S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling. S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling

KEAP1, a cytoplasmic repressor of the oxidative stress responsive transcription factor NRF2, senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remains undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2 dependent transcription. This work identifies a novel posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress sensing machinery of the cell. Overall design: To identify the transcriptional program activated by the small molecule NRF2 activator sAKZ692, we evaluated the transcriptional signature of compound treatment (20 µM, 24 hours) relative to DMSO in the IMR32 cell line.

KEAP1（KEAP1）是氧化应激应答转录因子NRF2（NRF2）的细胞质抑制因子，可通过其传感器半胱氨酸残基的修饰感知亲电试剂的存在。除外源性化学物外，已有研究证实多种活性代谢物可对KEAP1上的关键半胱氨酸残基进行共价修饰，但这类分子的完整种类及其对应的修饰模式仍未明确。本研究报道了小分子sAKZ692的发现——该分子通过高通量筛选获得，可通过抑制糖酵解酶丙酮酸激酶来激活细胞内NRF2的转录活性。经sAKZ692处理后，细胞内甘油醛-3-磷酸的积累量显著上升，该代谢物可介导KEAP1的半胱氨酸传感器残基发生S-乳酸化修饰，最终触发NRF2依赖的转录过程。本研究鉴定了一种源自活性中心碳代谢物的半胱氨酸残基新型翻译后修饰类型，并进一步阐明了细胞代谢与氧化应激感知系统之间的复杂关联。实验整体设计：为鉴定小分子NRF2激活剂sAKZ692所激活的转录程序，本研究对比分析了IMR32细胞系经20 μM sAKZ692处理24小时后的转录特征谱，以二甲基亚砜（DMSO）处理组作为空白对照。