Kim Sung Bae, Umezawa Yoshio, Kanno Kira A, Tao Hiroaki
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology, 16-1 Onogawa, Tsukuba 305-8569, Japan.
ACS Chem Biol. 2008 Jun 20;3(6):359-72. doi: 10.1021/cb800004s.
Bioactive small molecules, including steroids, activate multiple signaling pathways in mammalian cells. However, current technologies cannot illuminate such multiple effects of a ligand in mammalian cells. Here, we demonstrate integrated-molecule-format multicolor systems simultaneously visualizing bifacial activities of a ligand, where estrogen receptor alpha (ERalpha) was exemplified to demonstrate the present technology. First, we developed a single-molecule-format probe emitting red bioluminescence for imaging interaction between the phosphorylated ligand binding domain of ERalpha (ER LBD) and the Src homology-2 (SH2) domain of Src. The SH2 domain-linked ER LBD was sandwiched between dissected N- and C-terminal fragments of Pyrophorus plagiophthalamus (click beetle) luciferase emitting red bioluminescence. Second, another single-molecule-format bio-luminescent probe emitting green bioluminescence was constructed to visualize intramolecular interaction between ER LBD and LXXLL motifs. Mammalian cells carrying the two probes emit red and/or green light in response to agonistic and antagonistic activities of a ligand, which correspond to its genomic and nongenomic activities, respectively. Third, the two probes were assembled to make an single-molecule-format multicolor indicator, in which all of the components for ligand sensing and multiple-light emission were integrated. The probe emitted characteristic light spectra in response to various agonists and antagonists. This is the first example where (i) protein phosphorylation was recognized with a single bioluminescent probe and (ii) bifacial activities of a ligand, either agonistic or antagonistic, were simultaneously visualized with multiple colors.
包括类固醇在内的生物活性小分子可激活哺乳动物细胞中的多种信号通路。然而，目前的技术无法阐明配体在哺乳动物细胞中的这种多重效应。在此，我们展示了集成分子形式的多色系统，可同时可视化配体的双面活性，其中以雌激素受体α（ERα）为例来演示本技术。首先，我们开发了一种发射红色生物发光的单分子形式探针，用于成像ERα的磷酸化配体结合结构域（ER LBD）与Src的Src同源2（SH2）结构域之间的相互作用。与SH2结构域相连的ER LBD夹在发出红色生物发光的斜纹叩头虫荧光素酶的N端和C端片段之间。其次，构建了另一种发射绿色生物发光的单分子形式生物发光探针，以可视化ER LBD与LXXLL基序之间的分子内相互作用。携带这两种探针的哺乳动物细胞会根据配体的激动和拮抗活性分别发出红光和/或绿光，这分别对应其基因组和非基因组活性。第三，将这两种探针组装成一个单分子形式的多色指示剂，其中整合了用于配体传感和多光发射的所有组件。该探针会根据各种激动剂和拮抗剂发出特征光谱。这是第一个（i）用单个生物发光探针识别蛋白质磷酸化，以及（ii）用多种颜色同时可视化配体的激动或拮抗双面活性的例子。
