Measuring Intracellular H2O2 in Intact Human Cells Using the Genetically Encoded Fluorescent Sensor HyPer7

Jacobs, Lianne J H C; Hoehne, Martin; Riemer, Jan

doi:10.21769/bioprotoc.4538

Cited by 5 publications

(3 citation statements)

References 10 publications

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“…With the exception of NDUFS8, these subunits are all considered “accessory” or supernumerary subunits. We validated that NDUFA8 knockdown increased mitochondrial ROS, measured using mitochondrial matrix-targeted HyPer7(42) (Figure S2D). We isolated mitochondria from these cell lines and quantified supercomplex levels, subcomplex levels (complexes smaller than the size of fully assembled complex I), and complex I activity.…”

Section: Resultsmentioning

confidence: 67%

Systems-level analyses dissociate genetic regulators of reactive oxygen species and energy production

Bennett,

Lee,

Orr

et al. 2023

Preprint

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Respiratory chain dysfunction can decrease ATP and increase reactive oxygen species (ROS) levels. Despite the importance of these metabolic parameters to a wide range of cellular functions and disease, we lack an integrated understanding of how they are differentially regulated. To address this question, we adapted a CRISPRi- and FACS- based platform to compare the effects of respiratory gene knockdown on ROS to their effects on ATP. Focusing on genes whose knockdown is known to decrease mitochondria-derived ATP, we showed that knockdown of genes in specific respiratory chain complexes (I, III and CoQ10 biosynthesis) increased ROS, whereas knockdown of other low ATP hits either had no impact (mitochondrial ribosomal proteins) or actually decreased ROS (complex IV). Moreover, although shifting metabolic conditions profoundly altered mitochondria-derived ATP levels, it had little impact on mitochondrial or cytosolic ROS. In addition, knockdown of a subset of complex I subunits, including NDUFA8, NDUFB4, and NDUFS8, decreased complex I activity, mitochondria-derived ATP and supercomplex level, but knockdown of these genes had differential effects on ROS. Conversely, we found an essential role for ether lipids in the dynamic regulation of mitochondrial ROS levels independent of ATP. Thus, our results identify specific metabolic regulators of cellular ATP and ROS balance that may help dissect the roles of these processes in disease and identify therapeutic strategies to independently target energy failure and oxidative stress.

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Section: Resultsmentioning

confidence: 67%

Systems-level analyses dissociate genetic regulators of reactive oxygen species and energy production

Bennett,

Lee,

Orr

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

Section: Resultsmentioning

confidence: 76%

Systems-level analyses dissociate genetic regulators of reactive oxygen species and energy production

Bennett,

Lee,

Orr

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Respiratory chain dysfunction can decrease ATP and increase reactive oxygen species (ROS) levels. Despite the importance of these metabolic parameters to a wide range of cellular functions and disease, we lack an integrated understanding of how they are differentially regulated. To address this question, we adapted a CRISPRi- and FACS-based platform to compare the effects of respiratory gene knockdown on ROS to their effects on ATP. Focusing on genes whose knockdown is known to decrease mitochondria-derived ATP, we showed that knockdown of genes in specific respiratory chain complexes (I, III, and CoQ10 biosynthesis) increased ROS, whereas knockdown of other low ATP hits either had no impact (mitochondrial ribosomal proteins) or actually decreased ROS (complex IV). Moreover, although shifting metabolic conditions profoundly altered mitochondria-derived ATP levels, it had little impact on mitochondrial or cytosolic ROS. In addition, knockdown of a subset of complex I subunits—including NDUFA8 , NDUFB4 , and NDUFS 8—decreased complex I activity, mitochondria-derived ATP, and supercomplex level, but knockdown of these genes had differential effects on ROS. Conversely, we found an essential role for ether lipids in the dynamic regulation of mitochondrial ROS levels independent of ATP. Thus, our results identify specific metabolic regulators of cellular ATP and ROS balance that may help dissect the roles of these processes in disease and identify therapeutic strategies to independently target energy failure and oxidative stress.

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“…High-throughput imaging measurements of HEK293 cells expressing NAPStar3b from a pcDNA3.1(+) plasmid and data analysis were performed as previously described [76]. Experiments were performed using wildtype and GSR KO HEK293 cells.…”

Section: Methodsmentioning

confidence: 99%

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

Scherschel,

Niemeier,

Jacobs

et al. 2024

Preprint

Self Cite

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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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Measuring Intracellular H2O2 in Intact Human Cells Using the Genetically Encoded Fluorescent Sensor HyPer7

Cited by 5 publications

References 10 publications

Systems-level analyses dissociate genetic regulators of reactive oxygen species and energy production

Systems-level analyses dissociate genetic regulators of reactive oxygen species and energy production

Systems-level analyses dissociate genetic regulators of reactive oxygen species and energy production

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

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