The NAPstar family of NADP redox state sensors highlights glutathione as the primary mediator of anti-oxidative electron flux

Scherschel, M.; Niemeier, J.-O.; Jacobs, L.; Hoffmann, M.; Diederich, A.; Bell, C.; Hoehne, P.; Raetz, S.; Kroll, J.; Steinbeck, J.; Lichtenauer, S.; Multhoff, J.; Zimmermann, J.; Sadhanasatish, T.; Rothemann, R. A.; Grashoff, C.; Messens, J.; Ampofo, E.; Laschke, M. W.; Riemer, J.; Roma, L. P.; Schwarzlander, M.; Morgan, B.

doi:10.1101/2024.02.14.580349

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bioRxiv · 10.1101/2024.02.14.580349

The NAPstar family of NADP redox state sensors highlights glutathione as the primary mediator of anti-oxidative electron flux

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Abstract

The NADPH/NADP+ redox couple is central to metabolism and redox signalling. NADP redox state is differentially regulated by distinct enzymatic machineries at the subcellular compartment level. Nonetheless, a detailed understanding of subcellular NADP redox dynamics is limited by the availability of appropriate tools. Here, we introduce NAPstars, a family of genetically encoded, fluorescent protein-based NADP redox state biosensors. NAPstars offer real-time, specific, pH-resistant measurements, across a broad-range of NADP redox states, with subcellular resolution. We establish NAPstar measurements in yeast, plants and mammalian cell models, revealing a conserved robustness of cytosolic NADP redox homeostasis. NAPstars uncovered NADP redox oscillations linked to the cell cycle in yeast and illumination- and hypoxia-dependent NADP redox changes in plant leaves. By selectively impairing the glutathione and thioredoxin anti-oxidative pathways under acute oxidative challenge, NAPstars demonstrated an unexpected role for the glutathione system as the primary mediator of anti-oxidative electron flux that is conserved across eukaryotic kingdoms.

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Scherschel, M., Niemeier, J.-O., Jacobs, L., Hoffmann, M., Diederich, A., Bell, C., Hoehne, P., Raetz, S., Kroll, J., Steinbeck, J., Lichtenauer, S., Multhoff, J., Zimmermann, J., Sadhanasatish, T., Rothemann, R. A., Grashoff, C., Messens, J., Ampofo, E., Laschke, M. W., Riemer, J., Roma, L. P., Schwarzlander, M., Morgan, B.. 2024-02-15. The NAPstar family of NADP redox state sensors highlights glutathione as the primary mediator of anti-oxidative electron flux. https://doi.org/10.1101/2024.02.14.580349

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