Department of Chemistry, University of California, Berkeley, CA 94720.
Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
Proc Natl Acad Sci U S A. 2019 Sep 10;116(37):18285-18294. doi: 10.1073/pnas.1904610116. Epub 2019 Aug 26.
Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.
铜是生命所必需的，除了其作为酶功能的紧密结合、氧化还原活性活性位点辅助因子的既定能力外，新出现的数据表明，细胞内的铜也存在于不稳定的池（定义为与低分子量配体松散结合的铜）中，这些铜可以调节多种过渡金属信号转导过程，包括神经通讯和嗅觉、脂肪分解、休息-活动周期以及对致癌信号至关重要的激酶途径。为了帮助破译这一不断发展的生物学，我们报告了第一代比率荧光共振能量转移（FRET）铜探针 FCP-1，用于活细胞中不稳定 Cu（I）池的基于活性的传感。FCP-1 通过三（（2-吡啶基）甲基）胺桥将荧光素和罗丹明染料连接起来。受生物启发的 Cu（I）诱导的氧化裂解降低了荧光素供体和罗丹明受体之间的 FRET。FCP-1 对 Cu（I）具有高金属选择性和氧化态特异性的响应，并促进了比率测量，最大限度地减少了由于样品厚度、染料浓度和光强度变化引起的潜在干扰。FCP-1 能够对活细胞中不稳定 Cu（I）池的动态变化进行成像，以响应铜补充/耗竭、铜进口器 CTR1 的差异表达以及通过操纵细胞内谷胱甘肽水平和还原/氧化谷胱甘肽（GSH/GSSG）比来诱导氧化还原应激。FCP-1 成像揭示了由癌基因驱动的细胞转化引起的不稳定 Cu（I）缺乏，这促进了谷胱甘肽代谢的波动，其中较低的 GSH/GSSG 比降低了不稳定 Cu（I）的可用性，而不影响总铜水平。通过连接癌症中铜失调和谷胱甘肽应激，这项工作为使用基于活性的探针研究健康和疾病中金属和氧化还原途径之间更广泛的交叉对话提供了一个有价值的起点。
