Structure and Function of Drosophila NF-kappaB Signaling Pathways

描述（由申请人提供）：本研究的长期目标是通过控制调节蛋白的亚细胞定位来了解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易位。先天免疫应答中的通路功能广泛保守，在植物和哺乳动物中都有同源通路诱导抗微生物防御。特定病原体组的苍蝇感染激活Toll或Imd信号通路。Toll信号的目标是Dif和Dorsal，它们是NF-？B相关转录因子和仙人掌抑制蛋白，以及I？B同族体。Imd通路的目标是第三个NF-？B蛋白，它被保守的半胱天蛋白酶和IKK复合物的苍蝇对应物激活。事实上，这些途径已经在遗传和分子水平上被表征，并且可以在整个苍蝇和细胞培养系统中进行分析，这使得它们特别适合于实验研究。因此，现在有可能解决有关信号转导机制的基本问题，Toll和Imd通路功能的协调，通路结构的演变，以及免疫反应调节的总体方案。研究的重点将是获取和整合信号转导机制的知识，以全面调节和组织体液先天免疫防御。在开展这些研究时，我们将利用有关果蝇和哺乳动物Toll通路组分之间的同源关系的发现。我们将使用RNA干扰技术和磷酸特异性抗体来鉴定生理学上相关的仙人掌激酶。我们还将进行体外结合试验和定点诱变，以测试Toll通路中蛋白质-蛋白质相互作用的保护模型。使用基于果蝇转基因研究的最先进方法的方法，我们将测试关于结合位点数量在描述途径功能中的作用的假设。将利用位点定向诱变和报告基因研究的结合来完善我们对免疫调节的顺式调控位点的知识，并为识别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，建立先天免疫基因调控的预测模型。鉴于信号通路的保守性，拟议的研究结果应该对广泛生物体的先天免疫途径和防御具有重大意义。

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.