Development and Utilization of Activated STAT3 Detection Assays for Screening a Library of Secreted Proteins
Development and Utilization of Activated STAT3 Detection Assays for Screening a Library of Secreted Proteins
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DOI：
10.1089/adt.2010.0348
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发表时间：
2011-08-01
影响因子：
1.8
通讯作者：
Powers, Gordon
中科院分区：
文献类型：
--
作者：
Fursov, Natalie;Gates, Irina V.;Powers, Gordon
白细胞介素-6（IL-6）家族细胞因子是多功能蛋白质，其在宿主防御、急性期反应、免疫应答、造血和肿瘤发生中发挥重要作用。细胞因子由各种淋巴和非淋巴细胞产生，并通过与细胞表面受体的初始低亲和力结合介导其生物活性，细胞表面受体对其各自的配体具有特异性。配体特异性受体结合导致受体与普遍表达的信号转导跨膜组分gp 130的异源二聚化，随后激活gp 130相关的Janus激酶，其进而磷酸化信号转导子和转录激活子3（STAT 3）。磷酸化STAT 3（pSTAT 3）二聚化并易位到细胞核，在那里它激活基因转录。STAT 3的激活对于IL-6家族相关的生理效应是必需的。因此，评估STAT 3磷酸化的能力对于靶向IL-6家族细胞因子的药物发现工作是重要的。各种试剂和技术可用于检测IL-6型细胞因子在处理的细胞中的作用。本研究描述了两种pSTAT 3检测方法的发展：基于Meso-Scale Discovery技术的高通量筛选方法，该方法利用电化学发光信号测量来检测处理后细胞提取物中的pSTAT 3，以及基于荧光成像分析的二级表征方法，该方法监测激活后细胞中的pSTAT 3核转位。我们已经成功地利用这些测定筛选分泌蛋白的小文库，并鉴定了STAT 3磷酸化的诱导剂。在这项研究中获得的结果表明，这两种测定法是强大的，可靠的，并适合高通量筛选应用。
Interleukin-6 (IL-6) family of cytokines are multifunctional proteins that play an important role in host defenses, acute phase reactions, immune responses, hematopoiesis, and tumorigenesis. The cytokines are produced by various lymphoid and nonlymphoid cells and mediate their biological activity through initial low-affinity binding to cell surface receptors, which are specific for their respective ligands. Ligand-specific receptor binding results in the receptor heterodimerization with ubiquitously expressed signal-transducing transmembrane component gp130 followed by activation of the gp130-associated Janus kinase, which, in turn, phosphorylates signal transducer and activator of transcription 3 (STAT3). Phosphorylated STAT3 (pSTAT3) dimerizes and translocates to the nucleus, where it activates gene transcription. Activation of STAT3 is essential to IL-6 family-associated physiological effects. Therefore, the ability to assess STAT3 phosphorylation is important for drug discovery efforts targeting IL-6 family cytokines. Various reagents and technologies are available to detect the effect of IL-6 type cytokines in treated cells. The present study describes the development of two pSTAT3 detection assays: the high-throughput screening assay based on Meso-Scale Discovery technology, which utilizes electrochemoluminescent signal measurements for the detection of pSTAT3 in treated cell extracts, and the secondary characterization assay based on fluorescent imaging analysis, which monitors pSTAT3 nuclear translocation in cells after activation. We have successfully utilized these assays to screen a small library of secreted proteins and identified inducers of STAT3 phosphorylation. The results obtained in this study demonstrate that both assays are robust, reliable, and amenable to high-throughput screening applications.