Ehrlichia TRP120 HECT E3 ubiquitin ligase modulation of host cell pathways

项目总结/摘要 查菲埃立克体（Ehrlichia chaffeensis）; ch.）是一种革兰氏阴性专性胞内细菌，是人类的病原体 嗜单核细胞埃立克体病（HME），一种新出现的、危及生命的、蜱传的人畜共患病。E. ch.优先 感染单核吞噬细胞，并通过破坏 串联重复序列蛋白（TRP）效应子。TRP在感染期间通过1型 分泌系统，其中它们以环境依赖性方式与多种宿主细胞蛋白质相互作用 参与了重要的细胞过程。决定蛋白质-蛋白质连续体的分子特征 已知在宿主-病原体相互作用期间发生的相互作用，包括那些归因于埃里希体TRP的相互作用， 翻译后修饰（PTMs）。这项建议的科学前提是基于我们以前的 研究表明，E. ch.感染依赖于利用宿主PTM途径进行感染， 发现E. ch.TRP120兼职作为HECT型E3泛素（Ub）连接酶，其标记宿主蛋白， Ub介导的降解，新TRP 120-宿主蛋白相互作用的鉴定，感染增强 通过iRNA敲除选定的TRP 120靶点，并证明Wnt和Notch途径是 是埃立克体感染的关键这项研究的长期目标是了解宿主和病原体的作用， 衍生的PTM在宿主细胞重编程和E. ch.本提案的目标是确定 TRP 120 HECT E3 Ub连接酶活性在感染过程中功能作用，证明了TRP 120泛素化 和特异性宿主细胞靶标的降解，并进一步扩增和定义TRP 120 Ub连接酶的库 印刷受体.我们假设E. ch.TRP120 HECT E3连接酶调节宿主细胞信号传导和转录 阻遏物通过Ub介导的降解机制促进宿主细胞存活和埃里希体感染。目的 1是确定TRP 120 HECT E3 Ub连接酶在降解宿主细胞转录和信号传导中的作用 调节因子; Aim 2将鉴定在感染期间降解的新型TRP 120 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 基因组和有限数量的效应蛋白，通过PTM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因子的稳定性。理解相互作用 间E. ch. HECT型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质功能，以利用宿主细胞途径， 效益

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.