Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolism

Abstract

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.

Förster resonance energy transfer (FRET)-based small-molecule sensors and imaging agents

“…A FRET platform (probe 4) for the detection of labile Cu + was recently reported. 8 In probe 4, fluorescein and rhodamine moieties are used as the energy donor and acceptor respectively. These subunits were anchored to a tris[(2-pyridyl)methyl]amine (TPA) group that was designed to serve a dual roles as the linker and as the selective recognition site for Cu + (Fig.…”

Recent Endeavors on Molecular Imaging for Mapping Metals in Biology

“…Together with its 2-color response (F 526 /F 576 ), collective results indicate that oncogene-driven changes in the metabolism of GSH, a major cellular redox buffer, leads to a labile Cu ? deficiency with differential expression of CTR1 (Chung et al 2019). This work connects Cu ?…”

Activity-Based Sensing with a Metal-Directed Acyl Imidazole Strategy Reveals Cell Type-Dependent Pools of Labile Brain Copper

“…On the other hand, misregulation of copper homeostasis can lead to cellular malfunctions resulting from aberrant increases in reactive oxygen species (ROS) that can lead to oxidative damage to proteins, lipids, and DNA/RNA. 15,16 Such stress responses contribute to diseases including cancer, [17][18][19] neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases, [20][21][22][23][24] and genetic disorders such as Menkes and Wilson's diseases. [25][26][27] These correlations are intriguing, but the underlying causal contributions of copper homeostasis to function and disease in the brain and central nervous system remain insufficiently understood.…”

“…[25][26][27] These correlations are intriguing, but the underlying causal contributions of copper homeostasis to function and disease in the brain and central nervous system remain insufficiently understood. To this end, a number of chemical technologies have been developed to probe biological copper fluxes by molecular imaging, including fluorescent, [11][12][13][14]18,[28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] and magnetic resonance imaging (MRI), [48][49][50][51][52][53][54][55][56] and bioluminescent 57 copper indicators. These reporters can achieve high selectivity and signal-to-noise responses for copper ion imaging from cellular to tissue to whole animal settings.…”