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

我们研究的最终目标是了解蛋白质是如何在细菌的细胞质膜（CM）中转运的，从细胞质中蛋白质的合成开始，到CM另一侧的成熟结束。CM在细胞的定义中起着关键作用，因此我们对生命的定义。细胞有时被描述为“蛋白质袋”，其中CM是“袋”。它将细胞的内容物（细胞质）从细胞外环境中分离出来，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也提出了一个根本性的问题。许多蛋白质在细胞外执行（通常是必需的）功能。在大肠杆菌中，这些蛋白质占所有合成蛋白质的约30%。蛋白质合成只发生在细胞质中，但蛋白质不能自己穿过CM。因此，细胞已经进化出许多复杂的机制来运输蛋白质穿过CM。其中，SEC机器负责绝大多数蛋白质出口。Sec机制的中心组成部分是CM中普遍保守的通道。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通过该通道运输。通过Sec机器识别蛋白质底物是快速、高效且非常准确的，但是尽管经过数十年的研究，识别底物蛋白质的过程仍然是一个未解决的问题。然而，卫生署最近的研究为这个老问题提供了新的线索。这项研究表明，SecA本身识别底物蛋白，并且识别发生在底物蛋白仍在制造的时候-早在跨CM运输开始之前。本提案中所述的研究旨在了解：（i）SecA如何识别底物蛋白，（ii）底物蛋白如何随后递送至CM中的通道，以及（iii）识别和递送的时机如何影响CM另一侧底物蛋白的后续成熟。生物技术.例如，在他的博士研究期间，DH确定了一组信号，这些信号可以靶向通过比SecA依赖性途径更有效的第二平行途径跨CM转运的蛋白质，并且这些信号已被广泛用于分泌蛋白质，否则蛋白质转运难治。然而，通过这种平行途径的输出具有许多显著的局限性，包括低蛋白表达水平、细胞毒性，以及在某些情况下，有缺陷的蛋白成熟。对SecA依赖性转运机制的深入了解可能会导致蛋白质生产方法的改进。这项研究也有可能为对抗细菌感染的新方法做出贡献。一些细菌如E.大肠杆菌在细胞外含有第二层膜，作为许多抗微生物化合物的屏障，并且转运蛋白的适当成熟对于维持该屏障的完整性是重要的。因此，可以利用蛋白质被传递到CM中的通道和运输蛋白质的成熟之间的强有力的联系来增加这些细菌对许多其他无用的抗菌化合物的敏感性。例如，开发用于检查SecA-核糖体复合物结构的高通量测序（HTS）方法可用于检查其他蛋白质与其他大RNA或DNA分子的相互作用，并且使用HTS来鉴定涉及OM生物发生的新途径可扩展到研究其他生物途径。

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.