Mechanisms for Stress-Induced Transcriptional Reprogramming via Anti-Adaptors

通过反适配器进行应激诱导转录重编程的机制

• 批准号: 9229317
• 负责人: Alexandra M. Deaconescu
• 金额: $ 31.5万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9229317
• 关键词: Animals; Anti-Bacterial Agents; Antibiotics; Bacteria; Bacteria sigma factor KatF protein; Behavior; Binding; Binding Sites; Biochemistry; Biocide; Biological Assay; Cells; Communities; Complement; Complex; Cryoelectron Microscopy; Cues; DNA Damage; DNA-Directed RNA Polymerase; Desiccation; Development; Environment; Escherichia coli; Gammaproteobacteria; Genes; Genetic Transcription; Genome; Human; In Vitro; Individual; Infection; Knowledge; Laboratories; Magnesium; Mediating; Metals; Methods; Microbial Biofilms; Microscopy; Molecular Conformation; Molecular Genetics; Molecular Structure; Molecular Weight; Nutrient; Oxidative Stress; Pathway interactions; Phagocytosis; Phase; Phosphorylation; Plants; Play; Process; Protein Family; Proteins; Proteolysis; Regulation; Regulon; Reporting; Research Personnel; Resolution; Role; Sequence Homology; Signal Transduction; Starvation; Stress; Structural Models; Structure; System; Testing; Ubiquitin; Up-Regulation; Virulence; Virulence Factors; Work; X-Ray Crystallography; antimicrobial; biological adaptation to stress; biological systems; biophysical techniques; combat; in vivo; inhibitor/antagonist; inorganic phosphate; insight; logarithm; multicatalytic endopeptidase complex; novel; particle; pathogen; promoter; protein protein interaction; response; structural biology; toxic metal

PROJECT SUMMARY The dissociable promoter recognition subunit RpoS (also known as σs) is the master transcriptional regulator of the general stress response in γ-proteobacteria, and plays key roles in the virulence of many pathogens, including human, plant and animal pathogens. Under certain conditions, such as at the transition from the logarithmic to the stationary phase or in the presence of stress signals, RpoS redirects the core RNA polymerase machinery to a subset of promoters to reprogram transcription. However, intracellular RpoS levels are not steady – they are low in actively dividing cells, and substantially increased upon entering the stationary phase or upon encountering stress. To achieve proper regulation, there is tight control over RpoS levels, with the major point of regulation occurring at the level of RpoS proteolysis by the ATP-dependent ClpXP machine. Our central focus is to understand the mechanisms of RpoS proteolysis by ClpXP as well as its regulation by an emerging family of proteins collectively called anti-adaptors. In order to be degraded, RpoS is presented to ClpXP by a unique, highly specific adaptor called RssB, which acts catalytically, without being degraded. In turn, RssB itself is regulated by interactions with stress-specific anti-adaptors. Our work will focus on the structure and function of three-anti-adaptors: IraD (induced by oxidative stress and DNA damage), IraM (induced by magnesium starvation) and IraP (induced by phosphate starvation). These anti-adaptors share no sequence homology, and only weak homology with protein of known structure, warranting a structural biology effort aimed at deciphering the underlying mechanisms of RssB recognition. We will determine the structures of IraD, IraM and IraP, both in isolation and bound to RssB using X-ray crystallography. This will allow us to pinpoint, at atomic resolution, residues important for anti-adaptor/RssB interactions, and also regulation of the anti-adaptors themselves by oligomerization. We will complement these structural studies with molecular genetics, microscopy and functional assays for protein-protein interactions and RpoS degradation, which will allow us to correlate in vitro behavior with in vivo observations. We will also determine structures of a RpoS- RssB-ClpXP assembly using cutting-edge methods in electron cryo-microscopy, which will allow us to understand the RpoS and RssB conformational dynamics at the core of this paradigmatic mode of regulated proteolysis in bacteria. Overall, this work will not only bring fundamental, mechanistic insights, but will also open the way to the development of novel antibacterials that could target ClpXP directly, or, adaptor/anti- adaptor interfaces. The RpoS regulon has been reported to comprise up to 10% of the Escherichia coli genome, and RpoS itself plays important roles in bacterial persistence, host-pathogen interactions and biofilm formation, which underlie 80% of all infections.

项目摘要 可分离的启动子识别子单位RPO（也称为σs）是主转录调节器 γ-细菌的一般应激反应，并在许多病原体的病毒中起关键作用， 包括人，动植物病原体。在某些条件下，例如从 与固定相或应力信号存在的对数，RPO重定向核心RNA 聚合酶机械到启动子的子集进行重编程转录。但是，细胞内RPOS水平 不稳定 - 它们在积极分裂的细胞中很低，并且进入固定物时大幅增加 阶段或遇到压力。为了实现适当的调节，对RPOS水平有严格的控制， 由ATP依赖性CLPXP机器在RPOS蛋白水解水平上发生的主要调节点。 我们的核心重点是了解CLPXP的RPOS蛋白水解的机制以及其调节 一个新兴的蛋白质家族统称为抗自适应者。为了降级，将RPO提交给 CLPXP由一个称为RSSB的独特，高度特定的适配器，该适配器的作用，而不会降解。在 转弯，RSSB本身受到与应力特异性抗活体的相互作用的调节。我们的工作将集中在 三抗适应器的结构和功能：IRAD（由氧化应激和DNA损伤诱导），IRAM （由镁饥饿诱导）和IRAP（由磷酸盐饥饿诱导）。这些反适应性人共享 序列同源性，仅具有已知结构蛋白质的弱同源性，警告结构生物学 旨在破译RSSB识别的基本机制的努力。我们将确定结构 IRAD，IRAM和IRAP的属于X射线晶体学，并与RSSB结合。这将使我们能够 在原子分辨率下，确定对抗自适应/RSSB相互作用保留很重要，并且还调节了 反适应器本身是通过低聚的。我们将通过分子完成这些结构研究 蛋白质 - 蛋白质相互作用和RPOS降解的遗传学，显微镜和功能测定 允许我们将体外行为与体内观察结果相关联。我们还将确定RPOS-的结构 RSSB-CLPXP组装使用电子冷冻微观镜中的尖端方法，这将使我们得以 了解这种调节模式的核心的RPO和RSSB构象动力学 细菌中的蛋白水解。总体而言，这项工作不仅会带来基本的机械见解，而且还将带来 为可以直接靶向CLPXP的新型抗菌物的发展开辟道路，或 适配器接口。据报道，RPOS常规的大肠杆菌占10％ 基因组和RPO本身在细菌持久性，宿主 - 病原体相互作用和生物膜中起着重要作用 形成，这是所有感染的80％的基础。