High throughput assay for small G protein activation

描述(申请人提供)：小的GTP结合蛋白超家族(由小的单体GTP水解蛋白或GTP酶组成)调节不同的细胞过程，包括基因表达、细胞增殖、细胞骨架重组、囊泡运输和核质运输。在超过30%的人类肿瘤中发现了激活的Ras GTP酶的点突变，证明了这些蛋白在肿瘤发生中的关键作用。Rap GTP酶与RAS有大约50%的序列同源性，也被证明促进MAP激酶级联的激活，并在某些细胞系中促进细胞增殖。Rho GTP酶包括RhoA、CDC42和RAC-1，调节细胞增殖以及上述小GTP酶的许多不同细胞功能。为了更好地了解小GTP酶信号通路，以及促进发现特异性降低GTP酶活性并最终治疗几种形式的癌症的药物，开发检测小GTP酶活性的方法至关重要。我们建议开发一种新的技术，允许高通量和快速地分析小GTP酶的激活。将激活特异性探针与固定在96孔板上的谷胱甘肽S转移酶(GST)融合，以捕获细胞提取物中激活的小GTP酶。从下游GTPase效应蛋白中克隆出5种不同的激活特异性探针，以GST融合蛋白的形式表达，并进行纯化。利用纯化的重组小分子GTP酶可探索小分子GTP酶与激活特异性探针的结合条件。针对小GTP酶产生的抗体将在三个小时内识别结合蛋白并产生定量结果。最后，该程序将应用于相关样本，如用表皮生长因子(EGF)等细胞外有丝分裂配体处理的细胞提取物。这种分析将有助于阐明增殖信号转导途径的分子机制，并为筛选化合物文库提供手段，以寻找直接抑制致癌小GTP酶突变的新的癌症治疗候选药物。

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.