Abstract
NADPH-dependent antioxidant pathways have a critical role in scavenging hydrogen peroxide (H2O2) produced by oxidative phosphorylation. Inadequate scavenging results in H2O2 accumulation and can cause disease. To measure NADPH/NADP+ redox states, we explored genetically encoded sensors based on steady-state fluorescence anisotropy due to FRET (fluorescence resonance energy transfer) between homologous fluorescent proteins (homoFRET); we refer to these sensors as Apollo sensors. We created an Apollo sensor for NADP+ (Apollo-NADP+) that exploits NADP+-dependent homodimerization of enzymatically inactive glucose-6-phosphate dehydrogenase (G6PD). This sensor is reversible, responsive to glucose-stimulated metabolism and spectrally tunable for compatibility with many other sensors. We used Apollo-NADP+ to study beta cells responding to oxidative stress and demonstrated that NADPH is significantly depleted before H2O2 accumulation by imaging a Cerulean-tagged version of Apollo-NADP+ with the H2O2 sensor HyPer.
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Change history
02 March 2016
In the version of this article initially published online on 15 February 2016, the first two sentences incorrectly referred to hydroxyl radicals rather than superoxide. The original version stated, "Oxidative phosphorylation generates ATP in eukaryotic cells but also produces toxic hydroxyl radicals as a byproduct. Cells protect themselves by converting hydroxyl radicals first to H2O2 with superoxide dismutase enzymes and then to water using NADPH and glutathione and/or thioredoxin enzymes." This has now been corrected to the following: "Oxidative phosphorylation generates ATP in eukaryotic cells but also produces superoxide radicals as a byproduct. Cells protect themselves by converting superoxide first to H2O2 with superoxide dismutase enzymes and then to water using NADPH and glutathione and/or thioredoxin enzymes." The error has been corrected for the print, PDF and HTML versions of the article.
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Acknowledgements
This work was supported by the Natural Sciences and Engineering Research Council of Canada (Discovery grant RGPIN-371705 to J.V.R.) and the Canada Foundation for Innovation Leaders Opportunity Fund (grant to J.V.R.). Stipend support was provided to W.D.C. by a Banting and Best Diabetes Centre (BBDC)–University Health Network Graduate Award through the University of Toronto Faculty of Medicine, BBDC. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, the Canada Foundation for Innovation, Genome Canada, GlaxoSmithKline, the Innovative Medicines Initiative, Janssen, Lilly Canada, Merck & Co., the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, the São Paulo Research Foundation, Takeda and the Wellcome Trust. We thank Y. Chen and P. Silva for technical assistance on the project. We also thank R. Stein (Vanderbilt University Medical Center, Nashville, Tennessee, USA) for providing β-TC3 cells.
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W.D.C., C.V.B. and J.V.R. conceived the experiments, interpreted the data and wrote the paper. W.D.C. and C.V.B. performed the experiments. A.H., P.L. and S.G. performed the protein purification. All authors commented on the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Effect of scattering on anisotropy controls.
(a-b) Fluorescence intensity cross-sections of purified Venus monomer in the presence of a (a) low scattering media or (b) high scattering media. Scale bar represents 50 μm. (c) Fold change in anisotropy of purified Venus monomer and tandem-dimer in either low or high scattering media. High scattering media data was normalized to low scattering baselines.
Supplementary Figure 2 Effect of diamide on Apollo-NADP+ intensity.
(a-b) Average 2P-fluorescent intensity in the parallel and perpendicular channels of βTC3 cells expressing (a) Venus-tagged Apollo-NADP+ (n = 144 cells) or (b) Venus-tagged R198P (n = 73 cells) before and after treatment with 5mM diamide. Total intensity was calculated as .
Supplementary Figure 3 Enzymatic inactivation of Apollo-NADP+ through site-directed mutagenesis.
