Bacterial Regulation of Eukaryotic Signaling Enzymes: Structure and Function

真核信号酶的细菌调节：结构和功能

• 批准号: 8415960
• 负责人: Neal Mathew Alto
• 金额: $ 29.15万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8415960
• 关键词: Architecture; Bacteria; Bacterial Infections; Bacterial Model; Bacterial Toxins; Bacterial Typing; Binding; Biochemical; Biological Assay; Cell Polarity; Cell model; Cells; Communicable Diseases; Communication; Complex; Coupling; Crystallization; Data; Development; Disease Progression; Drug Targeting; Emerging Communicable Diseases; Enzymes; Escherichia coli; Escherichia coli EHEC; Event; Family; Glean; Golgi Apparatus; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Human; Immune response; In Vitro; Infection; Infectious Diseases Research; Knowledge; Lead; Liposomes; Membrane; Membrane Protein Traffic; Methods; Modeling; Molecular; Molecular Structure; Multienzyme Complexes; Nature; Pathway interactions; Peptides; Phosphotransferases; Property; Proteins; Regulation; Roentgen Rays; Series; Shigella; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Specificity; Structure; Surface; System; Testing; Virulence; Virulence Factors; X-Ray Crystallography; antimicrobial; base; combat; design; enzyme structure; improved; inhibitor/antagonist; innovation; insight; member; mimetics; novel; pathogen; reconstitution; scaffold; small molecule; theories; tool; trafficking

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.

描述（由申请人提供）：细菌III型“效应”蛋白是引导多种革兰氏阴性细菌传染病进展的主要毒力因子。最近的研究估计，单一病原体可将 10-50 个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并促进细菌复制、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持续存在的挑战。该提案旨在揭示对这些宿主-病原体关系的结构和功能理解。在此提议之前，我们鉴定了一类肠出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上的 ARF GTP 酶活性来劫持宿主货物运输途径（目标 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测定来研究新型 PAK 激酶激活机制（目标 2）。由此产生的基于结构的理论将用于直接比较众多细菌 III 型效应器同源物的作用，以将宿主酶组装成细菌效应器支架表面上的新信号传导电路（目标 3）。开发针对细菌效应子-宿主酶复合物的新药将是对抗新出现的传染病的创新方法。虽然这个想法具有巨大的潜力，但从毒力因子结构中收集的机制信息的缺乏迄今为止阻碍了它们作为合适药物靶点的开发。作为实现这一目标的一种手段，这些研究将使我们能够预测正在研究的细菌效应蛋白的新作用机制，并让我们了解相关病原体群基于结构的进化进程。