Bacterial Regulation of Eukaryotic Signaling Enzymes: Structure and Function

描述(由申请人提供)：细菌III型“效应蛋白”是指导许多革兰氏阴性细菌感染性疾病进展的主要毒力因素。最近的研究估计，单个病原体将10-50个独特的效应蛋白直接送入宿主细胞。总而言之，这些毒力因子劫持了宿主的先天性免疫反应，促进了细菌的复制、传播和疾病的发展。因此，确定细菌效应蛋白如何在结构、生化和生物物理水平上控制宿主细胞内的通讯通路是传染病研究中的一个持续挑战。这一建议试图揭示对这些宿主-病原体关系的结构和功能的理解。在此之前，我们鉴定了一类肠道嗜血性大肠杆菌效应蛋白，它通过独特的结构相互作用直接调节宿主膜运输GTP酶和细胞极性蛋白激酶。以这些结构为指导，我们将确定细菌调节人类信号酶的分子机制。这包括确定细菌如何通过直接调节膜表面的ARF GTP酶活性来劫持宿主货物运输路径(目标1)。我们还将通过一系列X射线结晶学、小分子抑制剂研究和酶分析来研究一种新的PAK激酶激活机制(目标2)。由此产生的基于结构的理论将被用于直接比较许多细菌III型效应器同系物将宿主酶组装成细菌效应器支架表面的新信号电路的作用(目标3)。开发针对细菌效应器-宿主酶复合体的新药将是对抗新出现的传染病的创新方法。虽然这一想法具有巨大的潜力，但迄今为止，从毒力因子结构中收集的机械性信息的匮乏阻碍了它们作为合适的药物靶点的发展。作为达到这一目的的一种手段，这些研究将使我们能够预测未被研究的细菌效应蛋白的新的作用机制，并提供对相关病原体群体基于结构的进化进展的一瞥。

DESCRIPTION (provided by applicant): Bacterial type III "effector" proteins are the primary virulence factors that guide the progression of numerous Gram-negative bacterial infectious diseases. Recent studies have estimated that a single pathogen delivers 10-50 unique effector proteins directly into host cells. Collectively, these virulence factors hijack host innate immune response and facilitate bacterial replication, dissemination, and disease progression. Therefore, determining how bacterial effector proteins control host intracellular communication pathways at the structural, biochemical, and biophysical level is an ongoing challenge in infectious disease research. This proposal seeks to reveal a structural and functional understanding of these host-pathogen relationships. Prior to this proposal, we identified a class of Enterohaemorhagic E. coli effector proteins that directly regulates host membrane trafficking GTPases and a cell polarity kinases through unique structural interactions. Using these structures as a guide, we will determine the molecular mechanism for bacterial regulation of human signaling enzymes. This includes determining how bacteria hijack host cargo trafficking pathways by directly regulating ARF GTPase activity on a membrane surface (Aim 1). We will also examine a novel PAK kinase activation mechanism through a series of X-ray crystallography, small molecule inhibitor studies, and enzymatic assays (Aim 2). The resulting structure-based theories will be used to directly compare the actions of numerous bacterial type III effector homologs to assemble host enzymes into new signaling circuits on the surface of bacterial effector scaffolds (Aim 3). Developing new drugs that target bacterial effector - host enzyme complexes would be an innovative approach to combat emerging infectious disease. While this idea holds great potential, the paucity of mechanistic information gleaned from virulence factor structures has so far hampered their development as suitable drug targets. As a means to this end, these studies will allow us to predict new mechanisms of action for understudied bacterial effector proteins, and provide a glimpse into the structural-based evolutionary progression of a related pathogen group.