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

项目概要/摘要 泛素（Ubiquitin，Ub）是一种由76个氨基酸组成的蛋白质，通过一系列的Ub激活作用与特定的蛋白质结合 酶E1、缀合酶E2和Ub连接酶E3。泛在化在广泛的 细胞过程的一个方面。泛素化系统的异常导致许多人类疾病， 例如神经变性疾病和癌症。 发现了一种独立于E1和E2酶发挥作用的非经典遍在蛋白化途径 最近来自细胞内细菌病原体嗜肺军团菌的SidE家族效应物是 发现在辅因子NAD+的存在下泛素化丝氨酸残基上的底物。这个不寻常的一面-- 催化的泛素化包括由其单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-核糖基化Ub（ADPR-Ub），其中SidE使用其mART结构域催化ADP-核糖基化Ub（ADPR-Ub）的转移。 从NAD+到Ub的Arg 42残基的核糖。在反应的第二步中，ADPR-Ub缀合至 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物化学研究，以及其他小组的结果， 这种新的磷酸核糖连接的丝氨酸泛素化（PR- 泛素化）。然而，关键的问题仍然没有解决，例如：如何可以mART域 特异性识别Ub和ADP-核糖基化Ub的Arg 42残基？有没有什么去泛素化 这些酶（DUB）可以特异性地使与细胞中的DUB类似的PR-泛素化物质去缀合， 典型的遍在化途径？SidE家族PR-Ub连接酶的特异性靶点是什么？的 该提案的首要目标是阐明这种新的Ub依赖性的机制， 探讨PR-泛素化在劫持真核细胞中的作用， 流程.具体而言，我们将追求以下三个目标：目标1：描绘分子 由SidE家族效应物介导的PR-泛素化的机制。目的2：识别和阐明 DUB对PR-泛素化缀合物的特异性机制。目的3：确定细胞靶点， SidE家族效应子及其在含军团菌空泡重塑中的作用。我们预计这些 对SidE家族PR-Ub连接酶的探索性研究将有助于阐明这种新型连接酶的分子机制 翻译后修饰的类型。更重要的是，我们建议进行的研究， 研究真核生物中潜在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.