Non-canonical phosphoribosyl ubiquitination and de-ubiquitination by legionella effectors

军团菌效应子的非典型磷酸核糖泛素化和去泛素化

• 批准号: 10592333
• 负责人: Yuxin Mao
• 金额: $ 32.06万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-08 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10592333
• 关键词: ADP ribosylation; Adenosine Diphosphate Ribose; Amino Acids; Autophagocytosis; Biochemical; Biological; Cell Culture Techniques; Cell Cycle Regulation; Cell physiology; Cells; Communicable Diseases; Complex; DNA Repair; Deubiquitinating Enzyme; Deubiquitination; Enzymes; Family; Family member; Generations; Genetic Transcription; Goals; Health; Homeostasis; Human; Immune response; Infection; Lead; Legionella; Legionella pneumophila; Ligase; Link; Malignant Neoplasms; Mediating; Membrane; Methods; Molecular; Mono(ADP-Ribose) Transferases; Names; Neurodegenerative Disorders; Organelles; Pathway interactions; Peptides; Play; Post-Translational Protein Processing; Process; Property; Proteins; Reaction; Role; Serine; Side; Signal Transduction; Stable Isotope Labeling; Structure; System; Ubiquitin; Ubiquitination; Vacuole; Western Blotting; cellular targeting; cofactor; combat; experimental study; forging; human disease; interdisciplinary approach; novel; pathogen; pathogenic bacteria; phosphoric diester hydrolase; tool; trafficking; ubiquitin isopeptidase; ubiquitin-protein ligase

PROJECT SUMMARY/ABSTRACT Ubiquitin (Ub), a 76 amino acid protein, is attached to specific proteins via a cascade of Ub activating enzyme E1, conjugating enzyme E2, and Ub ligase E3. Ubiquitination plays an essential role in a broad aspect of cellular processes. Aberrations in the ubiquitination system lead to a number of human diseases, such as neurodegenerative diseases and cancers. A non-canonical ubiquitination pathway that acts independently of E1 and E2 enzymes was discovered recently. The SidE family effectors from the intracellular bacterial pathogen Legionella pneumophila were found to ubiquitinate substrates on serine residues in the presence of co-factor NAD+. This unusual SidE- catalyzed ubiquitination involves two steps of reactions catalyzed by its mono-ADP-ribosyl transferase (mART) and phosphodiesterase (PDE) domains, respectively. The first step is the generation of mono- ADP-ribosylated Ub (ADPR-Ub), in which, SidE uses its mART domain to catalyze the transfer of ADP- ribose from NAD+ to the Arg42 residue of Ub. In the second step of reaction, ADPR-Ub is conjugated to a serine residue of substrate proteins via the PDE domain to generate serine ubiquitinated products with the releasing of AMP. Our recent structural and biochemical studies, as well as results from other groups, have shed light on the molecular mechanism underlying this novel phosphoribosyl-linked serine ubiquitination (PR- ubiquitination). However, key questions remain unaddressed, For example: How can the mART domain specifically recognize Ub and ADP-ribosylate the Arg42 residue of Ub? Are there any deubiquitinating enzymes (DUBs) that can specifically de-conjugate PR-ubiquitinated species similar to the DUBs in the canonical ubiquitination pathway? What are the specific targets of SidE family PR-Ub ligases? The overarching goal of this proposal is to elucidate the mechanism of this novel Ub-dependent posttranslational modification and to explore the role of PR-ubiquitination in hijacking eukaryotic cellular processes. Specifically, we will pursue the following three aims: Aim 1: To delineate the molecular mechanism of PR-ubiquitination mediated by SidE family effectors. Aim 2: To identify and elucidate the mechanism of DUBs specific for PR-ubiquitinated conjugates. Aim 3: To determine the cellular targets of SidE family effectors and their roles in the remodeling of Legionella-containing vacuoles. We expect these exploratory studies of SidE family PR-Ub ligases will shed light on the molecular mechanism of this novel type of posttranslational modification. More importantly, our proposed studies will also pave the way to investigate a potential PR-ubiquitination pathway in eukaryotic species.

项目概要/摘要 泛素 (Ub) 是一种由 76 个氨基酸组成的蛋白质，通过 Ub 激活级联与特定蛋白质结合 酶 E1、缀合酶 E2 和 Ub 连接酶 E3。泛素化在广泛的领域发挥着重要作用 细胞过程的一个方面。泛素化系统的异常会导致许多人类疾病， 例如神经退行性疾病和癌症。 发现了独立于 E1 和 E2 酶起作用的非经典泛素化途径 最近。来自细胞内细菌病原体嗜肺军团菌的 SidE 家族效应子是 发现在辅因子 NAD+ 存在的情况下，丝氨酸残基上的底物泛素化。这个不寻常的SidE- 催化泛素化涉及由其单-ADP-核糖基转移酶催化的两步反应 分别是（mART）和磷酸二酯酶（PDE）结构域。第一步是生成单 ADP-核糖基化 Ub (ADPR-Ub)，其中，SidE 使用其 mART 结构域来催化 ADP-核糖基化 Ub 的转移 核糖从 NAD+ 到 Ub 的 Arg42 残基。在反应的第二步中，ADPR-Ub 与 通过 PDE 结构域连接底物蛋白的丝氨酸残基，生成丝氨酸泛素化产物 AMP的释放。 我们最近的结构和生化研究以及其他小组的结果揭示了 关于这种新型磷酸核糖连接的丝氨酸泛素化（PR- 泛素化）。然而，关键问题仍未解决，例如：mART 域如何 特异性识别 Ub 并 ADP-核糖基化 Ub 的 Arg42 残基？是否存在去泛素化 酶（DUB），可以特异性地解偶联 PR 泛素化物种，类似于 典型的泛素化途径？ SidE 家族 PR-Ub 连接酶的具体靶点是什么？这 该提案的总体目标是阐明这种新型 Ub 依赖性机制 翻译后修饰并探讨 PR 泛素化在劫持真核细胞中的作用 流程。具体来说，我们将追求以下三个目标： 目标 1：描绘分子 SidE 家族效应子介导的 PR 泛素化机制。目标 2：识别并阐明 PR 泛素化缀合物特异的 DUB 机制。目标3：确定细胞目标 SidE 家族效应子及其在含军团菌液泡重塑中的作用。我们期待这些 SidE 家族 PR-Ub 连接酶的探索性研究将揭示这种新型分子机制 翻译后修饰的类型。更重要的是，我们提出的研究也将为 研究真核物种中潜在的 PR 泛素化途径。