IP-FCM measures physiologic protein-protein interactions modulated by signal transduction and small-molecule drug inhibition.
IP-FCM measures physiologic protein-protein interactions modulated by signal transduction and small-molecule drug inhibition.
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DOI：
10.1371/journal.pone.0045722
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Schrum AG
中科院分区：
文献类型：
--
作者：
Smith SE;Bida AT;Davis TR;Sicotte H;Patterson SE;Gil D;Schrum AG
蛋白质-蛋白质相互作用（PPI）介导控制生物信号的分子间网络的形成。出于这个原因，PPI在药理学中具有突出的意义，因为它们显示出高度的特异性，并且可能代表了大量潜在的可药用靶点。然而，生理PPI的研究可能会受到常规检测的限制，这些检测通常需要大量样本，灵敏度相对较低。在这里，我们建立了一种新的方法，免疫沉淀检测流式细胞术（IP-FCM），以评估PPI调制过程中的信号转导或药理学抑制两种不同类别的小分子化合物。首先，我们表明，IP-FCM可以检测具有定义的PPI变化低至10%的样品中的统计学显著差异。这种灵敏度允许IP-FCM检测在T细胞信号传导期间瞬时增加的PPI，ZAP 70和T细胞抗原受体（TCR）/CD 3复合物之间的抗原诱导的相互作用。相比之下，当T细胞在src家族激酶抑制剂PP 2存在下用抗原刺激时，IP-FCM检测到没有ZAP 70募集。此外，我们测试了IP-FCM是否具有足够的灵敏度来检测第二类罕见的化合物SMIPPI（PPI的小分子抑制剂）的作用。我们发现，第一代非优化SMIPPI，Ro-26-4550，抑制IL-2：CD 25的相互作用，通过IP-FCM检测。使用重组CD 25-Fc嵌合体或从T细胞裂解物捕获的生理全长CD 25可检测到这种抑制。因此，我们表明，IP-FCM是一个敏感的工具，用于测量生理PPI调制的信号转导和药理学抑制。
Protein-protein interactions (PPI) mediate the formation of intermolecular networks that control biological signaling. For this reason, PPIs are of outstanding interest in pharmacology, as they display high specificity and may represent a vast pool of potentially druggable targets. However, the study of physiologic PPIs can be limited by conventional assays that often have large sample requirements and relatively low sensitivity. Here, we build on a novel method, immunoprecipitation detected by flow cytometry (IP-FCM), to assess PPI modulation during either signal transduction or pharmacologic inhibition by two different classes of small-molecule compounds. First, we showed that IP-FCM can detect statistically significant differences in samples possessing a defined PPI change as low as 10%. This sensitivity allowed IP-FCM to detect a PPI that increases transiently during T cell signaling, the antigen-inducible interaction between ZAP70 and the T cell antigen receptor (TCR)/CD3 complex. In contrast, IP-FCM detected no ZAP70 recruitment when T cells were stimulated with antigen in the presence of the src-family kinase inhibitor, PP2. Further, we tested whether IP-FCM possessed sufficient sensitivity to detect the effect of a second, rare class of compounds called SMIPPI (small-molecule inhibitor of PPI). We found that the first-generation non-optimized SMIPPI, Ro-26-4550, inhibited the IL-2:CD25 interaction detected by IP-FCM. This inhibition was detectable using either a recombinant CD25-Fc chimera or physiologic full-length CD25 captured from T cell lysates. Thus, we demonstrate that IP-FCM is a sensitive tool for measuring physiologic PPIs that are modulated by signal transduction and pharmacologic inhibition.
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DOI：
10.4049/jimmunol.1003225
发表时间：
2011-02-15
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
de la Cruz J;Kruger T;Parks CA;Silge RL;van Oers NS;Luescher IF;Schrum AG;Gil D
通讯作者：
Gil D
影响因子：
4.8
作者：
Bida, Anya T.;Gil, Diana;Schrum, Adam G.
通讯作者：
Schrum, Adam G.
DOI：
10.1098/rstb.2003.1360
发表时间：
2004-01-29
影响因子：
6.3
作者：
Prasanth, SG;Méndez, J;Stillman, B
通讯作者：
Stillman, B
影响因子：
7.3
作者：
Raimundo, BC;Oslob, JD;Arkin, MR
通讯作者：
Arkin, MR
影响因子：
4.4
作者：
Gil, Diana;Schrum, Adam G.;Palmer, Ed
通讯作者：
Palmer, Ed