α-Ketoglutarate (α-KG) also known as 2-oxoglutarate (2-OG) is an intermediate metabolite in the tricarboxylic acid (TCA) cycle and is also produced by the deamination of glutamate. It is an indispensable cofactor for a series of 2-oxoglutarate-dependent oxygenases including epigenetic modifiers such as ten-eleven translocation DNA demethylases (TETs) and JmjC domain-containing histone demethylases (JMJDs). Since these epigenetic enzymes target genomic DNA and histone in the nucleus, the nuclear concentration of α-KG would affect the levels of transcription by modulating the activity of the epigenetic enzymes. Thus, it is of great interest to measure the nuclear concentration of α-KG to elucidate the regulatory mechanism of these enzymes. Here, we report a novel fluorescence resonance energy transfer (FRET)-based biosensor with multiple nuclear localization signals (NLSs) to measure the nuclear concentration of α-KG. The probe contains the α-KG-binding GAF domain of NifA protein from Azotobacter vinelandii fused with EYFP and ECFP. Treatment of 3T3-L1 preadipocytes expressing this probe with either dimethyl-2-oxoglutarate (dimethyl-2-OG), a cell-permeable 2-OG derivative, or citrate elicited time- and dose-dependent changes in the FRET ratio, proving that this probe functions as an α-KG sensor. Measurement of the nuclear α-KG levels in the 3T3-L1 cells stably expressing the probe during adipocyte differentiation revealed that the nuclear concentration of α-KG increased in the early stage of differentiation and remained high thereafter. Thus, this nuclear-localized α-KG probe is a powerful tool for real-time monitoring of α-KG concentrations with subcellular resolution in living cells and is useful for elucidating the regulatory mechanisms of epigenetic enzymes.

α-酮戊二酸 (α-KG) 也称为 2-氧代戊二酸 (2-OG) 是三羧酸 (TCA) 循环中的中间代谢物，也由谷氨酸脱氨基产生。它是一系列 2-氧代戊二酸依赖性加氧酶不可或缺的辅助因子，包括表观遗传修饰剂，例如 11 易位 DNA 去甲基化酶 (TET) 和含 JmjC 结构域的组蛋白去甲基化酶 (JMJD)。由于这些表观遗传酶靶向细胞核中的基因组 DNA 和组蛋白，因此 α-KG 的核浓度会通过调节表观遗传酶的活性来影响转录水平。因此，测量 α-KG 的核浓度以阐明这些酶的调节机制具有重要意义。这里，我们报告了一种基于荧光共振能量转移 (FRET) 的新型生物传感器，该传感器具有多个核定位信号 (NLS)，用于测量 α-KG 的核浓度。该探针包含来自 NifA 蛋白的 α-KG 结合 GAF 结构域Azotobacter vinelandii与 EYFP 和 ECFP 融合。用可渗透细胞的 2-OG 衍生物或柠檬酸盐处理表达该探针的 3T3-L1 前脂肪细胞，可引起 FRET 比率的时间和剂量依赖性变化，证明该探头用作 α-KG 传感器。在脂肪细胞分化过程中稳定表达探针的 3T3-L1 细胞中核 α-KG 水平的测量表明，α-KG 的核浓度在分化早期增加，此后保持较高水平。因此，这种核定位的 α-KG 探针是实时监测活细胞中具有亚细胞分辨率的 α-KG 浓度的强大工具，可用于阐明表观遗传酶的调节机制。