Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN, USA.
PLoS One. 2012;7(9):e45722. doi: 10.1371/journal.pone.0045722. Epub 2012 Sep 21.
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.
蛋白质-蛋白质相互作用（PPI）介导了控制生物信号的分子间网络的形成。出于这个原因，PPI 在药理学中非常重要，因为它们具有高度的特异性，并且可能代表着一个巨大的潜在可成药靶点库。然而，传统的测定方法通常需要大量的样本，并且相对灵敏度较低，这使得对生理 PPI 的研究受到了限制。在这里，我们基于一种新的方法，即免疫沉淀流式细胞术（IP-FCM），来评估两种不同类别的小分子化合物在信号转导或药理抑制过程中 PPI 调节的情况。首先，我们表明 IP-FCM 可以检测到具有明确 PPI 变化的样本中，统计学上差异显著的变化低至 10%。这种灵敏度使得 IP-FCM 能够检测到 T 细胞信号转导过程中瞬时增加的 PPI，即 ZAP70 和 T 细胞抗原受体（TCR）/CD3 复合物之间的抗原诱导相互作用。相比之下，当 T 细胞在 src 家族激酶抑制剂 PP2 的存在下被抗原刺激时，IP-FCM 未检测到 ZAP70 的募集。此外，我们测试了 IP-FCM 是否具有足够的灵敏度来检测第二种罕见的化合物，即 SMIPPI（PPI 的小分子抑制剂）的作用。我们发现第一代非优化的 SMIPPI，Ro-26-4550，抑制了 IP-FCM 检测到的 IL-2:CD25 相互作用。这种抑制作用可以使用重组 CD25-Fc 嵌合体或从 T 细胞裂解物中捕获的生理全长 CD25 来检测。因此，我们证明了 IP-FCM 是一种敏感的工具，可以测量受信号转导和药理抑制调节的生理 PPI。
