一种用于在活细胞中可视化乙酰辅酶A的基因编码荧光生物传感器。
A genetically encoded fluorescent biosensor for visualization of acetyl-CoA in live cells.
作者信息
Smith Joseph J, Valentino Taylor R, Ablicki Austin H, Banerjee Riddhidev, Colligan Adam R, Eckert Debra M, Desjardins Gabrielle A, Diehl Katharine L
机构信息
Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
ARC Tangents Analytics, Salt Lake City, UT 84108, USA.
出版信息
Cell Chem Biol. 2025 Feb 20;32(2):325-337.e10. doi: 10.1016/j.chembiol.2025.01.002. Epub 2025 Jan 27.
Acetyl-coenzyme A is a central metabolite that participates in many cellular pathways. Evidence suggests that acetyl-CoA metabolism is highly compartmentalized in mammalian cells. Yet methods to measure acetyl-CoA in living cells are lacking. Herein, we engineered an acetyl-CoA biosensor from the bacterial protein PanZ and circularly permuted green fluorescent protein (cpGFP). The sensor, "PancACe," has a maximum change of ∼2-fold and a response range of ∼10 μM-2 mM acetyl-CoA. We demonstrated that the sensor has a greater than 7-fold selectivity over coenzyme A, butyryl-CoA, malonyl-CoA, and succinyl-CoA, and a 2.3-fold selectivity over propionyl-CoA. We expressed the sensor in E. coli and showed that it enables detection of rapid changes in acetyl-CoA levels. By localizing the sensor to either the cytoplasm, nucleus, or mitochondria in human cells, we showed that it enables subcellular detection of changes in acetyl-CoA levels, the magnitudes of which agreed with an orthogonal PicoProbe assay.
乙酰辅酶A是一种参与多种细胞途径的核心代谢物。有证据表明，乙酰辅酶A代谢在哺乳动物细胞中高度分隔化。然而，目前缺乏在活细胞中测量乙酰辅酶A的方法。在此，我们利用细菌蛋白PanZ和环状排列的绿色荧光蛋白（cpGFP）构建了一种乙酰辅酶A生物传感器。该传感器“PancACe”的最大变化约为2倍，对乙酰辅酶A的响应范围约为10 μM至2 mM。我们证明，该传感器对辅酶A、丁酰辅酶A、丙二酰辅酶A和琥珀酰辅酶A的选择性大于7倍，对丙酰辅酶A的选择性为2.3倍。我们在大肠杆菌中表达了该传感器，并表明它能够检测乙酰辅酶A水平的快速变化。通过将该传感器定位于人类细胞的细胞质、细胞核或线粒体中，我们表明它能够进行亚细胞水平的乙酰辅酶A水平变化检测，其变化幅度与正交的PicoProbe检测结果一致。
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