DeADP-ribosylation of host targets mediated by a bacterial effector

由细菌效应子介导的宿主靶标的 DeADP-核糖基化

• 批准号: 10667971
• 负责人: Chittaranjan Das
• 金额: $ 22.93万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-19 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10667971
• 关键词: ADP ribosylation; Address; Adenosine Diphosphate Ribose; Adopted; Affinity; Arginine; Bacteria; Binding; Biochemical; Biological; Biological Assay; Biological Process; Biotinylation; Cell physiology; Cells; Chemistry; Complement; Complex; Coupled; Crystallization; Cytosol; Data Set; Deubiquitinating Enzyme; Deubiquitination; Development; Elements; Engineering; Enzyme Interaction; Enzymes; Event; Family; Glutaminase; Glutamine; Glycoside Hydrolases; Goals; Health; Human; Hydrolysis; Infection; Interferometry; Investigation; Label; Legionella; Legionella pneumophila; Legionnaires' Disease; Ligation; Link; Liquid Chromatography; Lysine; Measures; Mediating; Methods; Molecular; Mutagenesis; Organelles; Outcome; Pathogenesis; Pathogenicity; Pneumonia; Process; Protein Secretion; Proteins; Proteome; Proteomics; Research; Resistance; Role; Series; Serine; Side; Streptavidin; Structure; System; Ubiquitin; Ubiquitination; Vacuole; Virulence; Work; antimicrobial drug; candidate identification; cofactor; crosslink; enzyme model; improved; insight; mutant; novel; pathogen; pathogenic bacteria; polypeptide; tandem mass spectrometry; tool; ubiquitin isopeptidase; ubiquitin-protein ligase; unnatural amino acids

The pathogenic bacteria Legionella pneumophila (Lp), the causative agent of Legionnaires’ disease, injects nearly 330 effectors using its Dot/Icm protein secretion system into host cytosol to remodel cellular processes toward creating and maintaining a replicative organelle called the legionella-containing vacuole (LCV). Research in the last few years has shown some remarkable examples of ubiquitination chemistry catalyzed by some of these effectors that are unparalleled in the eukaryotic world. Two notable examples of ubiquitination of host targets have been identified where the mechanisms are independent of and radically different from the eukaryotic E1-E2-E3 system. This has added an exciting new layer of complexity to ubiquitin (Ub) manipulation by Lp, which was already known to possess several classical Ub E3 ligases and deubiquitinases among its effectors. This raises a question as to how far and deep Legionella’s manipulation of the host ubiquitin goes. Are there additional new Ub-manipulating proteins/enzymes in the large effector arsenal of the bacteria? We have performed an activity-based enrichment analysis to identify novel Ub-interacting proteins among Lp effectors to address this question. We have used Ub-derived activity-based probes (Ub-ABPs) to enrich proteins from the soluble proteome of Lp and have identified candidates using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This has led us to the Dot/Icm substrate MavL, with unknown biochemical and biological function, as a novel Ub-interacting effector. On further analysis, we find that MavL is a deADP-ribosyl glycohydrolase capable of removing ADP-ribose (ADPR) from an arginine side chain of ADP-ribosylated Ub (ADPR-Ub). We will use a proximity labeling biotinylation strategy implemented under infection conditions to capture MavL substrates. To complement this strategy, we will engineer an inactive mutant of the enzyme into an affinity tool for proteome-wide enrichment and identification of MavL interactors from host cells. Combining information from the proteomics datasets, we aim to arrive at a list of candidates which will be validated in cell-based assays under infection conditions. Further, we will try to provide the structural basis of Ub recognition by this novel pathogenic effector. Taken together, we will be able to characterize a unique Ub-recognizing deADP-ribosylating enzyme from a pathogenic bacterium and explore its host targets, thereby expanding the scope of reversible ADP-ribosylation in host-pathogen interaction.

致病菌嗜肺军团菌（Lp）是军团病的病原体， 使用其 Dot/Icm 蛋白分泌系统将近 330 个效应器注射到宿主细胞质中以重塑细胞 创建和维持称为含军团菌的复制细胞器的过程 过去几年的研究已经展示了一些值得注意的泛素化例子。 其中一些效应子催化的化学反应在真核世界中是无与伦比的，其中有两个值得注意。 已经确定了宿主靶点泛素化的例子，其中机制是独立的 与真核 E1-E2-E3 系统完全不同，这增加了一个令人兴奋的新层。 Lp 操纵泛素 (Ub) 的复杂性，众所周知，Lp 拥有几种经典的 Ub E3 连接酶和去泛素化酶在其效应子中的作用范围和深度如何？ 军团菌对宿主泛素的操纵是否存在其他新的 Ub 操纵？ 细菌大型效应器库中的蛋白质/酶？ 我们进行了基于活性的富集分析来鉴定新型 Ub 相互作用蛋白 为了解决这个问题，我们使用了 Ub 衍生的基于活性的探针 (Ub-ABP)。 富集 Lp 可溶性蛋白质组中的蛋白质，并使用液体鉴定了候选蛋白质 色谱法与串联质谱法 (LC-MS/MS) 相结合，使我们得到了 Dot/Icm。 底物 MavL 具有未知的生化和生物学功能，作为一种新型的 Ub 相互作用效应子。 进一步分析，我们发现MavL是一种去ADP-核糖基糖水解酶，能够去除ADP-核糖 (ADPR) 来自 ADP-核糖基化 Ub (ADPR-Ub) 的精氨酸侧链 我们将使用邻近标记。 在感染条件下实施生物素化策略以捕获 MavL 底物以补充。 通过这种策略，我们将把酶的无活性突变体设计成全蛋白质组的亲和工具 结合来自宿主细胞的信息来富集和鉴定 MavL 相互作用子。 蛋白质组学数据集，我们的目标是获得一份候选者列表，这些候选者将在基于细胞的测定中得到验证 此外，我们将尝试以此提供Ub识别的结构基础。 综合起来，我们将能够描述一种独特的 Ub 识别效应。 来自病原菌的 deADP-核糖基化酶，从而探索其宿主靶点，扩大 宿主-病原体相互作用中可逆 ADP-核糖基化的范围。