Genetic encoding of Post-translational modifications

翻译后修饰的基因编码

• 批准号: BB/R004692/1
• 负责人: Amit Sachdeva
• 金额: $ 50.79万
• 依托单位: University of East Anglia
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR004692%2F1
• 关键词: Genetic; encoding; Post; translational; modifications

Post-translational modifications (PTMs) are covalent modifications on side chains or termini of proteins installed by specific enzymes after ribosomal protein synthesis in cells. PTMs play an important role in modulating several biological processes and is one of the methods used by nature to increase the repertoire of chemical functional groups in proteins. Methylation of arginine is an important PTM that modulates the activity of numerous proteins involved in RNA processing, transcriptional regulation, signal transduction and DNA repair. Tyrosine phosphorylation, another PTM, that plays an important role in cell-cycle progression, cell proliferation, cellular homeostasis, neural activity, transcriptional activation and ageing. Many diseases, including cancer and neurological disorders, are associated with abnormal levels of PTMs. Despite the importance of PTMs in biology, it is often difficult to obtain a homogeneous sample of protein with specific PTMs. This is mainly because cells have competing mechanisms to install and remove PTMs, and samples of post-translationally modified proteins also contain unmodified proteins. It is often difficult to separate post-translationally modified proteins from their unmodified analogues. The problem is further compounded if the protein has multiple sites for PTMs. In addition, proteomics analyses have revealed numerous previously unidentified sites of PTMs on many proteins. Often enzymes that install these PTMs are not known, thus further limiting the access to these post-translationally modified proteins. To address these challenges, here we propose to develop methods for genetic site-specific incorporation of methylarginines and phosphotyrosine. This work would provide facile access to several important post-translationally modified proteins. Proteins are synthesized in all organisms by a biological machine called the ribosome. The ribosome uses messenger RNA (mRNA) as a template and transfer RNA (tRNA) as an adaptor molecule to catalyse protein synthesis using a set of rules defined by the genetic code. For each naturally occurring amino acid there are, 1) a unique set of triplet bases in the DNA or mRNA, called codons, which correspond to the amino acid, and 2) a unique set of aminoacyl-tRNA synthetases (aaRS)/ tRNA pairs. aaRSs catalyze the charging of their corresponding tRNAs with their corresponding amino acids. During protein synthesis, the charged tRNA is taken to the ribosome where it binds to its cognate codon on the mRNA in the small subunit of the ribosome, and concurrently an amino acid is added to the growing peptide chain in the large subunit. Genetic incorporation of unnatural amino acids in cells has been achieved by assigning an amber stop codon or quadruplet codons to unnatural amino acids and supplying the cells with orthogonal aaRS/ tRNA pairs. Orthogonal aaRS/ tRNA pairs do not interfere with the tRNA synthetases and the tRNAs of the host cell, and specifically recognize the unnatural amino acid. Using this approach, numerous unnatural amino acids have been genetically encoded in different organisms. However, only a small number of PTMs have been genetically encoded. In this proposal, we explore different approaches to identify new orthogonal aaRS/ tRNA pairs in E. coli to enable genetic encoding of methylarginines and phosphotyrosine.

翻译后修饰（Post-translational modification，PTM）是细胞内核糖体蛋白质合成后，由特定的酶在蛋白质的侧链或末端进行共价修饰。PTM在调节几种生物过程中起重要作用，并且是自然界用于增加蛋白质中化学官能团库的方法之一。精氨酸的甲基化是一种重要的PTM，其调节参与RNA加工、转录调节、信号转导和DNA修复的许多蛋白质的活性。酪氨酸磷酸化是另一种PTM，在细胞周期进程、细胞增殖、细胞稳态、神经活性、转录激活和衰老中起重要作用。许多疾病，包括癌症和神经系统疾病，都与PTM的异常水平有关。尽管PTM在生物学中很重要，但通常很难获得具有特定PTM的蛋白质的均匀样品。这主要是因为细胞具有安装和移除PTM的竞争机制，并且后修饰蛋白质的样品也包含未修饰的蛋白质。通常难以将后修饰的蛋白质与其未修饰的类似物分离。如果蛋白质具有多个PTM位点，则问题进一步复杂化。此外，蛋白质组学分析揭示了许多以前未鉴定的位点的PTM上的许多蛋白质。通常，安装这些PTM的酶是未知的，因此进一步限制了对这些后修饰蛋白质的获取。为了解决这些挑战，在这里，我们建议开发方法的遗传位点特异性纳入甲基精氨酸和磷酸酪氨酸。这项工作将为几种重要的后修饰蛋白质提供便利。蛋白质在所有生物体中都是由一种叫做核糖体的生物机器合成的。核糖体使用信使RNA（mRNA）作为模板，转移RNA（tRNA）作为衔接分子，使用遗传密码定义的一组规则催化蛋白质合成。对于每种天然存在的氨基酸，1）DNA或mRNA中对应于氨基酸的一组独特的三联体碱基，称为密码子，和2）一组独特的氨酰-tRNA合成酶（阿尔斯）/ tRNA对。aaRS催化其相应的tRNA与其相应的氨基酸的电荷。在蛋白质合成过程中，带电荷的tRNA被带到核糖体，在那里它与核糖体的小亚基中mRNA上的同源密码子结合，同时一个氨基酸被添加到大亚基中不断生长的肽链上。通过将琥珀终止密码子或四联体密码子分配给非天然氨基酸并向细胞提供正交阿尔斯/ tRNA对，已经实现了非天然氨基酸在细胞中的遗传掺入。阿尔斯/ tRNA对不干扰宿主细胞的tRNA合成酶和tRNA，并且特异性地识别非天然氨基酸。使用这种方法，许多非天然氨基酸已经在不同的生物体中进行了遗传编码。然而，只有少量的PTM已被遗传编码。在这个提议中，我们探索了不同的方法来确定新的正交阿尔斯/ tRNA对在E。大肠杆菌，使甲基精氨酸和磷酸酪氨酸的遗传编码。

项目成果

Site-Specific Encoding of Photoactivity in Antibodies Enables Light-Mediated Antibody-Antigen Binding on Live Cells

抗体中光活性的位点特异性编码使得光介导的抗体-抗原能够在活细胞上结合

• DOI: 10.1002/ange.201908655
• 发表时间: 2019
• 期刊: Angewandte Chemie
• 作者: Bridge T

Site-specific encoding of photoactivity and photoreactivity into antibody fragments.

• DOI: 10.1038/s41589-022-01251-9
• 发表时间: 2023-06
• 期刊: NATURE CHEMICAL BIOLOGY
• 影响因子: 14.8
• 作者: Bridge, Thomas;Wegmann, Udo;Crack, Jason C. C.;Orman, Kate;Shaikh, Saher A. A.;Farndon, William;Martins, Carlo;Saalbach, Gerhard;Sachdeva, Amit
• 通讯作者: Sachdeva, Amit