Ehrlichia TRP120 HECT E3 ubiquitin ligase modulation of host cell pathways

项目摘要/摘要 查菲埃利希菌Ehrlichiachaffesis(E.ch.)是一种革兰氏阴性、专性胞内细菌，也是人类的病原体 单核细胞增多性埃立克体病(HME)，一种新出现的、威胁生命的、由壁虱传播的人畜共患病。E.ch.优先考虑 感染单核巨噬细胞并通过颠覆介导的天然免疫防御在细胞内存活 部分通过串联重复蛋白(TRP)效应器。在感染过程中，TRP通过1型病毒在细胞内分泌 它们与多种宿主细胞蛋白以上下文相关的方式相互作用的分泌系统 参与了基本的细胞过程。决定蛋白质-蛋白质连续体的分子特征 在宿主-病原体相互作用过程中发生的相互作用，包括那些被归因于埃利希氏TRP的相互作用，是已知的。 涉及翻译后修饰(PTM)。这项提议的科学前提是基于我们之前的 研究表明，E.ch。感染依赖于宿主PTM感染途径的利用， 发现E.ch.TRP120兼职为Hect-type E3泛素(Ub)连接酶，标记宿主蛋白 UB介导的降解，鉴定新的TRP120与宿主蛋白的相互作用，增强感染 通过iRNA敲除选定的TRP120靶标，并证明Wnt和Notch通路是 对埃利希菌感染至关重要。这项研究的长期目标是了解宿主和病原体的作用 在宿主细胞重编程和E.ch感染中派生的pTMS。这项提案的目标是确定 TRP120 Hect E3 Ub连接酶在感染过程中的功能作用，证实TRP120泛素化 和降解特定的宿主细胞靶标，并进一步扩大和定义TRP120 Ub连接酶的谱系 底物。我们假设E.ch。TRP120 Hect E3连接酶调节宿主细胞信号和转录 抑制因子通过Ub介导的降解机制促进宿主细胞存活和埃立克病毒感染。目标 1是确定TRP120 Hect E3 Ub连接酶在降解宿主细胞转录和信号中的作用 Aim 2将鉴定在感染过程中降解的新型TRP120 Hect E3 Ub连接酶底物。这 这项研究意义重大，因为它将解决细胞内病原体，特别是那些小的 基因组和有限数量的效应蛋白，通过PTMS与真核细胞相互作用，并利用病原体- 编码的Ub连接酶操纵复杂的和进化上保守的宿主细胞途径以促进感染。 我们已经建立了一个很好的模型来研究细胞内病原体 通过病原体编码的Ub连接酶影响细胞调节剂的稳定性。了解相互作用 在E.ch.Hect-type E3 Ub连接酶和宿主过程/途径将进一步定义Trp兼职 在病理生物学中的作用，以及病原体如何模仿真核蛋白的功能来利用宿主细胞的途径 利益。

Project Summary/Abstract Ehrlichia chaffeensis (E. ch.) is a gram-negative, obligately intracellular bacterium and etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging, life-threatening, tick-borne zoonosis. E. ch. preferentially infects mononuclear phagocytes and survives intracellularly by subverting innate immune defenses mediated in part by tandem repeat protein (TRP) effectors. TRPs are secreted intracellularly during infection via a type-1 secretion system where they interact in a context-dependent manner with a diverse array of host cell proteins involved in essential cellular processes. The molecular features that determine the continuum of protein-protein interactions that occur during host-pathogen interplay, including those attributed to ehrlichial TRPs, are known to involve posttranslational modifications (PTMs). The scientific premise of this proposal is based on our previous research demonstrating that the E. ch. infection is dependent on exploitation of host PTM pathways for infection, the discovery that E. ch.TRP120 moonlights as a HECT-type E3 ubiquitin (Ub) ligase that tags host proteins for Ub-mediated degradation, identification of novel TRP120-host protein interactions, enhancement of infection through iRNA knockdown of selected TRP120 targets, and demonstration that Wnt and Notch pathways are critical for ehrlichial infection. The long-term goal of this research is to understand the role of host- and pathogen- derived PTMs in host cell reprogramming and infection by E. ch. The objectives of this proposal are to determine the functional role of TRP120 HECT E3 Ub ligase activity during infection, demonstrate TRP120 ubiquitination and degradation of specific host cell targets, and to further expand and define the repertoire of TRP120 Ub ligase substrates. We hypothesize that E. ch. TRP120 HECT E3 ligase modulates host cell signaling and transcriptional repressors via a Ub-mediated degradation mechanism to promote host cell survival and ehrlichial infection. Aim 1 is to determine the role of TRP120 HECT E3 Ub ligase in degrading host cell transcription and signaling regulators; Aim 2 will identify novel TRP120 HECT E3 Ub ligase substrates degraded during infection. This research is significant because it will addresses how intracellular pathogens, particularly those with small genomes and a limited number of effector proteins, interface with eukaryotic cells via PTMs and use pathogen- encoded Ub ligases to manipulate complex and evolutionarily conserved host cell pathways to promote infection. We have established an excellent model to investigate the mechanisms whereby intracellular pathogens influence the stability of cellular regulators though pathogen-encoded Ub ligases. Understanding the interactions between E. ch. HECT-type E3 Ub ligases and host processes/pathways will further define TRP moonlighting roles in pathobiology, and how pathogens mimic eukaryotic protein function to exploit host cell pathways for their benefit.