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子。最近的研究估计，一种病原体可以将10-50种独特的效应蛋白直接传递到宿主细胞中。总的来说，这些毒力因子劫持宿主先天免疫反应，促进细菌复制、传播和疾病进展。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通讯途径是传染病研究中的一个持续挑战。该建议旨在揭示这些宿主-病原体关系的结构和功能理解。在此之前，我们确定了一类肠出血性大肠杆菌效应蛋白，它们通过独特的结构相互作用直接调节宿主膜运输gtp酶和细胞极性激酶。利用这些结构作为指导，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上的ARF GTPase活性来劫持宿主货物运输途径（Aim 1）。我们还将通过一系列x射线晶体学、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（目的2）。由此产生的基于结构的理论将用于直接比较许多细菌III型效应物同源物在细菌效应支架表面将宿主酶组装成新的信号通路的作用（目的3）。开发针对细菌效应-宿主酶复合物的新药将是对抗新发传染病的一种创新方法。虽然这一想法具有很大的潜力，但迄今为止，从毒力因子结构中收集的机制信息的缺乏阻碍了它们作为合适的药物靶点的发展。作为实现这一目标的手段，这些研究将使我们能够预测尚未被研究的细菌效应蛋白的新的作用机制，并为相关病原体群的基于结构的进化进程提供一瞥。

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.