Morosky Pearl, Comyns Cody, Nunes Lance G A, Chung Christina Z, Hoffmann Peter R, Söll Dieter, Vargas-Rodriguez Oscar, Krahn Natalie
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, United States.
Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI, United States.
Front Mol Biosci. 2023 Jan 24;10:1096261. doi: 10.3389/fmolb.2023.1096261. eCollection 2023.
Post-translational modifications (PTMs) can occur on almost all amino acids in eukaryotes as a key mechanism for regulating protein function. The ability to study the role of these modifications in various biological processes requires techniques to modify proteins site-specifically. One strategy for this is genetic code expansion (GCE) in bacteria. The low frequency of post-translational modifications in bacteria makes it a preferred host to study whether the presence of a post-translational modification influences a protein's function. Genetic code expansion employs orthogonal translation systems engineered to incorporate a modified amino acid at a designated protein position. Selenoproteins, proteins containing selenocysteine, are also known to be post-translationally modified. Selenoproteins have essential roles in oxidative stress, immune response, cell maintenance, and skeletal muscle regeneration. Their complicated biosynthesis mechanism has been a hurdle in our understanding of selenoprotein functions. As technologies for selenocysteine insertion have recently improved, we wanted to create a genetic system that would allow the study of post-translational modifications in selenoproteins. By combining genetic code expansion techniques and selenocysteine insertion technologies, we were able to recode stop codons for insertion of -acetyl-l-lysine and selenocysteine, respectively, into multiple proteins. The specificity of these amino acids for their assigned position and the simplicity of reverting the modified amino acid mutagenesis of the codon sequence demonstrates the capacity of this method to study selenoproteins and the role of their post-translational modifications. Moreover, the evidence that Sec insertion technology can be combined with genetic code expansion tools further expands the chemical biology applications.
翻译后修饰（PTMs）几乎可发生在真核生物的所有氨基酸上，是调节蛋白质功能的关键机制。研究这些修饰在各种生物过程中的作用需要能够对蛋白质进行位点特异性修饰的技术。细菌中的遗传密码扩展（GCE）就是实现这一目的的一种策略。细菌中翻译后修饰的频率较低，这使其成为研究翻译后修饰的存在是否会影响蛋白质功能的理想宿主。遗传密码扩展采用经过工程改造的正交翻译系统，以便在指定的蛋白质位置掺入修饰氨基酸。含硒代半胱氨酸的硒蛋白也已知会发生翻译后修饰。硒蛋白在氧化应激、免疫反应、细胞维持和骨骼肌再生中发挥着重要作用。其复杂的生物合成机制一直是我们理解硒蛋白功能的障碍。随着最近硒代半胱氨酸插入技术的改进，我们希望创建一个能够研究硒蛋白翻译后修饰的遗传系统。通过结合遗传密码扩展技术和硒代半胱氨酸插入技术，我们能够分别将终止密码子重新编码，以便将ε-乙酰基-L-赖氨酸和硒代半胱氨酸插入多种蛋白质中。这些氨基酸在其指定位置的特异性以及通过密码子序列诱变恢复修饰氨基酸的简便性，证明了该方法研究硒蛋白及其翻译后修饰作用的能力。此外，硒代半胱氨酸插入技术可与遗传密码扩展工具相结合的证据进一步拓展了化学生物学的应用。
