蛋白质翻译后修饰定点展示系统的开发及其应用

The content of this research project corresponds to the research focus of the guide: new technologies and new methods for the development of precise labeling, detection, and identification for novel dynamic modification of biological macromolecules. .Dynamic post-translational modification of proteins plays a central role in almost every process of the cellular physiological activity. Therefore, understanding the dynamic post-translational modification process of proteins at the molecular level is the key to many fields such as cell signal transduction and cell cycle regulation. The use of chemical and biological methods to site-specifically and efficiently assemble proteins with authentic post-translational modifications and their analogs in vivo and in vitro, at the molecular level, to decipher the function of dynamic post-translational modification in controlling of protein structure, cell localization, substrate interactions and The effects of physiological functions. This project uses genetic code extension technology, combined with protein and nucleic acid engineering evolution methods, to develop several novel bio-orthogonal protein translation systems; and use this system to incorporate a variety of natural post-translational modifications and their analogs in forms of unnatural amino acid to be installed in both prokaryotes and eukaryotes; finally, using this chemical biology system to install post-translational modifications of proteins on important signal transduction proteins, To study its mechanism in the process of disease occurrence and development.

本项目的研究内容对应指南的研究重点：针对生物大分子新型动态修饰，发展精准标记、检测和鉴定的新技术、新方法。. 蛋白质动态翻译后修饰在细胞生理活动的各个步骤扮演着核心的作用。因此在分子层面上深入理解蛋白质动态翻译后修饰过程是细胞信号转导，细胞周期调控等众多领域的关键。利用化学生物学方法，在体内和体外位点特异性地、高效地组装带有天然翻译后修饰及其类似物的蛋白质，在分子层面上阐释了蛋白质动态翻译后修饰对于蛋白质结构，细胞定位，作用底物和生理功能的影响。本项目利用遗传密码拓展技术，结合蛋白和核酸工程进化手段，拟开发新型生物正交的蛋白质翻译系统；并利用新型系统将多种天然翻译后修饰及其类似物以非天然氨基酸的形式位点特异性的遗传编码，实现其在原核生物和真核生物的在体组装；最后，利用这个化学生物学系统，将蛋白质翻译后修饰展示在重要的信号转导蛋白质上，用于研究其在疾病发生、发展过程中的机制。

蛋白质动态翻译后修饰在细胞生理活动的各个步骤扮演着核心的作用。因此在分子层面上深入理解蛋白质动态翻译后修饰过程是细胞信号转导，细胞周期调控等众多领域的关键。本项目利用化学生物学方法，在体内和体外位点特异性地、高效地组装带有天然翻译后修饰及其类似物的蛋白质，并取得系列研究进展。项目提出了全新的正交移植策略，开发了多套具有广谱正交性的嵌合翻译系统，实现了系列色氨酸精准修饰和酪氨酸羟基化修饰的定点引入；以接近天然氨基酸的编码效率，高效、高特异地引入蛋白质精准修饰，并构建了依赖非天然氨基酸生长的合成微生物；首次发现Cation-Pi相互作用可被精准地工程改造，开发了亲和力显著提升的超级读码蛋白用于组蛋白甲基化修饰的富集和检测；实现了赖氨酸氨酰化修饰的精准在体合成，并利用在体合成技术，发现了细胞中存在新型氨酰化赖氨酸泛素化修饰，并探索了新修饰的生物学功能和书写酶。