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: 8235694
• 负责人: Neal Mathew Alto
• 金额: $ 30.15万
• 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/8235694
• 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 Delivery Systems; 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. PUBLIC HEALTH RELEVANCE: Human kinases and GTPases are major targets of bacterial toxins and effector proteins. This proposal examines the ability of a family of bacterial type III effector proteins to hijack human signaling enzymes. A deeper understanding of the enzymatic and biochemical interface between these bacterial effectors and human kinases and GTPases by X-ray crystallography will lead to a more complete knowledge of numerous pathogenic mechanisms and may reveal new aspects of signal transduction in the human host cell.

描述（由申请人提供）：细菌III型“效应子”蛋白是指导许多革兰氏阴性细菌传染病的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传递到宿主细胞中。总的来说，这些毒力因子劫持了先天免疫反应，并促进细菌复制，传播和疾病进展。因此，确定细菌效应蛋白控制如何在结构，生化和生物物理水平的细胞内通信途径是传染病研究的持续挑战。该建议旨在揭示对这些宿主病原关系的结构和功能理解。在进行此提案之前，我们确定了一类肠内大肠杆菌效应子蛋白，该蛋白可以通过独特的结构相互作用直接调节宿主膜运输GTPase和细胞极性激酶。使用这些结构作为指导，我们将确定人类信号传导酶细菌调节的分子机制。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促测定来检查一种新型的PAK激酶激活机制（AIM 2）。最终的基于结构的理论将直接比较许多细菌III型效应子同源物的作用，以将宿主酶组装到细菌效应子支架表面上的新信号电路中（AIM 3）。开发靶向细菌效应子 - 宿主酶复合物的新药将是打击新兴的传染病的一种创新方法。尽管这个想法具有巨大的潜力，但从毒力因子结构中收集的机械信息的匮乏已迄今已成为合适的药物目标的发育。作为这一目的的一种手段，这些研究将使我们能够预测细菌效应蛋白的新作用机理，并瞥见相关病原体基的基于结构的进化进程。 公共卫生相关性：人类激酶和GTPases是细菌毒素和效应蛋白的主要靶标。该建议研究了一个细菌III型效应蛋白家族对劫持人类信号酶的能力。通过X射线晶体学对这些细菌效应子与人类激酶和GTPase之间的酶促和生化界面的更深入了解将使人们对众多致病机制有更完整的了解，并可能揭示人类宿主细胞中信号转导的新方面。