Rapidly Reversible Manipulation of Molecular Activity with Dual Chemical Dimerizers
Rapidly Reversible Manipulation of Molecular Activity with Dual Chemical Dimerizers
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DOI：
10.1002/anie.201301219
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发表时间：
2013-01-01
影响因子：
16.6
通讯作者：
Inoue, Takanari
中科院分区：
文献类型：
--
作者：
Lin, Yu-Chun;Nihongaki, Yuta;Inoue, Takanari
细胞信号传导事件的四个主要特征是快速、局部、特异性和可逆。凭借这些特征，细胞在时空上编排动态信号传导。特别地，可逆性使得小区能够调整信令事件的持续时间并有效地利用它们的有限资源。这一特征的例子是小 GTP 酶（水解三磷酸鸟苷 (GTP) 的酶）和磷脂酰肌醇脂质，它们触发不同的细胞过程，包括增殖、转化、迁移和凋亡。 [1]为了生成每个功能的精确命令，这些信号分子受到一对打开或关闭其活性的酶的严格调节：鸟嘌呤核苷酸交换因子 (GEF) 和小 GTP 酶的 GTP 酶激活蛋白，以及磷脂酰肌醇脂质的磷脂酰肌醇激酶和磷酸酶。 [1b, 2] 二聚化（CID）技术已被广泛用于快速操纵分子活动。 [3]在 CID 系统中，化学二聚剂，例如雷帕霉素 (Scheme1a)，会诱导两种蛋白质的二聚化：FK506 结合蛋白 (FKBP) 和 FKBP12-雷帕霉素结合蛋白 (FRB)。当 FKBP 预定位于质膜且 FRB 融合目标蛋白 (FRB-POI) 预定位于细胞质时，雷帕霉素诱导的二聚化导致胞质 FRB-POI 重新定位至质膜（方案 1b 中的左图和中图）。 [4] POI 在质膜上的积累随后会引发与特定 POI 分子相关的生物效应。整个过程可以在完整的活细胞中在几秒的时间内诱导。由于信号传导的快速、局部和特异性诱导，CID 技术已被证明是一种强大且通用的实验扰动工具。为了满足信号传导的第四个特征，即可逆性，可以考虑洗掉雷帕霉素以解离二聚复合物。然而，雷帕霉素从细胞中的清除速度极其缓慢。 [5]此外，雷帕霉素和 FKBP 之间的结合亲和力非常高 (200pm)。[3b, c, 6] 因此，一旦开启雷帕霉素诱导的操作，在相当的时间尺度上将其关闭就具有挑战性。[3b, c, 7] 然而，雷帕霉素解离动力学应该是实验条件的函数，例如洗脱方案、细胞 FKBP 和 FRB 构建体的类型、二聚体浓度以及蛋白质构型和表达水平。因此，我们开始使用我们实验室先前开发的一系列CID探针来评估CID的可逆性。具体来说，我们用荧光标记的 FKBP 和 FRB 蛋白共转染 COS-7 细胞，这些蛋白各自位于细胞内不同的区室中：CFP–FRB（CFP= 青色荧光蛋白）是胞浆，而 YFP–FKBP（YFP= 黄色荧光蛋白）方案 1. a) 二聚体雷帕霉素 (Rapa) 和 GA3-AM 的结构 本研究中使用。 b) 双 CID 系统对分子活性的快速、局部、特异性和可逆调节的示意图：雷帕霉素与 FKBP 结合并捕获 FRB，从而导致 CFP-FRB-POI 从胞质溶胶重新定位到 GAIs-YFP-FKBP-C2 (LACT) 标记的质膜，并激活 POI 依赖性的质膜。 质膜上的信号事件（如“ON”所示）。随后添加 GA3-AM 会诱导 GAI 和 GID1 之间的二聚化，从而导致 GAIs-YFP-FKBP-C2 (LACT)/雷帕霉素/CFP-FRB-POI 复合物作为一个整体从质膜重新定位到 Tom20-mCherry-GID1 标记的…
The four main characteristics of cellular signaling events are that they are rapid, local, specific, and reversible. With these features, cells spatiotemporally choreograph dynamic signaling. In particular, reversibility enables cells to adjust the duration of a signaling event and efficiently utilize their finite resources. This characteristic is exemplified by small GTPases (enzymes that hydrolyze guanosine triphosphate, GTP) and phosphatidylinositol lipids, which trigger diverse cellular processes, including proliferation, transformation, migration, and apoptosis.[1] To generate the precise command for each function, these signaling molecules are tightly regulated by a pair of enzymes that switch their activity on or off: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins for small GTPases, and phosphatidylinositol kinases and phosphatases for phosphatidylinositol lipids.[1b, 2] The chemically inducible dimerization (CID) technique has been widely used to rapidly manipulate molecular activities.[3] In a CID system, a chemical dimerizer, such as rapamycin (Scheme1a), induces the dimerization of two proteins: FK506 binding protein (FKBP) and the FKBP12–rapamycin binding protein (FRB). When FKBP is prelocalized to the plasma membrane and the FRB-fused protein of interest (FRB–POI) to the cytosol, rapamycin-induced dimerization results in the relocation of cytosolic FRB–POI to the plasma membrane (left and middle panels in Scheme 1b).[4] The accumulation of the POI at the plasma membrane subsequently triggers a biological effect that is pertinent to the specific POI molecule. The entire process can be induced on a timescale of seconds in intact living cells. Owing to the rapid, local, and specific induction of signaling, the CID technique has proven powerful and versatile as an experimental perturbation tool. To fulfill the fourth characteristic of signaling, namely, reversibility, one may consider washing rapamycin out to dissociate the dimerized complex. However, the clearance of rapamycin from cells is extremely slow.[5] Furthermore, the binding affinity between rapamycin and FKBP is extremely high (200pm).[3b, c, 6] Accordingly, once rapamycin-induced manipulation has been turned on, it is challenging to turn it off on a comparable timescale.[3b, c, 7] Nevertheless, the rapamycin-dissociation kinetics should be a function of experimental conditions, such as the washout protocol, cell type, dimerizer concentration, and the protein configuration and expression level of both the FKBP and FRB constructs. Therefore, we began to evaluate the reversibility of CID by using a series of CID probes previously developed in our laboratory. Specifically, we co-transfected COS-7 cells with fluorescently tagged FKBP and FRB proteins that each reside in a distinct compartment within the cell: CFP–FRB (CFP= cyan fluorescent protein) is cyotosolic, whereas YFP–FKBP (YFP= yellow fluorescentScheme 1. a) Structure of the dimerizers rapamycin (Rapa) and GA3-AM used in this study. b) Schematic representation of the rapid, local, specific, and reversible modulation of molecular activity by dual CID systems: Rapamycin binds to FKBP and traps FRB, and thus causes the relocation of CFP–FRB–POI from the cytosol to the GAIs–YFP–FKBP–C2 (LACT)-labeled plasma membrane and the activation of the POI-dependent signaling event at the plasma membrane (as indicated by “ON”). The subsequent addition of GA3-AM induces dimerization between the GAIs and GID1 and thus results in relocation of the GAIs–YFP–FKBP–C2 (LACT)/rapamycin/CFP–FRB–POI complex as a whole from the plasma membrane to the Tom20–mCherry–GID1-labeled …