(a-b) (a) Single point mutations were made in Venus-tagged WT-G6PD to the NADPH (1, S40A; and 2, R72Q) and G6P (3, H201N; 4,K205T) catalytic sites as well as to create two clinical point mutants (R198P-G6PD, Canton; and R459L-G6PD, Santiago). These constructs were subsequently imaged in βTC3 cells before (black bars) and after (hatched bars) addition of 5 mM diamide (n = 20-45). (b) Combinations of the single point mutations were imaged in βTC3 cells again before (black bars) and after (hatched bars) the addition of 5mM diamide (n = 7-19). The * indicates p<0.05 as determined using one-way ANOVA followed by Tukey test. (c-d) Initial enzymatic velocities of 1 ng/μL of the purified proteins in response to varying NADP+. The reactions were initiated by the addition of 2 mM G6P. (c) Comparison between initial velocities of Venus-tagged WT-G6PD (blue triangles), quadruple mutant-G6PD (black circles) and the R198P-G6PD (brown triangles). The Vmax values were calculated to be 174±12, 1.67±0.39, and 0.00±0.58 s-1 for the WT-G6PD, R198P-G6PD, and quadruple mutant-G6PD, respectively. (d) The initial rate of reaction of a mixture of 1 ng/μL WT-G6PD and 0, 1 and 4 ng/μL quadruple mutant-G6PD. The Michaelis-Menten Km constants were calculated to be 0.097±0.014, 0.095±0.015 and 0.113±0.010 mM, respectively.
Supplementary Figure 4 Effect of critical factors on the NADP+ dose response curve.
(a) Steady-state fluorescence anisotropy of purified (50 ng/μL) Venus-tagged Apollo-NADP+, Wild-Type G6PD, and R198P-G6PD as well as Venus monomer and tandem dimer in the presence of 0.1 to 100 μM NADP+ (n = 6). (b-c) Steady-state fluorescence anisotropy of purified Apollo-NADP+ (50 ng/μL) in the presence of 0.01 to 1000 μM NADP+ and (b) 1mM of critical metabolites (n = 10) or (c) variable pH (n = 10). Hill fits were performed on the curves and no significant difference was found between the Hill coefficients in the presence of these factors (NT: 0.917±.046, G6P: 0.997±.073, ATP: 1.102±.070, NADH: 0.896±.064, NAD+: 0.930±.075) or by altering pH (7:1.050±.044, 7.5: 0.969±.064, 8.0: 1.113±.077). Hill coefficients are presented as mean±standard deviation.
Supplementary Figure 6 Hydrogen peroxide dose response curve.
(a-c) H2O2 dose response measured using (a) Apollo-NADP+ in the presence of high (15mM – black) or low (1mM – blue) glucose (n = 3 samples, 49-68 cells). Controls used were R198P-G6PD (54 cells) and diamide-treated cells (145-cells) as indicated. (b) NAD(P)H autofluorescence (1mM glucose, n = 3 samples, 135 cells) and (c) an NADP+/NADPH biochemical assay in high (15mM) glucose (n = 3 samples). The * indicates p<0.05 relative to the baseline values (combined 0μM and 0.1μM H2O2).
Supplementary Figure 7 Effect of HyPer on the Apollo-NADP+ response.
(a-c) Temporal response of Apollo-NADP+ to a bolus of 20 μM H2O2. (a) Representative curves of two cells co-expressing Apollo-NADP+ with either high or low levels of HyPer (peak intensities 280/85 A.U., respectively). (b) Fluorescence image of HyPer at peak fluorescence intensity. (c) 2P steady-state anisotropy image of these cells at that time-point. (d-f) Temporal response of Apollo-NADP+ to a bolus of 0.25 mM diamide. (d) Representative curves of two cells co-expressing Apollo-NADP+ and either high or low levels of HyPer (peak intensities 200/70 A.U., respectively). (e) Fluorescence image of HyPer at peak fluorescence intensity. (f) 2P steady-state anisotropy image of these cells at that timepoint. Scale bars represent 10 μm.
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Cameron, W., Bui, C., Hutchinson, A. et al. Apollo-NADP+: a spectrally tunable family of genetically encoded sensors for NADP+. Nat Methods 13, 352–358 (2016). https://doi.org/10.1038/nmeth.3764
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DOI: https://doi.org/10.1038/nmeth.3764
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