Integration of RNAi, proteomic and chemical genetic approaches to identify specif

描述(申请人提供)：Wnt/Wingless(WG)通路是一组进化上保守的核心信号通路之一，它调节后生动物发育的许多方面。Wnt途径的错误调节可能是有害的，因为几种成分的突变与肝脏、结肠、乳房和皮肤的肿瘤发生有关。因此，开发和实施新的技术以产生可用于调节Wnt/WG信号通路活性的分子工具是至关重要的。转录因子β-catenin(？-cat)/Aradillo(ARM)是Wnt途径最重要的效应因子之一。由于连环蛋白反应转录(CRT)与许多癌症的发生有关，因此它是开发调节β-CAT核活动的治疗药物的良好靶点。最近，我们采用了一种新的方法，将“敏化”的化学遗传高通量筛选(HTS)与RNA干扰(RNAi)筛选技术相结合，以在果蝇细胞中鉴定Wnt途径的特异性小分子抑制剂。目标/假设：我们假设我们的初步化学遗传筛选将识别专门针对稳定的？-CAT池的活性的小分子抑制剂。在这项建议中，我们概述了实验，以研究候选小分子影响稳定的β-CAT活性的分子机制(S)，并确定它们的蛋白质靶标。此外，由于传统上小分子的靶标识别一直很困难，我们还建议使用质谱学来识别？-CAT的全面的蛋白质相互作用组网络。最后，我们提出，对已知和新鉴定的？-CAT相互作用蛋白与候选小分子相互作用蛋白的dsRNA介导的击倒进行表型比较分析，将为我们提供一种新的方法来鉴定从化学遗传筛选中分离出来的抑制剂。具体目的：1)研究初级筛选中发现的小分子影响培养细胞CRT和WNT反应表型的分子机制。2)寻找可能调节β-CAT活性/稳定性的新的β-CAT/ARM蛋白相互作用伙伴。3)对候选小分子和dsRNA介导的β-CAT相互作用蛋白的敲除进行表型比较分析，并评估候选小分子是否改变了β-CAT与其已知和新鉴定的同源蛋白伙伴的结合。研究设计：对于化学遗传学筛选，我们将通过dsRNA介导的负性调控因子Axin的敲除来激活Wnt途径，从而稳定和激活？-CAT的胞液池。这些被激活的细胞将被大的小分子文库处理，以测试是否有任何单独的化合物可以抑制？-CAT介导的转录激活(CRT)，这是通过Wnt反应荧光素酶报告基因(DTF12)的活性来判断的。候选小分子将使用FRET和免疫共沉淀分析来测试它们改变？-CAT与其已知蛋白质伙伴，如Tcf、Bcl9/Lewless(Lgs)、pygopus(PYGO)、APC和Axin的相互作用的能力。我们还将使用RNAi结合候选小分子来进行上位性分析(在细胞中)。这将使我们能够确定候选小分子影响Wnt途径的位置/阶段。此外，我们将使用TAP-TAG(串联亲和纯化)技术寻找新的β-CAT蛋白相互作用伙伴，目的是寻找其他可能调节稳定的β-CAT池活性的伙伴蛋白。此外，我们将测试候选小分子是否可以取消β-CAT与TAP屏幕中确定的新的相互作用蛋白之间的相互作用。最后，我们将使用比较表型分析来测试从小分子或dsRNA介导的敲除β-CAT蛋白伙伴获得的表型之间的相似性。我们将使用基于细胞和体内的报告分析、免疫细胞化学和基于形态的分析来进行表型分析。项目简介Wnt/Wingless(WG)信号通路是一条进化保守的通路，参与细胞生物学和动物发育的许多方面的调节。Wnt途径的错误调控也与多种人类疾病有关，包括肝癌、结肠癌、乳腺癌和皮肤癌。该项目的主要目标是开发和实施新技术，以产生可用于调节Wnt信号通路活性的分子工具。

DESCRIPTION (provided by applicant): The Wnt/wingless (wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Misregulation of the Wnt pathway can be detrimental since mutations in several components are associated with tumorigenesis of the liver, colon, breast and skin. It is therefore crucial to develop and implement new technologies in order to generate molecular tools that may be used to modulate the activity of the Wnt/wg signaling pathway. One of the most important effectors of the Wnt pathway is encoded by the transcription factor, ?-catenin (?-cat)/armadillo (arm). Since Catenin Responsive Transcription (CRT) has been implicated in the genesis of many cancers, it makes a good target for developing therapeutics that could modulate the nuclear activity of ?-cat. Recently, we employed a novel methodology of integrating a "sensitized" chemical genetic high-throughput screen (HTS) with RNA-interference (RNAi) screening technology in order to identify specific small molecule inhibitors of the Wnt pathway in Drosophila cells. Objective/hypothesis: We hypothesize that our primary chemical genetic screen will identify small molecule inhibitors that specifically target the activity of the stabilized pool of ?-cat. In this proposal, we outline experiments to investigate the molecular mechanism(s) by which the candidate small molecules impact the activity of stabilized ?-cat and also identify their protein targets. Moreover, since target identification of small molecules has been traditionally difficult, we also propose to identify the comprehensive protein "interactome" network of ?