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.

致病细菌肺炎军团菌（LP），是军团疾病的致病药物， 使用其点/ICM蛋白质分泌系统注射几乎330个效果，以重塑宿主细胞质 创建和维护复制细胞器的过程称为含传说 吸尘器（LCV）。过去几年的研究显示了一些显着的泛素化例子 在真核生物世界中无与伦比的这些作用中的某些作用催化了化学。两个值得注意的 已经确定了机制独立的宿主目标泛素化的例子 与真核E1-E2-E3系统的根本不同。这增加了令人兴奋的新层 LP对泛素（UB）操纵的复杂性，该操作已经众所周知 UB E3连接酶和去泛素酶的作用。这就提出了一个问题，即 Legionella对宿主泛素操纵的操纵。还有其他新的UB操作吗 细菌的大效应库中的蛋白质/酶？ 我们已经进行了基于活动的富集分析，以识别新型的UB相互作用蛋白 在LP效应中以解决这个问题。我们使用了基于UB的活动探针（UB-ABP） 从LP的固体蛋白质组中富集蛋白质，并使用液体鉴定了候选物 色谱与串联质谱法（LC-MS/MS）结合。这使我们进入了点/ICM 具有未知的生化和生物学功能的底物MAVL是一种新型的UB相互作用效应子。在 进一步的分析，我们发现MAVL是一种能够去除ADP-核糖的DeadP-核糖基糖基因多醇 （ADPR）来自ADP-核糖化UB（ADPR-UB）的精氨酸侧链。我们将使用接近标签 在感染条件下实施的生物素化策略以捕获MAVL底物。完成 这种策略，我们将把酶的无活跃突变体设计成蛋白质组范围的亲和力工具 来自宿主细胞的MAVL相互作用者的富集和鉴定。结合信息 蛋白质组学数据集，我们的目标是获得将在基于细胞的测定中验证的候选者列表 在感染条件下。此外，我们将尝试提供UB识别的结构性基础 新型致病效应子。综上所述，我们将能够表征独特的UB识别 来自致病细菌的DeadP-核糖基化酶并探索其宿主靶标，从而扩展 宿主 - 病原体相互作用中可逆ADP-核糖化的范围。