The Roles of Host Factors in the AMPylation Activity of a Bacterial Effector

描述(申请人提供)：AMP修饰是新近发现的一种翻译后修饰，用于通过几种病原体的毒力因子稳定地修饰AMP蛋白。在所有已描述的AMP化因子中，具有(HPFX[D/E]GN[G/K]R)序列的保守功能基序Fic(环腺苷单磷酸诱导的丝状化)催化酶反应，因此不变的组氨酸残基是必不可少的。AMP部分的生化修饰改变了靶分子的活性，表明这种信号机制代表了一种新的翻译后修饰，其功能类似于其他成熟的修饰，如磷酸化、泛素化和糖基化(27)。已知嗜肺乳杆菌效应子AnkX通过一种依赖于位于其N-末端结构域的Fic基序的机制干扰宿主膜转运途径。然而，关于AnkX活动的两个最重要的问题尚未得到解决。首先，目前尚不清楚AnkX是否具有AMP化活性。其次，AnkX的细胞靶点是未知的。我们最近的研究揭示了AnkX的一些非常有趣的特性：AnkX的自我AMP化活性需要一个或多个真核来源的因素。此外，我们观察到AnkX对酵母是有毒的，这种毒性可以通过过度表达与囊泡对接到顺式高尔基体中的复合体的成分来抑制。因此，我们假设激活AnkX的AMP化活性需要一个或多个宿主因素，并且AnkX以参与内质网(ER)和高尔基体之间顺行膜运输的一个或多个蛋白质为靶标。在拟议的研究中，我们计划：1.鉴定和鉴定AnkX AMP化活性所需的宿主因子(S)。2.分析AnkX对宿主信号通路和嗜肺乳杆菌胞内生长的影响。我们还计划通过研究它们在细胞过程中的作用和确定相关的结合蛋白来分析可能的辅助因子(S)。这些研究将揭示不需要Fic基序的假定的AMP化酶，这可能会导致对这个令人兴奋的领域的新的研究路线。 公共卫生相关性：宿主蛋白的AMP化被许多重要的病原体利用。因此，了解这种细菌如何影响宿主细胞过程可能会为我们研究与这些过程异常相关的其他疾病提供线索。我们的研究可以为预防和/或治疗由嗜肺军团菌引起的感染提供新的策略。

DESCRIPTION (provided by applicant): AMPylation is a newly discovered posttranslational modification used to stably modify proteins with AMP by virulence factors of several pathogens. In all described AMPylation factors, a conserved functional motif called Fic (filamentation induced by cyclic adenosine monophosphate) with the sequences (HPFx[D/E]GN[G/K]R) catalyzes the enzymatic reaction whereby the invariant histidine residue is essential. Biochemical modification by the AMP moiety alters the activity of the target molecules, suggesting that this signaling mechanism represents a novel posttranslational modification with functions similar to other well established modifications, such as phosphorylation, ubiquitylation and glycosylation (27). The L. pneumophila effector AnkX is known to interfere with host membrane trafficking pathways via a mechanism that is dependent upon a Fic motif located in its N-terminal domain. However, the two most important questions about the activity of AnkX have not been addressed. First, it is not clear whether AnkX possesses AMPylation activity. Second, the cellular targets of AnkX are unknown. Our recent studies have revealed some highly intriguing properties of AnkX: The self-AMPylation activity of AnkX requires one or more factors of eukaryotic origin. Moreover, we observed that AnkX is toxic to yeast and such toxicity can be suppressed by overexpressing components of the complex involved in docking vesicles to the cis-Golgi compartment. Therefore, we hypothesize that one or more host factors are required to activate the AMPylation activity of AnkX and that AnkX targets one or more proteins involved in anterograde membrane transport between the endoplasmic reticulum (ER) and the Golgi apparatus. In the proposed study, we plan to: 1. Identify and characterize the host factor(s) required for the AMPylation activity of AnkX. 2. Analyze the effect of AnkX on host signaling pathways and intracellular growth L. pneumophila. We also plan to analyze the putative co-factor(s) by studying their roles in cellular processes and by identifying the relevant binding proteins. These studies will shed light on the putative AMPylation enzymes that do not require a Fic motif, which could lead to novel lines of research into this exciting field. PUBLIC HEALTH RELEVANCE: AMPylation of host proteins is employed by many important pathogens. Thus, understanding of how this bacterium affects host cell processes could lead to clues in our study of other diseases associated with abnormalities of these processes. Our study can provide novel strategies for the prevention and/or treatment of infections caused by Legionella pneumophila other important pathogens.