Imaging Cytosolic NADH-NAD+ Redox State with a Genetically Encoded Fluorescent Biosensor

Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
Cell metabolism (Impact Factor: 17.57). 10/2011; 14(4):545-54. DOI: 10.1016/j.cmet.2011.08.012
Source: PubMed


NADH is a key metabolic cofactor whose sensitive and specific detection in the cytosol of live cells has been difficult. We constructed a fluorescent biosensor of the cytosolic NADH-NAD(+) redox state by combining a circularly permuted GFP T-Sapphire with a bacterial NADH-binding protein, Rex. Although the initial construct reported [NADH] × [H(+)] / [NAD(+)], its pH sensitivity was eliminated by mutagenesis. The engineered biosensor Peredox reports cytosolic NADH:NAD(+) ratios and can be calibrated with exogenous lactate and pyruvate. We demonstrated its utility in several cultured and primary cell types. We found that glycolysis opposed the lactate dehydrogenase equilibrium to produce a reduced cytosolic NADH-NAD(+) redox state. We also observed different redox states in primary mouse astrocytes and neurons, consistent with hypothesized metabolic differences. Furthermore, using high-content image analysis, we monitored NADH responses to PI3K pathway inhibition in hundreds of live cells. As an NADH reporter, Peredox should enable better understanding of bioenergetics.

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    • "Neg shRNA, SPG7, and PPIF KD 293T cells were transiently transfected with NADH-NAD + redox sensor peredox m-cherry (Hung et al., 2011). Confocal images were acquired at 405 and 561 nm excitation every 3 s using Carl Zeiss 710 Meta NLO. "
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    Molecular Cell 09/2015; DOI:10.1016/j.molcel.2015.08.009 · 14.02 Impact Factor
    • "The need to oxidize nutrients to generate biomass may result in electron acceptor insufficiency and limit proliferation even in the presence of oxygen. Notably, the NAD+/NADH ratio in normoxic cancer cells is more reduced than previously assumed (Hung et al., 2011; Zhao et al., 2015), consistent with oxidation capacity being constrained. Because the NAD+/NADH ratio and the pyruvate/lactate ratio are tightly coupled in cells (Williamson et al., 1967), a low NAD+/NADH ratio is expected to drive increased pyruvate to lactate conversion. "
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    Cell 07/2015; 162(3):552-563. DOI:10.1016/j.cell.2015.07.017 · 32.24 Impact Factor
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    • "These Frex sensors (Zhao et al., 2011) specifically report NADH levels over a large dynamic range; however, they do not adapt an optimal tertiary structure in some cells, and their fluorescence is pH sensitive. Peredox sensors (Hung et al., 2011) are much more pH resistant and partially reflect the more physiologically relevant NAD + /NADH ratio; however , they have a limited dynamic range, and their affinity appears too high to be useful under physiological conditions. Importantly, neither Frex nor Peredox sensors show obvious fluorescence response to NAD "
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    ABSTRACT: The altered metabolism of tumor cells confers a selective advantage for survival and proliferation, and studies have shown that targeting such metabolic shifts may be a useful therapeutic strategy. We developed an intensely fluorescent, rapidly responsive, pH-resistant, genetically encoded sensor of wide dynamic range, denoted SoNar, for tracking cytosolic NAD(+) and NADH redox states in living cells and in vivo. SoNar responds to subtle perturbations of various pathways of energy metabolism in real time, and allowed high-throughput screening for new agents targeting tumor metabolism. Among > 5,500 unique compounds, we identified KP372-1 as a potent NQO1-mediated redox cycling agent that produced extreme oxidative stress, selectively induced cancer cell apoptosis, and effectively decreased tumor growth in vivo. This study demonstrates that genetically encoded sensor-based metabolic screening could serve as a valuable approach for drug discovery. Copyright © 2015 Elsevier Inc. All rights reserved.
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