Ohata Jun, Krishnamoorthy Lakshmi, Gonzalez Monica A, Xiao Tong, Iovan Diana A, Toste F Dean, Miller Evan W, Chang Christopher J
Department of Chemistry, Department of Molecular and Cell Biology, Helen Wills Neuroscience Institute, University of California, Berkeley, California 94720, United States.
ACS Cent Sci. 2020 Jan 22;6(1):32-40. doi: 10.1021/acscentsci.9b01038. Epub 2020 Jan 7.
Chemical probes that report on protein activity, rather than protein abundance, with spatial and temporal resolution can enable studies of their native function in biological contexts as well as provide opportunities for developing new types of biochemical reporters. Here we present a sensing platform, termed proximity-activated imaging reporter (PAIR), which combines activity-based methionine bioconjugation and antibody labeling with proximity-dependent oligonucleotide-based amplification to monitor dynamic changes of a given analyte in cells and animals through context-dependent methionine labeling of specific protein targets. We establish this PAIR method to develop sensors for imaging reactive oxygen species (ROS) and calcium ions through oxaziridine-directed labeling of reactive methionine residues on β-actin and calmodulin (CaM), respectively, where the extent of methionine bioconjugation on these protein targets can serve as an indicator of oxidative stress or calcium status. In particular, application of PAIR to activity-based CaM detection provides a method for imaging integrated calcium activity in both in vitro cell and in vivo zebrafish models. By relying on native protein biochemistry, PAIR enables redox and metal imaging without introduction of external small molecules or genetically encoded indicators that can potentially buffer the natural/existing pools. This approach can be potentially generalized to target a broader range of analytes by pairing appropriate activity-based protein probes with protein detection reagents in a proximity-driven manner, providing a starting point not only for designing new sensors but also for monitoring endogenous activity of specific protein targets in biological specimens with spatial and temporal fidelity.
能够以空间和时间分辨率报告蛋白质活性而非蛋白质丰度的化学探针，可用于研究其在生物环境中的天然功能，并为开发新型生化报告分子提供机会。在此，我们展示了一种传感平台，称为邻近激活成像报告分子（PAIR），它将基于活性的甲硫氨酸生物共轭和抗体标记与基于邻近依赖性寡核苷酸的扩增相结合，通过对特定蛋白质靶点进行依赖于环境的甲硫氨酸标记，来监测细胞和动物中给定分析物的动态变化。我们建立了这种PAIR方法，以开发用于成像活性氧（ROS）和钙离子的传感器，分别通过恶唑烷对β-肌动蛋白和钙调蛋白（CaM）上的反应性甲硫氨酸残基进行定向标记，其中这些蛋白质靶点上甲硫氨酸生物共轭的程度可作为氧化应激或钙状态的指标。特别地，将PAIR应用于基于活性的CaM检测，为在体外细胞和体内斑马鱼模型中成像整合钙活性提供了一种方法。通过依赖天然蛋白质生物化学，PAIR能够进行氧化还原和金属成像，而无需引入可能缓冲天然/现有池的外部小分子或基因编码指示剂。这种方法有可能通过以邻近驱动的方式将适当的基于活性的蛋白质探针与蛋白质检测试剂配对，推广到针对更广泛的分析物，不仅为设计新传感器提供了起点，也为在生物样本中以空间和时间保真度监测特定蛋白质靶点的内源性活性提供了起点。
