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

整合 RNAi、蛋白质组学和化学遗传学方法来识别特异性

基本信息

批准号：
7934325
负责人：
Ramanuj Dasgupta
金额：
$ 15.98万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2010-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7934325
关键词：
Affect Affinity Chromatography Animals Binding Biological Assay Breast Cancer cell line Cancerous Candidate Disease Gene Cell Line Cell Nucleus Cell Proliferation Cells Cellular biology Cessation of life Chemicals Co-Immunoprecipitations Colon Complex Cultured Cells Development Differentiation and Growth Dissection Double-Stranded RNA Drosophila genus Family Dasypodidae Felis catus Fluorescence Resonance Energy Transfer Gene Targeting Genetic Epistasis Genetic Screening Genetic Transcription Goals Individual Ligands Liquid Chromatography Liver Luciferases Malignant Neoplasms Malignant neoplasm of liver Mammalian Cell Mass Spectrum Analysis Mediating Metallothionein Methodology Methods Molecular Morphology Mutation Nuclear Pathway interactions Phenotype Proteins Proteomics RNA Interference Regulation Reporter Reporter Genes Research Design Screening procedure Signal Pathway Site Skin Specificity Staging Stimulus Technology Testing Therapeutic Transcriptional Activation Upper arm base chemical genetics comparative genetic regulatory protein high throughput screening human disease in vivo inhibitor/antagonist new technology novel promoter research study small molecule small molecule libraries stable cell line tandem mass spectrometry tool transcription factor tumorigenesis

项目摘要

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.

描述（由申请人提供）：Wnt/Wingless（WG）途径是一组进化保守的信号通路，可调节后生动物发展的许多方面。 Wnt途径的不正调可能是有害的，因为几个成分的突变与肝脏，结肠，乳腺和皮肤的肿瘤发生有关。因此，开发和实施新技术至关重要，以生成可用于调节WNT/WG信号通路活动的分子工具。 Wnt途径的最重要效应子之一是由转录因子？-Catenin（？-cat）/armadillo（ARM）编码。由于Catenin反应式转录（CRT）与许多癌症的起源有关，因此它是开发可以调节核活性的疗法的良好靶标。最近，我们采用了一种新的方法，将“敏化”化学遗传高通量筛选（HTS）与RNA干扰（RNAI）筛选技术整合在一起，以鉴定果蝇细胞中WNT途径的特定小分子抑制剂。客观/假设：我们假设我们的主要化学遗传筛选将鉴定出小分子抑制剂，这些抑制剂专门针对？-CAT的稳定池的活性。在此提案中，我们概述了研究分子机制的实验，候选者小分子会影响稳定的活性？-CAT并确定其蛋白质靶标。此外，由于传统上很难对小分子的目标识别，因此我们还建议使用质谱法确定？-CAT的全面蛋白质“ Interactome”网络。最后，我们建议对已知和新鉴定的dsRNA介导的敲低的比较表型分析与候选小分子的cAT相互作用蛋白相比，将为我们提供一种新的方法，以靶向鉴定在化学遗传筛选中分离的抑制剂。具体目的：1）研究培养细胞中主要筛选中鉴定出的小分子会影响CRT和WNT反应性表型的分子机制。 2）确定可能调节？-CAT的活性/稳定性的新型蛋白质相互作用伴侣。 3）对候选小分子和dsRNA介导的？ - 猫交织蛋白的敲低进行比较表型分析，并评估候选小分子是否改变了？cat的结合与其已知且新鉴定的认知蛋白伴侣的结合。研究设计：对于化学遗传筛选，我们将使用DSRNA介导的负调节剂的敲低AXIN激活Wnt途径，AXIN会导致稳定和激活？-CAT的胞质池。这些“活化”的细胞将用小的小分子文库处理，以测试任何单个化合物是否可以抑制？-CAT介导的转录激活（CRT），这是由Wnt响应性荧光素酶报告基因（DTF12）的活性所判断的。候选小分子将针对其改变其已知蛋白质伴侣的相互作用的能力进行测试，例如TCF，Bcl9/Legless（LGS），Pygopus（Pygo），APC和AXIN，使用FRET和共释放药物抗沉淀分析。我们还将使用该途径的已知调节剂与候选小分子一起进行上学分析（在细胞中）。这将使我们能够确定候选小分子影响WNT途径的位点/阶段。此外，我们将使用TAP-TAG（串联亲和力纯化）技术来确定？-CAT的新型蛋白质相互作用伙伴，目的是找到可能调节稳定池的其他伴侣蛋白质。此外，我们将测试候选小分子是否可以消除水龙头屏幕上鉴定的新型相互作用蛋白之间的相互作用。最后，我们将采用比较表型分析来测试从小分子或dsRNA介导的cat蛋白伴侣敲低的表型之间的相似性。我们将采用基于细胞的和体内报告基因测定，基于免疫化学和形态的测定法，以进行表型分析。项目叙述Wnt/Wingless（WG）信号通路是一种进化保守的途径，它参与了细胞生物学和动物发育的许多方面的调节。 Wnt途径的不正体也与多种人类疾病有关，包括肝脏，结肠，乳腺癌和皮肤癌。该项目的主要目标是开发和实施新技术，以生成可用于调节Wnt信号通路活动的分子工具。