遗传密码子拓展技术在赖氨酸酰化修饰研究中的应用  

Applications of genetic code expansion in the study of lysine acylation

作　　者：孔影 叶慧[1] 邵畅[1,2] 郝海平 KONG Ying;YE Hui;SHAO Chang;HAO Haiping(Key Laboratory of Drug Metabolism and Pharmacokinetics,China Pharmaceutical University,Nanjing 210009;Department of Pharmacy,Shenzhen Luohu People's Hospital,Shenzhen 518005,China)

机构地区：[1]中国药科大学药物代谢动力学重点实验室,南京210009 [2]深圳市罗湖区人民医院药学部,深圳518005

出　　处：《中国药科大学学报》2023年第5期519-526,共8页Journal of China Pharmaceutical University

基　　金：国家重点研发计划资助项目(No.2021YFA1301300);国家自然科学基金资助项目(No.82173783);江苏省自然科学基金资助项目(No.BK20220088);中国博士后科学基金资助项目(No.2020M681785);广东省基础与应用基础研究基金项目(No.2021A1515111100)。

摘　　要：赖氨酸酰化修饰在细胞中普遍存在,控制着蛋白质的多种功能;然而,在活细胞中进行特定位点酰化修饰的生物学功能研究还存在困难。近年来发展的遗传密码子拓展(genetic code expansion,GCE)技术通过正交的氨酰基-tRNA合成酶/tRNA能够在活细胞内定向插入与天然酰化修饰结构一致的非天然氨基酸(unnatural amino acids,UAAs),实现在精准引入酰化修饰的基础上研究目的蛋白的理化性质和生物学行为的改变。此外,GCE技术还能定点引入无法被去酰化酶识别的模拟酰化修饰的UAAs,从而提高目的蛋白赖氨酸酰化修饰产物的稳定性。在目的蛋白特定位点插入“光交联”型UAA则被用于阐明酰化修饰蛋白的互作蛋白质组。根据不同结构和功能的酰化修饰分类,分别阐述了GCE技术结合上述3类UAAs的新颖设计,及其在研究蛋白酰化修饰对目的蛋白的活性、稳定性、细胞定位、蛋白质-DNA相互作用和蛋白质-蛋白质相互作用等功能影响中的应用。最后,展望了GCE技术在蛋白质酰化修饰研究中的局限和应用前景。Lysine acylation is a ubiquitous protein modification that controls various aspects of protein function.However,it can be challenging to decipher the biological function of site-specific acylation modifications in living cells.The recently developed genetic code expansion(GCE)technology has enabled site-specific incorporation of unnatural amino acids(UAAs)that are structurally consistent with the natural acylation modifications in vivo through orthogonal aminoacyl-tRNA synthetase/tRNA pairs,thus facilitating the study of physicochemical properties and biological behaviors of homogeneously acylated proteins.Besides,GCE technology allows for the targeted introduction of UAAs that mimic acylation modifications but cannot be recognized by deacylases,which improves the stability of lysine acylation modification products.Moreover,the insertion of photo-crosslinked UAAs at specific sites of the target protein has been used to elucidate the reciprocal proteome of acylated modified proteins.Based on the introduction of different structural and functional acylation modifications,we described the novel design of GCE technology combined with three types of UAAs,and their application in studying the functional effects of protein acylation modifications on the enzyme activity,protein stability,cellular localization,protein-DNA interactions and protein-protein interactions of target proteins,with a description of the limitations and prospects of GCE technology in studying protein acylation modification.

关 键 词：非天然氨基酸 赖氨酸酰化修饰 遗传密码子拓展技术 蛋白组学技术 

分 类 号：Q755[生物学—分子生物学]