Structure and Function of Drosophila NF-kappaB Signaling Pathways

果蝇 NF-κB 信号通路的结构和功能

• 批准号: 7529407
• 负责人: Steven Alexander Wasserman
• 金额: $ 34.2万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7529407
• 关键词: Adaptor Signaling Protein; Address; Animals; Architecture; Arthritis; Arts; Bacteria; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Cactaceae; Caspase; Cell Culture System; Cell Surface Receptors; Complex; DNA; Data; Death Domain; Development; Disease; Dorsal; Drosophila genome; Drosophila genus; Elements; Embryo; Event; Evolution; Gene Expression; Gene Expression Regulation; Genome; Genomics; Goals; Health; Heart Diseases; Homologous Gene; Human; Immune; Immune response; Immune system; Immunity; In Vitro; Infection; Insecta; Investigation; Knowledge; Malignant Neoplasms; Mammals; Mediating; Mediator of activation protein; Methods; Modeling; Molecular; Molecular Genetics; Monitor; NF-kappa B; Natural Immunity; Nature; Nuclear; Nuclear Translocation; Numbers; Organism; Pathway interactions; Pattern; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Plants; Positioning Attribute; Protein Kinase; Proteins; Public Health; RNA Interference; Range; Regulation; Reporter; Reporter Genes; Research; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Site-Directed Mutagenesis; Specificity; Structure; Technology; Testing; Trans-Activators; Transgenes; Transgenic Organisms; Tube; antimicrobial; antimicrobial peptide; base; embryonic cell culture; fly; fungus; genetic regulatory protein; genome sequencing; human TYRP1 protein; inhibitor/antagonist; insight; interest; microbial; mutant; pathogen; predictive modeling; programs; protein protein interaction; research study; response; transcription factor

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand signaling pathways that regulate programs of innate immune gene expression by controlling the subcellular localization of regulatory proteins. In the fruit fly Drosophila, signal transduction by the cell surface receptor Toll promotes nuclear translocation of transcription factors governing both innate immunity and embryonic patterning. Pathway function in innate immune responses has been widely conserved, with homologous pathways inducing antimicrobial defenses in both plants and mammals. Infection of flies with particular sets of pathogens activates either the Toll or the Imd signaling pathway. The targets of Toll signaling are Dif and Dorsal, which are NF-?B related transcription factors, and the inhibitory protein Cactus, an I?B homolog. The target of the Imd pathway is a third NF-?B protein, Relish, which is activated by a conserved caspase and by the fly counterpart of the IKK complex. The fact that these pathways have been characterized at both the genetic and molecular level and can be assayed in whole fly and cell culture systems makes them particularly amenable to experimental investigation. It is now possible, therefore, to address fundamental questions about the mechanisms for signal transduction, the coordination of Toll and Imd pathway function, the evolution of pathway architecture, and the overall program for regulation of immune responses. The focus of the proposed research will be to acquire and integrate knowledge of signal transduction mechanism into the context of overall regulation and organization of humoral innate immune defenses. In carrying out these studies, we will take advantage of discoveries regarding the orthologous relationships between fly and mammalian Toll pathway components. We will use RNA interference technology and phosphospecific antibodies to identify the physiologically relevant Cactus kinase. We will also carry out in vitro binding assays and site-directed mutagenesis to test a model for conservation of protein-protein interactions in the Toll pathway. Using an approach based on a state-of-the-art method for transgenic studies in Drosophila, we will test hypotheses regarding the role of binding site number in delineating pathway functions. A combination of site-directed mutagenesis and reporter gene studies will be exploited to refine our knowledge of a cis-regulatory site for immune regulation and to provide the basis for identification of the trans-acting factors. Lastly, we will combine functional studies, expression data, and sequence comparisons across twelve Drosophila genomes to develop a predictive model for innate immune gene regulation. Given the conserved nature of the signaling pathways, the results of the proposed research should be of substantial interest with regard to innate immunity pathways and defenses in a broad range of organisms. PUBLIC HEALTH RELEVANCE Humans and other animals rely on innate immune defenses to recognize and respond to infection by a range of microbial pathogens. Furthermore, abnormal function of innate immune systems contributes to a range of human disorders, including arthritis, heart disease, and cancer. By studying the mechanism and regulation of such response pathways, we will therefore obtain knowledge of broad and substantial significance to human health.

描述（由申请人提供）：本研究的长期目标是了解通过控制调节蛋白的亚细胞定位来调节先天免疫基因表达程序的信号通路。在果蝇中，细胞表面受体 Toll 的信号转导促进了控制先天免疫和胚胎模式的转录因子的核转位。先天免疫反应中的途径功能已广泛保守，在植物和哺乳动物中均具有诱导抗菌防御的同源途径。果蝇感染特定病原体后会激活 Toll 或 Imd 信号通路。 Toll 信号传导的目标是 Dif 和 Dorsal（它们是 NF-κB 相关转录因子）以及抑制蛋白 Cactus（IκB 同源物）。 Imd 途径的目标是第三个 NF-κB 蛋白 Relish，它由保守的 caspase 和 IKK 复合物的果蝇对应物激活。事实上，这些途径已在遗传和分子水平上得到表征，并且可以在整个果蝇和细胞培养系统中进行测定，这使得它们特别适合实验研究。因此，现在有可能解决有关信号转导机制、Toll 和 Imd 通路功能的协调、通路结构的进化以及免疫反应调节的总体程序等基本问题。拟议研究的重点是获取信号转导机制的知识并将其整合到体液先天免疫防御的整体调节和组织背景中。在进行这些研究时，我们将利用有关果蝇和哺乳动物 Toll 途径成分之间的直系同源关系的发现。我们将使用 RNA 干扰技术和磷酸化特异性抗体来鉴定生理相关的 Cactus 激酶。我们还将进行体外结合测定和定点诱变，以测试 Toll 途径中蛋白质-蛋白质相互作用的保守模型。使用基于果蝇转基因研究最先进方法的方法，我们将测试有关结合位点数量在描述途径功能中的作用的假设。将利用定点诱变和报告基因研究的结合来完善我们对免疫调节顺式调节位点的了解，并为鉴定反式作用因子提供基础。最后，我们将结合 12 个果蝇基因组的功能研究、表达数据和序列比较，开发先天免疫基因调控的预测模型。鉴于信号通路的保守性，拟议研究的结果对于广泛生物体的先天免疫通路和防御应该具有重大意义。 公共卫生相关性人类和其他动物依靠先天免疫防御来识别和应对一系列微生物病原体的感染。此外，先天免疫系统的异常功能会导致一系列人类疾病，包括关节炎、心脏病和癌症。通过研究此类反应途径的机制和调节，我们将获得对人类健康具有广泛和实质性意义的知识。