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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=708144
• 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.

生物细胞使用储存在基因和DNA中的遗传信息，从20种不同的氨基酸或构建模块构建蛋白质。蛋白质在细胞中发挥着重要的结构和功能作用，例如，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量，甚至复制或复制遗传信息传递给下一代。类似的过程发生在各种各样的生命中，从单细胞微生物到复杂的多细胞生物，包括人类。 尽管蛋白质仅由20种典型的氨基酸组成，但在成熟蛋白质中发现了400多种不同的氨基酸。这些非典型氨基酸（ncAA）是生命所必需的，因为参与基本细胞过程的蛋白质经历了显着的翻译后修饰（PTM）。在这个过程中，某些（很多时候未知的）被称为酶的蛋白质在原始蛋白质形成后对这20个氨基酸进行化学修饰。PTM传递改变细胞命运和基因表达的化学信号。PTM或PTM的特定组合的确切作用目前尚不清楚，因为没有方法可用于合成具有多个ncAA的蛋白质。这种方法是迫切需要的，并确定在蛋白质和途径的结构和功能作用的PTM是必不可少的。 一些涉及PTM的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。在信号通路中，带有磷酸盐的氨基酸从一个蛋白质级联转移到下一个蛋白质（就像电话的分子游戏）。这个过程告诉细胞周围环境发生了什么，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，其他细胞）。表观遗传途径是另一个应用领域，涉及改变基因表达的化学修饰的AA，而实际上不改变基因组中的DNA序列。事实上，这些表观遗传变化可以遗传给下一代。最后，依赖于ncAA硒代半胱氨酸的氧化应激途径形成细胞的天然抗氧化防御系统。由于硒代半胱氨酸是一种高度化学反应性的ncAA，拟议的工作还将探索使用这种ncAA来创造有效的生物催化剂（加速化学反应的试剂）。 为了研究这些蛋白质修饰在细胞途径中的作用，并创造具有新特性的酶，拟议的研究将建立合成具有多种ncAA的蛋白质的系统。拟议的工作将涉及工程蛋白质合成系统，以位点特异性硬连线蛋白质与多个ncAA，创造新的生物合成和化学合成路线ncAA，并设计含硒半胱氨酸的蛋白质的工业相关应用。该技术将是基础性的，不限于这里概述的项目，因为ncAA，包括翻译后修饰，在自然界中无处不在，并在从微生物到人类细胞的各种生物系统中发挥重要作用。