Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions
Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions
复制标题
DOI：
10.1111/j.1600-0854.2005.00298.x
复制
发表时间：
2005-07-01
期刊：
影响因子：
4.5
通讯作者：
Stauber, RH
中科院分区：
文献类型：
--
作者：
Knauer, SK;Moodt, S;Stauber, RH
调节的核质转运对于细胞内稳态至关重要，并且依赖于蛋白质相互作用网络。此外，细胞核和细胞质的空间划分标志着两个细胞内区室，可以很容易地通过显微镜区分。因此，我们结合调节核质转运的规则和自发荧光蛋白，开发了新型的细胞生物传感器组成的谷胱甘肽S-转移酶，突变的绿色荧光蛋白和合理的组合的核输入和输出信号。添加调节序列导致三类生物传感器适用于识别信号特异性核输出和输入抑制剂，干扰蛋白酶活性的小分子和防止活细胞中特定蛋白质-蛋白质相互作用的化合物。作为一个独特的功能，我们的系统利用细胞质生物传感器的核积累作为所有检测的可靠读数。系统地研究了生物传感器的有效性，并通过使用全自动平台进行高通量筛选（HTS）显微镜和测定分析来证明。引入的模块化生物传感器不仅具有进一步剖析核质转运途径的潜力，而且还可用于许多筛选应用，用于潜在候选药物的早期评估。
Regulated nucleo-cytoplasmic transport is crucial for cellular homeostasis and relies on protein interaction networks. In addition, the spatial division into the nucleus and the cytoplasm marks two intracellular compartments that can easily be distinguished by microscopy. Consequently, combining the rules for regulated nucleo-cytoplasmic transport with autofluorescent proteins, we developed novel cellular biosensors composed of glutathione S-transferase, mutants of green fluorescent protein and rational combinations of nuclear import and export signals. Addition of regulatory sequences resulted in three classes of biosensors applicable for the identification of signal-specific nuclear export and import inhibitors, small molecules that interfere with protease activity and compounds that prevent specific protein-protein interactions in living cells. As a unique feature, our system exploits nuclear accumulation of the cytoplasmic biosensors as the reliable readout for all assays. Efficacy of the biosensors was systematically investigated and also demonstrated by using a fully automated platform for high throughput screening (HTS) microscopy and assay analysis. The introduced modular biosensors not only have the potential to further dissect nucleocytoplasmic transport pathways but also to be employed in numerous screening applications for the early stage evaluation of potential drug candidates.