小柯机器人

工程化正交信号通路揭示序列空间的稀疏占用性
2019-10-25 10:44

美国麻省理工学院Michael T. Laub课题组近日取得一项新成果。他们通过构建工程正交信号通路,揭示了序列空间的稀疏占用性。相关论文2019年10月23日在线发表于《自然》杂志。

研究人员通过使用结合了深度测序的细胞分选技术在大肠杆菌中产生新型的两组分信号蛋白,来直接研究此序列空间的拥挤程度,从而分析基于协同进化模式设计的大型文库。研究人员产生了58条由功能性激酶与底物对组成的绝缘通路,这些激酶与底物对的特异性与其母体蛋白不同,并且证明了其中的几对与大肠杆菌中所有27个旁系通路正交。

此外,从生成的激酶与底物对中,研究人员鉴定出由彼此相互正交的六对组成的集合,这大大增加了大肠杆菌的两组分信号传导能力。这些结果表明序列空间没有被密集地占据。序列空间中旁系同源物的相对稀疏性表明,在进化过程中很容易出现新的绝缘途径,或者可以重新设计。研究人员通过在大肠杆菌中响应植物细胞分裂素的信号传导途径来证明后者,而没有对现有途径的干扰。

这项工作还证明了协同进化指导的诱变和序列空间的映射能够如何用于设计大组正交蛋白质间相互作用。

据了解,基因复制是细胞产生新的信号传导途径的常见而强大的机制,但是最近复制的蛋白质通常必须彼此绝缘,并与其他旁系同源物绝缘,以防止有害的串扰。当新的传感器或合成信号通路在细胞内进行工程改造或在基因组之间转移时,也会出现类似的挑战。将新途径引入细胞的难易程度取决于序列空间中旁源途径的密度和分布,该序列空间由它们的特异性决定残基所定义。

附:英文原文

Title: Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space

Author: Conor J. McClune, Aurora Alvarez-Buylla, Christopher A. Voigt, Michael T. Laub

Issue&Volume: 2019-10-23

Abstract: Gene duplication is a common and powerful mechanism by which cells create new signalling pathways1,2, but recently duplicated proteins typically must become insulated from each other and from other paralogues to prevent unwanted crosstalk3. A similar challenge arises when new sensors or synthetic signalling pathways are engineered within cells or transferred between genomes. How easily new pathways can be introduced into cells depends on the density and distribution of paralogous pathways in the sequence space that is defined by their specificity-determining residues4,5. Here we directly investigate how crowded this sequence space is, by generating novel two-component signalling proteins in Escherichia coli using cell sorting coupled to deep sequencing to analyse large libraries designed on the basis of coevolutionary patterns. We produce 58 insulated pathways comprising functional kinase–substrate pairs that have different specificities than their parent proteins, and demonstrate that several of these new pairs are orthogonal to all 27 paralogous pathways in E. coli. Additionally, from the kinase–substrate pairs generated, we identify sets consisting of six pairs that are mutually orthogonal to each other, which considerably increases the two-component signalling capacity of E. coli. These results indicate that sequence space is not densely occupied. The relative sparsity of paralogues in sequence space suggests that new insulated pathways can arise easily during evolution, or be designed de novo. We demonstrate the latter by engineering a signalling pathway in E. coli that responds to a plant cytokinin, without crosstalk to extant pathways. Our work also demonstrates how coevolution-guided mutagenesis and the mapping of sequence space can be used to design large sets of orthogonal protein–protein interactions.

DOI: 10.1038/s41586-019-1639-8

Source: https://www.nature.com/articles/s41586-019-1639-8

Nature:《自然》,创刊于1869年。隶属于施普林格·自然出版集团,最新IF:43.07
官方网址:http://www.nature.com/
投稿链接:http://www.nature.com/authors/submit_manuscript.html


本期文章:《自然》:Online/在线发表

分享到:

0