FK506-binding proteins (FKBPs) belong to the immunophilin family and are linked to various disease states, including the inflammatory response. The inhibition of cytokine and chemokine expression in addition to positive effects of FKBPs on corneal inflammation in animal models suggests that they may be used for ophthalmic delivery in the treatment of dry eye disease. To pass the effective barriers protecting eye tissues, testing the transduction domains of FKBPs is essential. However, monitoring their transduction efficiencies is not a simple task. The quantitative measurement of FKBP interactions was performed using a cell model with a specific G protein-coupled receptor, as FKBPs had been known to act at the inositol 1,4,5-trisphosphate receptor (IP₃R) leading to the inhibition of intracellular calcium mobilization. Because of its luminescence amplitude and stability, human urotensin II receptor was expressed in aequorin parental cells to measure the action of selected FKBPs. This luminescence-based functional assay platform exhibited a high signal-to-background ratio of more than 100 and a Z’ factor at 0.6204. As expected, changes in the sequence of the transduction domain affected the function of the FKBPs. The intracellular calcium mobilization assay with selected FKBPs represented a robust and reliable platform to screen initial candidates. Although the precise nature of the control that FKBPs exert on the IP₃R is uncertain, this approach can be used to develop innovative anti-inflammatory treatments for dry eye disease by optimizing protein transduction domain sequences.

中文翻译：

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利用细胞内钙动员试验筛选转导的 FK506 结合蛋白

FK506 结合蛋白 (FKBPs) 属于亲免素家族，与各种疾病状态有关，包括炎症反应。除了 FKBPs 在动物模型中对角膜炎症的积极作用外，细胞因子和趋化因子表达的抑制表明它们可用于治疗干眼病的眼科给药。为了通过保护眼组织的有效屏障，测试 FKBPs 的转导域是必不可少的。然而，监测它们的转导效率并不是一项简单的任务。使用具有特定 G 蛋白偶联受体的细胞模型进行 FKBP 相互作用的定量测量，因为已知 FKBP 作用于肌醇 1,4,5-三磷酸受体 (IP ₃R) 导致细胞内钙动员的抑制。由于其发光幅度和稳定性，人尿紧张素 II 受体在水母发光蛋白亲代细胞中表达，以测量选定的 FKBP 的作用。这种基于发光的功能测定平台表现出超过 100 的高信号背景比和 0.6204 的Z ' 因子。正如预期的那样，转导域序列的变化影响了 FKBPs 的功能。使用选定的 FKBP 进行的细胞内钙动员测定代表了筛选初始候选者的强大而可靠的平台。尽管 FKBP 对 IP _{3施加的控制的精确性质}R 不确定，这种方法可用于通过优化蛋白质转导域序列来开发干眼病的创新抗炎治疗。