Jacqui Wood Cancer Centre, Division of Cellular Medicine, School of Medicine , University of Dundee , Dundee DD1 9SY , Scotland , United Kingdom.
Dundee Imaging Facility, School of Life Sciences , University of Dundee , Dundee DD1 5EH , Scotland , United Kingdom.
Chem Res Toxicol. 2019 Mar 18;32(3):500-512. doi: 10.1021/acs.chemrestox.8b00354. Epub 2019 Mar 11.
Transcription factor NF-E2 p45-related factor 2 (Nrf2) and its principal negative regulator, Kelch-like ECH-associated protein 1 (Keap1), comprise a molecular effector and sensor system that robustly responds to perturbations of the cellular redox homeostasis by orchestrating a comprehensive cytoprotective program. Under homeostatic conditions, Nrf2 is a short-lived protein, which is targeted for ubiquitination and proteasomal degradation. Upon encounter of electrophiles, oxidants, or pro-inflammatory stimuli, the cysteine sensors in Keap1 are chemically modified, rendering Keap1 unable to target Nrf2 for degradation, and consequently leading to accumulation of the transcription factor and enhanced transcription of cytoprotective genes. A detailed understanding of the protein-protein interactions between Nrf2 and Keap1 has been achieved by use of various in vitro systems, but few assays are available to assess these interactions in the context of the living cell. We previously developed an imaging-based FLIM/FRET methodology to visualize and measure the interaction between Nrf2 and Keap1 in single cells. Here, our goal was to improve this methodology in order to increase throughput and precision, and decrease cell-to-cell variability. To eliminate the possibility of orientation bias, we incorporated a flexible linker between Keap1 and the FRET acceptor fluorescent protein tag. To ensure the correct image capture of Nrf2 fused to the FRET donor fluorescent protein tag, we matched the maturation time of the fluorescent tag to the half-life of the endogenous Nrf2, by using sfGFP as the FRET donor. Using a global binning approach increased the assay throughput, whereas including the measured instrument response function in the analysis improved precision. The application of this methodology revealed a strong covariation of the results with the expression level of the acceptor. Taking the acceptor level into account circumvented cell-to-cell variability and enhanced sensitivity of the measurements of the Keap1-Nrf2 interaction in live cells.
转录因子 NF-E2 p45 相关因子 2（Nrf2）及其主要负调控因子 Kelch 样 ECH 相关蛋白 1（Keap1），构成了一个分子效应器和传感器系统，通过协调全面的细胞保护程序，对细胞氧化还原稳态的干扰做出强烈反应。在稳态条件下，Nrf2 是一种短寿命蛋白，可被泛素化和蛋白酶体降解。当遇到亲电子体、氧化剂或促炎刺激时，Keap1 中的半胱氨酸传感器会发生化学修饰，使 Keap1 无法将 Nrf2 靶向降解，从而导致转录因子积累并增强细胞保护基因的转录。通过使用各种体外系统，已经对 Nrf2 和 Keap1 之间的蛋白质-蛋白质相互作用有了详细的了解，但很少有测定方法可用于评估活细胞中这些相互作用。我们之前开发了一种基于成像的 FLIM/FRET 方法，用于在单细胞中可视化和测量 Nrf2 和 Keap1 之间的相互作用。在这里，我们的目标是改进该方法，以提高通量和精度，并降低细胞间变异性。为了消除取向偏差的可能性，我们在 Keap1 和 FRET 受体荧光蛋白标签之间引入了一个柔性接头。为了确保融合到 FRET 供体荧光蛋白标签的 Nrf2 的正确图像捕获，我们通过使用 sfGFP 作为 FRET 供体，使荧光标签的成熟时间与内源性 Nrf2 的半衰期相匹配。使用全局分箱方法提高了测定的通量，而在分析中包含测量的仪器响应函数则提高了精度。该方法的应用揭示了结果与受体表达水平之间的强烈相关性。考虑到受体水平，可以避免细胞间变异性，并提高活细胞中 Keap1-Nrf2 相互作用测量的灵敏度。
