Protein synthesis with multiple non-canonical amino acids

多种非规范氨基酸的蛋白质合成

• 批准号: RGPIN-2014-04282
• 负责人: ODonoghue, Patrick
• 金额: $ 3.79万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744049
• 关键词: Protein; synthesis; multiple; non; canonical

The biological cell uses genetic information stored in genes and DNA to build proteins from 20 different amino acids or building blocks. Proteins play important structural and functional roles in the cell by, for example, establishing communication between cells, converting food molecules (like sugar) into chemical energy that the cell can use, and even copying or replicating the genetic information that is passed on to the next generation. Similar processes occur across the great diversity of life, from single-celled microbes to complex multicellular organisms, including humans.Despite the fact that proteins are made from only 20 typical amino acids, over 400 different kinds of amino acids are found in mature proteins. These non-canonical amino acids (ncAAs) are essential to life because proteins involved in fundamental cellular processes undergo significant posttranslational modification (PTM). In this process, certain (many times unknown) proteins called enzymes chemically modify those 20 building block amino acids after the original protein is made. PTMs relay chemical signals that alter cell fate and gene expression. The precise role of PTMs or particular combinations of PTMs is currently unknown because no methods are available to synthesize proteins with multiple ncAAs. Such a method is urgently needed and essential to define the structural and functional role of PTMs in proteins and pathways. Some of the most complex and interesting pathways involving PTMs include signalling, epigenetic and oxidative stress pathways. In signalling pathways, amino acids with attached phosphates are transferred in cascades from one protein to the next (like a molecular game of telephone). This process tells the cell what is going on in its environment, where energy sources are and whether predators (such as viruses) or prey (in some cases other cells) are present. Epigenetic pathways are another application area and involve chemically modified AAs that alter gene expression, without actually changing the sequence of DNA in the genome. These epigenetic changes can in fact be inherited by the next generation. Finally, oxidative stress pathways that rely on the ncAA selenocysteine form the cell's natural antioxidant defense system. Because selenocysteine is a highly chemically reactive ncAA, the proposed work will also explore using this ncAA to create efficient biocatalysts (agents that speed up chemical reactions). To investigate the role of these protein modifications in cellular pathways and to create enzymes with novel properties, the proposed research will establish systems that synthesize proteins with multiple ncAAs. The proposed work will involve engineering protein synthesis systems to site-specifically hardwire proteins with multiple ncAAs, creating new biosynthetic and chemical synthetic routes to ncAAs, and designing selenocysteine containing proteins for industrially relevant applications. The technology will be fundamental and not limited to the projects outlined here, because ncAAs, including posttranslational modifications, are ubiquitous in nature and play important roles in diverse biological systems from microorganisms to human cells.

生物细胞利用储存在基因和DNA中的遗传信息，从20种不同的氨基酸或构建块中构建蛋白质。蛋白质在细胞中扮演着重要的结构和功能角色，例如，通过建立细胞之间的通信，将食物分子（如糖）转化为细胞可以使用的化学能，甚至复制或复制遗传信息传递给下一代。从单细胞微生物到复杂的多细胞生物（包括人类），类似的过程发生在各种各样的生命中。尽管蛋白质只由20种典型的氨基酸组成，但在成熟的蛋白质中发现了400多种不同的氨基酸。这些非规范氨基酸（ncAAs）对生命至关重要，因为参与基本细胞过程的蛋白质经历了重要的翻译后修饰（PTM）。在这个过程中，某些（很多时候是未知的）被称为酶的蛋白质在原始蛋白质合成后，用化学方法修饰了这20个基本氨基酸。PTMs传递改变细胞命运和基因表达的化学信号。PTMs或PTMs的特定组合的确切作用目前尚不清楚，因为没有方法可以合成含有多个ncAAs的蛋白质。这种方法对于确定PTMs在蛋白质和通路中的结构和功能作用是迫切需要的。涉及PTMs的一些最复杂和最有趣的途径包括信号传导、表观遗传和氧化应激途径。在信号传导途径中，氨基酸与附着的磷酸盐以级联的方式从一个蛋白质转移到另一个蛋白质（就像电话的分子游戏）。这个过程告诉细胞在其环境中发生了什么，能量来源在哪里，是否存在捕食者（如病毒）或猎物（在某些情况下是其他细胞）。表观遗传途径是另一个应用领域，涉及化学修饰的AAs改变基因表达，而不实际改变基因组中的DNA序列。这些表观遗传变化实际上可以被下一代遗传。最后，依赖于ncAA硒代半胱氨酸的氧化应激途径形成了细胞的天然抗氧化防御系统。由于硒代半胱氨酸是一种具有高度化学活性的ncAA，因此该研究还将探索利用这种ncAA来制造高效的生物催化剂（加速化学反应的试剂）。为了研究这些蛋白质修饰在细胞通路中的作用，并创造具有新特性的酶，拟议的研究将建立具有多个ncaa合成蛋白质的系统。拟议的工作将涉及工程蛋白质合成系统，以定位特定的硬线蛋白与多个ncAAs，创建新的生物合成和化学合成途径ncAAs，并设计含硒半胱氨酸的蛋白质用于工业相关应用。这项技术将是基础的，并不局限于这里概述的项目，因为ncAAs，包括翻译后修饰，在自然界中无处不在，在从微生物到人类细胞的各种生物系统中发挥重要作用。