Article

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: 16.75). 10/2011; 14(4):545-54. DOI: 10.1016/j.cmet.2011.08.012
Source: PubMed

ABSTRACT 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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    Cell 07/2015; 162(3):552-563. DOI:10.1016/j.cell.2015.07.017 · 33.12 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.
    Cell metabolism 05/2015; 21(5):777-89. DOI:10.1016/j.cmet.2015.04.009 · 16.75 Impact Factor
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    • "Measurement of mitochondrial and cytosolic NADH levels Mitochondrial and cytosolic NADH levels were measured after calibration of Frex and Peredox fluorescence in living cells expressing recombinant Frex and Peredox protein, respectively (Hung et al, 2011; Zhao et al, 2011). After transfection for 30 h, Panc-1 cells were harvested by trypsinization and were washed and resuspended in PBS. "
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    ABSTRACT: The malate-aspartate shuttle is indispensable for the net transfer of cytosolic NADH into mitochondria to maintain a high rate of glycolysis and to support rapid tumor cell growth. The malate-aspartate shuttle is operated by two pairs of enzymes that localize to the mitochondria and cytoplasm, glutamate oxaloacetate transaminases (GOT), and malate dehydrogenases (MDH). Here, we show that mitochondrial GOT2 is acetylated and that deacetylation depends on mitochondrial SIRT3. We have identified that acetylation occurs at three lysine residues, K159, K185, and K404 (3K), and enhances the association between GOT2 and MDH2. The GOT2 acetylation at these three residues promotes the net transfer of cytosolic NADH into mitochondria and changes the mitochondrial NADH/NAD(+) redox state to support ATP production. Additionally, GOT2 3K acetylation stimulates NADPH production to suppress ROS and to protect cells from oxidative damage. Moreover, GOT2 3K acetylation promotes pancreatic cell proliferation and tumor growth in vivo. Finally, we show that GOT2 K159 acetylation is increased in human pancreatic tumors, which correlates with reduced SIRT3 expression. Our study uncovers a previously unknown mechanism by which GOT2 acetylation stimulates the malate-aspartate NADH shuttle activity and oxidative protection. © 2015 The Authors.
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