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

描述（由申请人提供）：AMP化是一种新发现的翻译后修饰，用于通过多种病原体的毒力因子用AMP稳定修饰蛋白质。在所有描述的AMP化因子中，具有序列（HPFx[D/E]GN[G/K]R）的被称为Fic（由环腺苷一磷酸诱导的腺苷化）的保守功能基序催化酶促反应，由此不变的组氨酸残基是必需的。AMP部分的生物化学修饰改变了靶分子的活性，表明这种信号传导机制代表了一种新的翻译后修饰，其功能与其他公认的修饰相似，如磷酸化、泛素化和糖基化（27）。洛杉矶已知嗜肺菌效应子AnkX通过依赖于位于其N-末端结构域中的Fic基序的机制干扰宿主膜运输途径。然而，关于AnkX活动的两个最重要的问题尚未得到解决。首先，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AMPylation活性需要一个或多个真核生物来源的因子。此外，我们观察到，AnkX是有毒的酵母和这样的毒性可以抑制过表达的复杂组件参与对接囊泡的顺式高尔基体隔室。因此，我们假设需要一种或多种宿主因子来激活AnkX的AMP化活性，并且AnkX靶向一种或多种参与内质网（ER）和高尔基体之间的顺行膜转运的蛋白质。在拟议的研究中，我们计划：1。鉴定和表征AnkX的AMP化活性所需的宿主因子。2.分析AnkX对宿主信号通路和细胞内生长的影响。嗜肺菌我们还计划通过研究它们在细胞过程中的作用和鉴定相关的结合蛋白来分析推定的辅因子。这些研究将揭示不需要Fic基序的假定AMP化酶，这可能会导致这一令人兴奋的领域的新的研究路线。 公共卫生相关性：许多重要病原体都利用宿主蛋白的腺苷酸化。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/或治疗嗜肺军团菌等重要病原体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.