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。先天免疫应答中的途径功能已被广泛保守，在植物和哺乳动物中具有诱导抗微生物防御的同源途径。用特定的病原体感染苍蝇会激活Toll或Imd信号通路。Toll信号传导的靶点是Dif和Dorsal，它们是NF-？B相关的转录因子，和抑制蛋白仙人掌，一个I？B同系物。IMD途径的目标是第三个NF-？B蛋白，Relish，由保守的半胱天冬酶和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的特点，并可以在整个苍蝇和细胞培养系统进行测定，使他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、通路结构的演变以及调节免疫应答的总体方案的基本问题。拟议的研究的重点将是获得和整合的信号转导机制的知识到体液先天免疫防御的整体调节和组织的背景下。在进行这些研究中，我们将利用有关果蝇和哺乳动物Toll通路组分之间的正交关系的发现。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途径中蛋白质-蛋白质相互作用的保守模型。使用的方法基于一个国家的最先进的方法在果蝇转基因研究，我们将测试假设的结合位点数的作用，描绘通路功能。定点诱变和报告基因的研究相结合，将利用完善我们的知识的顺式调节位点的免疫调节，并提供识别的反式作用因子的基础。最后，我们将结合联合收割机的功能研究，表达数据，并在12个果蝇基因组序列比较，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，拟议研究的结果应该对广泛生物体中的先天免疫通路和防御具有重大意义。 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有助于一系列人类疾病，包括关节炎，心脏病和癌症。通过研究这些反应途径的机制和调节，我们将因此获得对人类健康具有广泛和实质性意义的知识。

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.