Ehrlichia Notch SLiM-activated oncoprotein inhibition of apoptosis

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，是 人类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. 优先感染单核巨噬细胞，并通过颠覆天然免疫防御在细胞内存活 部分由串联重复蛋白(TRP)效应器介导。在过去的十年里，我们的实验室已经识别出 大量分子上的色氨酸-宿主相互作用，其中许多是科学上新发现的，照亮了呼吸 以及在感染期间发生的病原体-宿主相互作用动力学的复杂性。我们已经证明了E.ch。是 感染依赖于包括Notch和Wnt在内的保守的真核信号通路的激活。 然而，理解Notch激活事件的分子基础，从而使E.ch。调整凹槽的用途 感染信号仍然是我们知识中的一个主要缺口。因此，这次调查的目的是为了确定 从分子和细胞机制方面阐述了E.已经进化到改变Notch信号的用途以应对感染。我们建议 那个E.ch。TRP120具有不同的真核蛋白质相互作用模块，称为短线性基序(SLIMs)， 模仿Notch配体的功能，并直接与同源受体结合，利用Notch信号来感染。这 研究将解决我们对蛋白质相互作用模块在功能上的不同作用的有限理解 在宿主-病原体相互作用和细胞重新编程的连续体中。这项研究的长期目标是 是确定埃立克体细胞拟态的分子基础，以及感染和感染的机制 免疫逃避。这项建议的目标是确定所涉及的分子相互作用和功能 机制使E.ch.TRP120 Notch配体模拟通过抑制宿主建立和促进感染 细胞凋亡。我们假设E.ch。TRP120有一个Notch Slim模拟物，可以激活Notch信号来 上调抗凋亡调节因子(MCL1和NICD)，从而抑制线粒体凋亡信号转导和 半胱氨酸酶的激活。目标1将定义E.ch。TRP120 Notch Slim模拟物及其受体结合研究 和信号；目标2将检查E.ch。TRP120 Notch上调MCL1对线粒体凋亡的抑制作用； 目标3将调查E.ch的角色。XIAP的缺口稳定化和caspase活性的抑制。这 研究将扩展我们对E.ch。TRP120表面蛋白 利用Notch信号抑制细胞凋亡，促进宿主细胞存活和感染。这件事的意义 研究正在定义一种机制策略，即具有小基因组和有限基因组的细胞内病原体 许多效应器蛋白，已经进化出宿主模仿模块，以改变宿主细胞信号的用途来操纵 下游宿主对感染的防御机制。对E.ch.的分子认识病理生物学也会 促进埃利希氏菌新治疗方法的开发。以及细胞内的病原体利用 细长的模仿或利用保守的细胞途径来感染和免疫逃避。

ABSTRACT Ehrlichia chaffeensis (E. ch.) is a gram-negative, obligately intracellular bacterium and the 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. Within the last decade, our laboratory has identified a multitude of molecular ehrlichial TRP-host interactions, many that are new to science, illuminating the breath and complexity of pathogen-host interaction dynamics that occur during infection. We have shown that E. ch. is dependent on activation of conserved eukaryotic signaling pathways including Notch and Wnt for infection. However, understanding the molecular basis of Notch activation events, whereby E. ch. repurposes Notch signaling for infection remains a major gap in our knowledge. Thus, the purpose of this investigation is to define the molecular and cellular mechanisms E. ch. has evolved to repurpose Notch signaling for infection. We propose that E. ch. TRP120 has distinct eukaryotic protein interaction modules known as short linear motifs (SLiMs) that mimic Notch ligand function and directly engage cognate receptors to exploit Notch signaling for infection. This investigation will address our limited understanding of the functionally diverse roles of protein interaction modules in the continuum of host-pathogen interactions and cellular reprogramming. The long-term goal of this research is to define the molecular basis of Ehrlichia host cell mimicry, and the mechanisms involved in infection and immune evasion. The objective of this proposal is to determine the molecular interactions involved and functional mechanisms whereby E. ch. TRP120 Notch ligand mimicry establishes and promotes infection by inhibiting host cell apoptosis. We hypothesize that E. ch. TRP120 has a Notch SLiM mimetic that activates Notch signaling to upregulate anti-apoptotic regulators (MCL1 and NICD), thereby inhibiting mitochondrial apoptotic signaling and caspase activation. Aim 1 will define the E. ch. TRP120 Notch SLiM mimetic and investigate receptor binding and signaling; Aim 2 will examine E. ch. TRP120 Notch-upregulated MCL1 inhibition of mitochondrial apoptosis; and Aim 3 will investigate the role of E. ch. Notch stabilization of XIAP and inhibition of caspase activation. This investigation will extend our knowledge of the molecular interactions by which E. ch. TRP120 surface protein exploits Notch signaling to inhibit apoptosis and promote host cell survival and infection. The significance of this research is defining the mechanistic strategies whereby intracellular pathogens with small genomes and a limited number of effector proteins, have evolved host mimicry modules to repurpose host cell signaling to manipulate downstream host defense mechanisms for infection. A molecular understanding of E. ch. pathobiology will also facilitate development of novel therapeutic approaches for Ehrlichia spp. and intracellular pathogens that utilize SLiM mimicry or exploit conserved cellular pathways for infection and immune evasion.