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

项目摘要/摘要 泛素(Ub)是一种由76个氨基酸组成的蛋白质，它通过一系列的Ub激活与特定的蛋白质结合 酶E1、结合酶E2和Ub连接酶E3。泛素化在广泛的 细胞过程的一个方面。泛素化系统中的异常会导致许多人类疾病， 比如神经退行性疾病和癌症。 发现了一种非典型的泛素化途径，其作用不依赖于E1和E2酶 最近。细胞内致病菌嗜肺军团菌的副家族效应是 发现在辅因子NAD存在的情况下泛素化丝氨酸残基上的底物。这不同寻常的一面- 催化泛素化涉及其单-ADP-核糖基转移酶催化的两步反应 (MART)和磷酸二酯酶(PDE)结构域。第一步是产生单声道- ADP-核糖化Ub(ADPR-Ub)，其中Side利用其Mart结构域催化ADP-Ub的转移。 核糖从NAD到Ub的Arg42残基。在第二步反应中，ADPR-Ub偶联到 底物蛋白的丝氨酸残基通过PDE结构域产生丝氨酸泛素化产物 AMP的释放。 我们最近的结构和生化研究，以及来自其他小组的结果，都揭示了 关于这种新的磷酸核糖连接丝氨酸泛素化(PR-R)的分子机制 泛素化)。然而，关键问题仍然没有得到解决，例如：Mart域如何 特异性识别Ub和ADP-核糖化Ub的Arg42残基？有没有去泛素化的 酶(DUBS)可以特异性地去结合PR泛素化物种，类似于DUBS在 经典的泛素化途径？副家族PR-Ub连接酶的具体靶点是什么？这个 这一建议的首要目标是阐明这种新的Ub依赖的机制 翻译后修饰及PR泛素化在劫持真核细胞中的作用 流程。具体地说，我们将追求以下三个目标：目标1：描绘分子 副家族效应物介导的PR泛素化机制。目标2：确定和阐明 PR泛素化结合物的DUBS专一性机制。目标3：确定细胞靶点 副家族效应器及其在含军团菌空泡重塑中的作用。我们期待着这些 对副家族PR-Ub连接酶的探索性研究将有助于阐明这一新的分子机制 翻译后修饰的类型。更重要的是，我们建议的研究也将为 研究真核生物中潜在的PR泛素化途径。

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.