-cat using mass-spectrometry. Finally, we propose that comparative phenotypic analysis of dsRNA-mediated knockdown of the known and newly identified ?-cat-interacting proteins with that of the candidate small molecules will provide us with a novel method for target identification of the inhibitors isolated in the chemical genetic screen. Specific aims: 1) Investigate the molecular mechanisms by which the small molecules identified in the primary screen impact CRT and Wnt-responsive phenotypes in cultured cells. 2) Identify novel protein interaction partners of ?-cat/arm that may regulate the activity/stability of ?-cat. 3) Perform comparative phenotypic analysis of candidate small molecules and dsRNA-mediated knockdown of ?-cat-interacting proteins and assess whether the candidate small molecules alter the binding of the ?-cat to its known and newly identified cognate protein partners. Study design: For the chemical genetic screen, we will activate the Wnt pathway using the dsRNA-mediated knockdown of the negative regulator, Axin which results in the stabilization and activation of the cytosolic pool of ?-cat. These "activated" cells will be treated with large small molecule libraries to test if any of the individual compounds could inhibit ?-cat mediated activation of transcription (CRT) as judged by activity of the Wnt- responsive luciferase reporter gene (dTF12). The candidate small molecules will be tested for their ability to alter ?-cat's interaction with its known protein partners, such as Tcf, Bcl9/legless (lgs), pygopus (pygo), APC and Axin using FRET and co-immunoprecipitation assays. We will also perform epistasis analysis (in cells) using RNAi of known regulators of the pathway in conjunction with candidate small molecules. This will enable us to determine the site/stage at which the candidate small molecules affect the Wnt pathway. Additionally, we will identify novel protein interaction partners of ?-cat using the TAP-tag (Tandem Affinity Purification) technology with the purpose of finding additional partner proteins that might regulate the activity of the stabilized pool of??-cat. Moreover, we will test if the candidate small molecules could abrogate the interaction between ?-cat and the novel interacting proteins identified in the TAP screen. Finally, we will employ comparative phenotypic analysis to test for similarities between phenotypes obtained from small molecules or dsRNA-mediated knockdown of??-cat protein partners. We will employ cell-based and in vivo reporter assays, immuno- cytochemical and morphology-based assays in order to conduct the phenotypic analysis. Project narrative The Wnt/wingless (wg) signaling pathway is an evolutionarily conserved pathway, which is involved in the regulation of many aspects of cell biology and animal development. Misregulation of the Wnt pathway has also been implicated in a variety of human diseases including cancer of the liver, colon, breast and the skin. The primary goal of this project is to develop and implement new technologies in order to generate molecular tools that may be used to modulate the activity of the Wnt signaling pathway.