High throughput assay for small G protein activation

小 G 蛋白激活的高通量测定

• 批准号: 6932935
• 负责人: JACQUES T WEISSMAN
• 金额: $ 9.96万
• 依托单位: ACTIVE MOTIF, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6932935
• 关键词: antibody; antineoplastics; biological signal transduction; cell line; colorimetry; drug discovery /isolation; drug screening /evaluation; enzyme activity; glutathione transferase; guanosinetriphosphatase activating protein; guanosinetriphosphatases; high throughput technology; immunologic assay /test; immunologic substance development /preparation; laboratory mouse; laboratory rabbit; molecular probes; recombinant proteins; technology /technique development

DESCRIPTION (provided by applicant): The small GTP binding protein superfamily (consisting of small, monomeric GTP hydrolyzing proteins or GTPases) regulates diverse cellular processes including gene expression, cellular proliferation, cytoskeletal reorganization, vesicle trafficking, and nucleocytoplasmic transport. Activated point mutants of Ras GTPases have been found in over 30% of all human tumors, demonstrating a key role for these proteins in carcinogenesis. The Rap GTPases, which share about 50% sequence identity with Ras, have also been shown to promote activation of MAP kinase cascades and in certain cell lines promote cellular proliferation. The Rho GTPases, including RhoA, Cdc42, and Rac-1, regulate cell proliferation as well as many of the various cell functions mentioned above for small GTPases. It is critical to develop assays for measuring small GTPase activation in order to better understand small GTPase signaling pathways as well as to facilitate discovery of drugs that specifically decrease GTPase activation and ultimately treat several forms of cancer. We propose to develop a new technology that allows for high-throughput and fast analysis of small GTPase activation. An activation specific probe will be fused to glutathione S-transferase (GST) immobilized onto a 96-well microplate to capture activated small GTPases in an extract from cells. Five different activation specific probes from downstream GTPase effecter proteins will be cloned, expressed as GST-fusion proteins, and purified. The binding conditions of small GTPases to the activation specific probes will be developed by using purified recombinant small GTPases. Antibodies raised specifically against small GTPases will identify the bound protein and yield a quantitative result in three hours. Finally, the procedure will be applied to relevant samples such as extracts from cells treated with extracellular mitogenic ligands such as epidermal growth factor (EGF). Such an assay will help to elucidate the molecular mechanisms of proliferative signal transduction pathways and provide means to screen compound libraries to find novel cancer therapy candidates that are direct inhibitors of cancer-causing small GTPase mutants.

描述（由申请人提供）：小GTP结合蛋白超家族（由小单体GTP水解蛋白或GTP酶组成）调节多种细胞过程，包括基因表达、细胞增殖、细胞骨架重组、囊泡运输和核质转运。在超过30%的人类肿瘤中发现了Ras GTP酶的激活点突变体，证明了这些蛋白质在癌发生中的关键作用。与Ras共有约50%序列同一性的Rap GTP酶也已显示促进MAP激酶级联的活化，并且在某些细胞系中促进细胞增殖。 Rho GTP酶，包括RhoA、Cdc 42和Rac-1，调节细胞增殖以及上述小GTP酶的多种细胞功能。开发用于测量小GTdR活化的测定法是至关重要的，以便更好地理解小GTdR信号传导途径以及促进发现特异性降低GTdR活化并最终治疗几种形式的癌症的药物。我们建议开发一种新的技术，允许高通量和快速分析的小GTdR激活。将活化特异性探针与固定在96孔微孔板上的谷胱甘肽S-转移酶（GST）融合，以捕获细胞提取物中的活化小GTP酶。将克隆来自下游GT3效应蛋白的五种不同的活化特异性探针，表达为GST融合蛋白，并纯化。将通过使用纯化的重组小GTP酶开发小GTP酶与活化特异性探针的结合条件。针对小GTP酶的特异性抗体将识别结合的蛋白质，并在三小时内产生定量结果。最后，该程序将应用于相关样品，如用细胞外促有丝分裂配体（如表皮生长因子（EGF））处理的细胞提取物。这种测定将有助于阐明增殖信号转导途径的分子机制，并提供筛选化合物文库的手段，以发现作为致癌小GT3突变体的直接抑制剂的新型癌症治疗候选物。