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The mechanism of SecA-dependent substrate recognition and delivery in Escherichia coli

大肠杆菌中 SecA 依赖性底物识别和递送机制

基本信息

批准号：
BB/L019434/1
负责人：
Damon Huber
金额：
$ 50.66万
依托单位：
University of Birmingham
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2014
资助国家：
英国
起止时间：
2014 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FL019434%2F1
关键词：
mechanism SecA dependent substrate recognition

项目摘要

The ultimate goal of our research is to understand how proteins are transported across the cytoplasmic membrane (CM) in bacteria starting with synthesis of a protein in the cytoplasm and ending with its maturation on the other side of the CM.The CM plays a critical role in the definition of a cell and thus our definition of life. Cells are sometimes described as "bags of proteins", in which the CM is the "bag". It divides the contents of the cell (the cytoplasm) from the extracellular milieu, and it protects the cell from changes in the environment. Without the bag, there is no cell. However, the CM also poses a fundamental problem. A large number of proteins carry out (often essential) functions outside the cell. In the bacterium Escherichia coli, these proteins comprise ~30% of all proteins synthesized. Protein synthesis occurs exclusively in the cytoplasm, but proteins cannot cross the CM on their own. Thus, the cell has evolved a number of complex machineries to transport proteins across the CM. Of these, the Sec machinery is responsible for the vast majority of protein export. The central component of the Sec machinery is a universally conserved channel in the CM. In bacteria, most proteins are transported through this channel by a pump protein, SecA, only after they have been fully (or nearly fully) synthesized. Recognition of protein substrates by the Sec machinery is rapid, efficient, and very accurate, but despite decades of research, the process by which substrate proteins are recognized remains an unsolved problem. However, recent research by DH sheds new light on this old problem. This research suggests that SecA itself recognizes substrate proteins, and recognition occurs as the substrate protein is still being made-long before transport across the CM commences. The research described in this proposal aims to understand: (i) how substrate proteins are recognized by SecA, (ii) how substrate proteins are subsequently delivered to the channel in the CM, and (iii) how the timing of recognition and delivery affects the subsequent maturation of the substrate protein on the other side of the CM.Previous research in this area has been directly applied in the development of new tools for biotechnology. For example, during his PhD research, DH identified a set of signals that could target proteins to be transported across the CM by a second parallel pathway that is more efficient than the SecA-dependent pathway, and these signals have been widely used to secrete proteins that are otherwise refractory to protein transport. However, export by this parallel pathway has a number of significant limitations, including low protein expression levels, cellular toxicity, and in some cases, defective protein maturation. Greater insight into the mechanism of SecA-dependent transport could lead to improved methods for protein production.This research also has the potential to contribute to new approaches for combating bacterial infections. Some bacteria such as E. coli contain a second membrane outside the cell that serves as a barrier to many antimicrobial compounds, and the proper maturation of transported proteins is important for maintaining the integrity this barrier. Thus, the strong link between route by which proteins are delivered to the channel in the CM and the maturation of the transported protein could be exploited to increase the sensitivity of these bacteria to many otherwise useless antimicrobial compounds.Finally, many of the techniques developed in this grant can be adapted for use in other studies. For example, the high-throughput sequencing (HTS) methods developed to examine the structure of SecA-ribosome complex can be used to examine the interaction of other proteins with other large RNA or DNA molecules, and the use of HTS to identify novel pathways involved in OM biogenesis can be extended to research into other biological pathways.

我们研究的最终目标是了解细菌中蛋白质是如何通过细胞质膜(CM)运输的，从细胞质中蛋白质的合成开始，到CM另一边的成熟结束。CM在细胞的定义中起着关键作用，因此我们对生命的定义也是如此。细胞有时被描述为“蛋白质袋”，其中CM是“袋”。它将细胞内容物(细胞质)与细胞外环境分开，并保护细胞免受环境变化的影响。没有袋子，就没有牢房。然而，CM也提出了一个根本问题。大量蛋白质在细胞外执行(通常是必不可少的)功能。在大肠杆菌中，这些蛋白质约占所有合成蛋白质的30%。蛋白质的合成只发生在细胞质中，但蛋白质本身不能穿过细胞质。因此，细胞进化出了许多复杂的机制来通过CM运输蛋白质。其中，SEC负责绝大多数蛋白质出口。SEC机制的中心组成部分是CM中一个普遍保守的通道。在细菌中，大多数蛋白质只有在完全(或几乎完全)合成后，才由泵蛋白SecA通过这一通道运输。SEC机器对蛋白质底物的识别是快速、高效和非常准确的，但尽管进行了数十年的研究，底物蛋白质的识别过程仍然是一个尚未解决的问题。然而，卫生署最近的研究为这个古老的问题提供了新的线索。这项研究表明，SECA本身识别底物蛋白，当底物蛋白仍在制造时-早在跨CM运输开始之前-识别就发生了。这项建议中描述的研究旨在了解：(I)底物蛋白质是如何被SecA识别的，(Ii)底物蛋白质如何随后被输送到CM中的通道，以及(Iii)识别和输送的时机如何影响CM另一侧的底物蛋白质随后的成熟。以前在这方面的研究已经直接应用于开发新的生物技术工具。例如，在他的博士研究期间，他发现了一组信号，这些信号可以针对蛋白质，通过第二条平行的途径通过CM运输，这条途径比依赖SecA的途径更有效，这些信号已经被广泛用于分泌本来难以进行蛋白质运输的蛋白质。然而，这种平行途径的出口有许多显著的限制，包括低蛋白表达水平、细胞毒性，在某些情况下，有缺陷的蛋白成熟。对SecA依赖的运输机制的更深入的了解可能会导致改进蛋白质生产的方法。这项研究也有可能为抗击细菌感染的新方法做出贡献。一些细菌，如大肠杆菌，在细胞外含有第二层膜，作为许多抗菌化合物的屏障，运输蛋白质的适当成熟对于维持这一屏障的完整性非常重要。因此，蛋白质被输送到CM中的通道和运输的蛋白质成熟之间的强烈联系可以被利用来增加这些细菌对许多原本无用的抗菌化合物的敏感性。最后，这项资助中开发的许多技术可以用于其他研究。例如，用来检测SecA-核糖体复合体结构的高通量测序(HTS)方法可以用来检测其他蛋白质与其他大RNA或DNA分子的相互作用，使用HTS来识别OM生物发生的新途径可以扩展到对其他生物途径的研究。