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

描述（由申请人提供）：ampyation是一种新发现的翻译后修饰，用于稳定地修饰几种病原体的毒力因子与AMP的蛋白质。在所有描述的氨酰化因子中，一个保守的功能基序Fic（由环磷酸腺苷诱导的丝化）具有序列（HPFx[D/E]GN[G/K]R）催化酶促反应，其中不变的组氨酸残基是必不可少的。AMP片段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，其功能类似于其他已建立的修饰，如磷酸化、泛素化和糖基化（27）。已知嗜肺乳杆菌效应物AnkX通过依赖于位于其n端结构域的Fic基元的机制干扰宿主膜运输途径。然而，关于AnkX活性的两个最重要的问题还没有得到解决。首先，尚不清楚AnkX是否具有ampyation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ampylation活性需要一个或多个真核起源的因子。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参与对接囊泡到顺式高尔基室的复合物的成分来抑制。因此，我们假设需要一个或多个宿主因子来激活AnkX的amppyation活性，并且AnkX靶向参与内质网（ER）和高尔基体之间顺行膜运输的一个或多个蛋白质。在拟进行的研究中，我们计划：1。鉴定和表征AnkX的ampyation活性所需的宿主因子。2. 分析AnkX对嗜肺乳杆菌宿主信号通路和细胞内生长的影响。我们还计划通过研究它们在细胞过程中的作用和鉴定相关的结合蛋白来分析假定的辅助因子。这些研究将揭示假定的不需要Fic基序的ampyation酶，这可能会为这个令人兴奋的领域带来新的研究方向。

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.