Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing, China.
Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing, China.
Nat Commun. 2022 Nov 17;13(1):7031. doi: 10.1038/s41467-022-34850-0.
An enhanced NADH/NAD ratio, termed reductive stress, is associated with many diseases. However, whether a downstream sensing pathway exists to mediate pathogenic outcomes remains unclear. Here, we generate a soluble pyridine nucleotide transhydrogenase from Escherichia coli (EcSTH), which can elevate the NADH/NAD ratio and meantime reduce the NADPH/NADP ratio. Additionally, we fuse EcSTH with previously described LbNOX (a water-forming NADH oxidase from Lactobacillus brevis) to resume the NADH/NAD ratio. With these tools and by using genome-wide CRISPR/Cas9 library screens and metabolic profiling in mammalian cells, we find that accumulated NADH deregulates PRPS2 (Ribose-phosphate pyrophosphokinase 2)-mediated downstream purine biosynthesis to provoke massive energy consumption, and therefore, the induction of energy stress. Blocking purine biosynthesis prevents NADH accumulation-associated cell death in vitro and tissue injury in vivo. These results underscore the pathophysiological role of deregulated purine biosynthesis in NADH accumulation-associated disorders and demonstrate the utility of EcSTH in manipulating NADH/NAD and NADPH/NADP.
增强的 NADH/NAD 比值，称为还原性应激，与许多疾病有关。然而，是否存在下游感应途径来介导致病结果尚不清楚。在这里，我们从大肠杆菌 (EcSTH) 中产生了一种可溶性吡啶核苷酸转氢酶，它可以提高 NADH/NAD 比值，同时降低 NADPH/NADP 比值。此外，我们将 EcSTH 与之前描述的 LbNOX（来自短乳杆菌的形成水的 NADH 氧化酶）融合，以恢复 NADH/NAD 比值。利用这些工具，并通过在哺乳动物细胞中进行全基因组 CRISPR/Cas9 文库筛选和代谢谱分析，我们发现积累的 NADH 会使 PRPS2（磷酸核糖焦磷酸激酶 2）介导的下游嘌呤生物合成失去调节，从而引发大量能量消耗，因此会引发能量应激。阻断嘌呤生物合成可防止体外 NADH 积累相关细胞死亡和体内组织损伤。这些结果强调了嘌呤生物合成失调在 NADH 积累相关疾病中的病理生理作用，并证明了 EcSTH 在操纵 NADH/NAD 和 NADPH/NADP 方面的实用性。
