Identification of purine biosynthesis as an NADH-sensing pathway to mediate energy stress, 2022, Ronghui Yang et al

Abstract
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+.

Results
Generation of tools for manipulation of cellular NADH/NAD+ and NADPH/NADP+
Electrons in NADH and NADPH can be translocated or trans hydrogenated between each other6,14. Therefore, the couples of NADH/NAD+ and NADPH/NADP+ are tightly metabolically connected in cells (Fig. 1a). Upon reductive stress, the elevated NADH/NAD+ ratio can rewire cellular transformations to dissipate electrons mainly by promoting glutamine-initiated lipogenesis, thus it often leads to a concomitant decrease in the NADPH/NADP+ ratio6.

Typically, hypoxia and ETC inhibition by antimycin A enhanced the cellular level of NADH and ratio of NADH/NAD+, concomitantly reducing the cellular level of NADPH and ratio of NADPH/NADP+ (Supplementary Fig. 1a, b). If we used α-ketobutyrate, a pyruvate analog that can be reduced to excretory α-hydroxybutyrate and thus reduce NADH accumulation8, to treat cells, it simultaneously restored the ratios of NADH/NAD+ and NADPH/NADP+ and cell proliferation under the conditions of hypoxia and ETC inhibition


https://www.nature.com/articles/s41467-022-34850-0