进行大规模的 探索解决基本重要的生物学问题。在过去，PI的实验室已经设计了一个 用于合成具有翻译后赖氨酸的蛋白质的多种基于ncAA诱变的方法 修饰（赖氨酸PTM）。一种允许泛素和泛素样蛋白直接官能化的新方法， PI的实验室还开发了用于与其他蛋白质结合的蛋白质。所有这些方法 即将实现，PI的实验室正在转移其研究重点，使用他们开发的技术来研究基础和 重要的生物学问题。将采取五个具体方向。第一个方向是利用 ncAA诱变技术产生设计的核小体（具有限定的赖氨酸PTM的核小体）， 探测由表观遗传擦除器靶向的组蛋白赖氨酸位点和PTM类型，包括SIRT 6、SIRT 7、HDAC 1和 LSD 1（HDAC 1和LSD 1的天然复合物）。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它们的结合配偶体， 基于质谱的蛋白质组学分析。第三个方向是对设计人员进行低温电磁分析 与SIRT 6、SIRT 7、HDAC 1和LSD 1结合的核小体（HDAC 1和LSD 1在其天然复合物中）， 阐明了四种酶在识别靶向赖氨酸位点和PTM中的结构基础。 染色质第四个方向是合成不同的triubiquitin异构体，并将其作为探针富集 来自细胞的结合配偶体，其身份将通过基于质谱的蛋白质组学分析来确认。 最后但并非最不重要的方向是合成环GMP-AMP合酶（cGAS），其是细胞内的前线传感器。 检测来自病原体的双链DNA并触发先天免疫反应的人类细胞，含赖氨酸 目的：研究赖氨酸PTMs在调节细胞活性、细胞定位和细胞半定量中的功能作用。 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， research.

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.