Legionella effectors reveal novel signaling function for the ribosome quality control (RQC) pathway

摘要/项目摘要 蛋白质合成是细胞代谢的一个关键步骤，受到来自内部刺激的干扰 或有时可能导致信使RNA上核糖体翻译暂停的外部因素， 这种现象被定义为核糖体停滞。有趣的是，革兰氏阴性细菌嗜肺军团菌 （L.p.）操纵几个关键的宿主过程，如蛋白质合成和膜转运， 在内质网（ER）样空泡中建立其复制小生境。L. P.通过注入 效应蛋白通过其IV型分泌系统，Dot/Icm。了解细菌如何破坏宿主 过程和操纵他们自己的优势提供了宝贵的见解疾病， 哺乳动物细胞生物学最近的研究强调，L.p分泌毒素进入感染细胞， 抑制蛋白质合成并诱导核糖体停滞。细胞已经进化出一种保守的机制， 核糖体相关质量控制（RQC），有助于检测和解析停滞的核糖体， 维持蛋白质稳态。然而，核糖体停滞应激对细胞的影响仍然存在 不太了解。通过用L. p.或次优剂量的感染来挑战细胞翻译装置 翻译延伸抑制剂，我们已经确定，在细胞中停滞的核糖体的传感激活 导致转录激活因子3上调的转录和翻译程序 （ATF 3）。我们已经确定，这个反动的程序是独立于其他典型的压力 反应途径，如未折叠蛋白反应（UPR）或整合应激反应（ISR）。 引人注目的是，ATF 3受L. p.衍生的蛋白质SidI和SusF（毒素-抗毒素对）调节。的目标 细胞中的SidI和SusF未定义。此外，ATF 3的上调依赖于多个 RQC途径的组成部分。此外，我们的观察表明，ATF 3信使是选择性的， 即使在抑制新生蛋白质合成的条件下也能翻译。伴随激活 在核糖体失速诱导的反应中，L. p.还招募了一群具有免疫活性的核糖体， 细胞内的复制生态位。 该建议旨在利用来自L. p.的分子工具来了解RQC的联系机制 与细胞核中ATF 3的激活有关。我们预计，从拟议的一组实验的结果将 阐明了维持细胞内蛋白质合成稳态的基本机制， 产生新的工具来研究和剖析RQC途径。我们组建了一支非常强大的队伍 专家和令人信服的初步数据，突出了我们实现目标的能力。

Abstract/Project Summary Protein synthesis is a key metabolic step in cells that is subjected to perturbations from internal stimuli or external agents that may sometimes lead to the pausing of translating ribosomes on messenger RNAs, a phenomenon defined as ribosome stalling. Interestingly, the gram-negative bacterium Legionella pneumophila (L.p.) manipulates several critical host processes such as protein synthesis and membrane transport to establish its replicative niche in an endoplasmic reticulum (ER)-like vacuole. L.p. achieves this by injecting effector proteins via its type IV secretion system, Dot/Icm. Understanding how bacteria sabotage host processes and manipulate them to their own advantage provides invaluable insights into disease and mammalian cell biology. Recent studies have highlighted that L.p secretes toxins into infected cells that can inhibit protein synthesis and induce ribosome stalling. Cells have evolved a conserved mechanism called ribosome-associated quality control (RQC) that aids in the sensing and resolution of stalled ribosomes and the maintenance of protein homeostasis. The consequences of ribosome stalling stress to cells however remain poorly understood. By challenging the cellular translation apparatus with infections of L.p. or sub-optimal doses of translation elongation inhibitors, we have determined that the sensing of stalled ribosomes in cells activates a transcriptional and translational program that leads to the upregulation of the activating transcription factor 3 (ATF3). We have determined that this reactionary program is independent from other canonical stress response pathways such as the unfolded protein response (UPR) or the integrated stress response (ISR). Strikingly, ATF3 is regulated by L.p. derived proteins SidI and SusF, a toxin-antitoxin couple. The targets of SidI and SusF in cells are undefined. Furthermore, the upregulation of ATF3 was dependent on multiple components of the RQC pathway. Moreover, our observations indicate that ATF3 messengers are selectively translated even under conditions where nascent protein synthesis is suppressed. Concomitant to the activation of the ribosome stall induced response, L.p. also recruits a pool of translationally active ribosomes to its replicative niche within cells. This proposal aims to utilize the molecular tools derived from L.p. to understand the mechanisms that link RQC to the activation of ATF3 in the nucleus. We expect that the results from the proposed set of experiments will shed light onto fundamental mechanisms that maintain the homeostasis of protein synthesis in cells and generate new tools to study and dissect the RQC pathway. We have assembled an exceptionally strong team of experts and compelling preliminary data that highlight our ability to accomplish our goals.