Structure and Function of Drosophila NF-kappaB Signaling Pathways

描述(申请人提供)：这项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，由细胞表面受体Toll进行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被广泛保守，在植物和哺乳动物中都有诱导抗菌防御的同源途径。感染特定病原体的果蝇会激活Toll或IMD信号通路。Toll信号的靶标是Dif和DRADAL，它们是与NF-B相关的转录因子，以及抑制蛋白仙人掌，它是一种I？B同源物。IMD途径的靶标是第三种核因子-？B蛋白，ARISE，它被保守的半胱氨酸天冬氨酸酶和IKK复合体的Fly对应物激活。事实上，这些途径已经在遗传和分子水平上进行了表征，并且可以在整个苍蝇和细胞培养系统中进行测试，这使得它们特别适合于实验研究。因此，现在有可能解决基本的问题，如信号转导机制，Toll和IMD途径功能的协调，途径结构的进化，以及免疫反应调节的整体程序。拟议研究的重点将是获得信号转导机制的知识，并将其整合到体液天然免疫防御的整体调节和组织的背景下。在进行这些研究时，我们将利用有关苍蝇和哺乳动物Toll通路组件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上相关的仙人掌激酶。我们还将进行体外结合分析和定点突变，以测试Toll途径中蛋白质-蛋白质相互作用的保守模型。使用一种基于最先进的方法进行果蝇转基因研究的方法，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。将利用定点突变和报告基因研究相结合的方法来完善我们对免疫调节的顺式调控位点的知识，并为鉴定反式作用因子提供基础。最后，我们将结合功能研究、表达数据和12个果蝇基因组的序列比较来开发一个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大范围生物体中的先天性免疫途径和防御机制产生实质性的兴趣。 与公共卫生相关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。因此，通过研究这些反应途径的机制和调控，我们将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.