news and views
Andreas Wiederkehr & Nicolas Demaurex
Genetically encoded NAD and NADP sensors will revolutionize the study of redox biology. Most metabolic pathways depend on either of the two important cofactors, nicotinamide adenine dinucleotide (NAD+) or nicotinamide
a
adenine dinucleotide phosphate (NADP+), that exist as redox couples with their corresponding reduced forms, NADH and NADPH.
NAD+
NADP+
O
O NH2
NH2
+ N
O
+ N
O
NADH
NADPH NH2
O NH2 N
O
P
N
O–
O– O
O HO
NH2 O
N
O
P
NH2
N
O
O
N
N
O
P
P
O
O HO
OH
HO
N
O–
O– O
O
OH
N
O
O
O
OH
HO
O
O P O–
482 nm
Rex + + +
NADP+
+ + Rex cpY FP
407 nm
+
FP
Rex + 407 nm 482 nm
cp
YF
NADPH
P
528 nm
iNap
iNap
O–
+ + NADPH + Rex
+ +
528 nm
Debbie Maizels/Springer Nature
b
cpY
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
Illuminating redox biology using NADH- and NADPH-specific sensors
P
YF
cp
Figure 1 | Sensing changes in the NADP+/NADPH ratio using the genetically encoded sensor iNap. (a) Chemical structure of the cofactors NAD and NADP in their reduced and oxidized forms. Blue circles indicate the functional group of the cofactor in its reduced and oxidized form; green circle indicates the phosphate group that distinguishes NAD from NADP. (b) Conformational changes in iNap following NADPH binding change the spectral properties of cpYFP (alterations in cpYFP indicated by shape changes). Andreas Wiederkehr is in the Mitochondrial Function group at the Nestlé Institute of Health Sciences, Lausanne, Switzerland, and Molecular Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. Nicolas Demaurex is in the Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland. e-mail: [email protected]
Alterations in the NAD+/NADH or NADP+/ NADPH ratios reflect changes in the redox status of cells or tissues. Such changes have also been found to be associated with pathological conditions and aging1–3. In this issue, Tao et al.4 describe the development and applications of a novel fluorescent indicator protein for NADPH, iNAP, to selectively follow kinetic changes of the NADP+/NADPH ratio in situ. Glycolysis and mitochondrial oxidative metabolism reduce NAD+ to NADH, which is used as an electron donor by the mitochondrial respiratory chain to initiate mitochondrial ATP synthesis. NADPH is regenerated from NADP+ by the pentose phosphate pathway2,4 and used, for example, in the reduction of glutathione, thioredoxin and peroxiredoxin as part of the cellular antioxidant system. Biochemical analytical methods do not capture the transient intracellular redox changes associated with metabolic activation or dysfunction. Therefore, cell biologists rely on the autofluorescence of NADH and NADPH to obtain kinetic information on the intracellular redox state of these cofactors. Unfortunately, the fluorescence spectra of NADH and NADPH are identical, and autofluorescence imaging fails to discriminate the two cofactors. Structurally, NAD and NADP differ only in a phosphate group at position 2′ of the ribose carrying the adenine group in NADP (Fig. 1). Yet most enzymes show high selectivity for either NAD or NADP, enabling these two cofactors to regulate different biochemical reactions. Specific probes for NAD and NADP are highly desirable, as these cofactors are involved in distinct biological processes and catalyze different enzymatic reactions. Today, only one fluorescent sensor designed to follow intracellular NADP+ concentrations has been described2. The Apollo-NADP+ sensor measures a reduction of fluorescence aniso tropy polarization due to NADP+-dependent homodimerization of a fluorescent glucose-6phosphate dehydrogenase fusion protein2. However, a number of genetically encoded sensors measuring NADH or the related NAD+/NADH ratio have been developed5. These probes are derived from Rex, a bacterial transcriptional repressor and natural NAD+/ NADH sensor. Rex alternatively binds either one molecule of NAD+ or two molecules of nature methods | VOL.14 NO.7 | JULY 2017 | 671
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
news and views NADH. Binding of NADH causes a conformational change that results in the release of Rex from its specific promoter regions. In the NAD+/NADH sensor SoNar, the nucleotidebinding domain of Rex from Thermus aquaticus (T-Rex) is fused to a circularly permutated YFP (cpYFP). Conformational changes of the T-Rex domain alter the chromophore microenvironment of cpYFP and thereby the spectral properties of SoNar. Interestingly, binding of NADH and NAD+ have opposite effects on SoNar fluorescence emission; as a result large ratiometric responses to changes in the NAD+/ NADH redox state are recorded. To develop iNap, Tao et al.4 converted the bright ratiometric NAD+/NADH-selective probe SoNar into a specific NADP+/NADPH sensor (Fig. 1). Applying the structural informatics tool CPASS (comparison of protein active site structures) to compare a large collection of proteins binding either NAD or NADP, the researchers established structural rules for proteins to accommodate either NAD or NADP. Based on these rules, SoNar mutants with switched cofactor selectivity were engineered. Several iNap variants with different affinities for NADPH that retained the spectral properties of SoNar were created. NADPH binding to iNAP sensors caused a nine-fold ratiometric fluorescence change when the probe was excited with 420 and 485 nm light. Importantly, the iNaps are NADP+/NADPH specific, insensitive to NADH and other nucleotides. The authors demonstrate the usefulness of iNap to study redox changes in situ. They confirm earlier results, which showed that NAD+ kinase is a key enzyme for modulating cytosolic
672 | VOL.14 NO.7| JULY 2017 | nature methods
NADPH levels4,6 and, interestingly, also the mitochondrial NADPH pool4. Such clear demonstration of organelle-specific NADPH responses has only become possible through the development of genetically encoded, compartment-specific, fluorescent-based probes. In vitro, rapid and transient increases of the NADP+/NADPH ratio in response to oxidants were observed. These time-resolved recordings highlight the robustness of the cellular NADP+/NADPH redox system in its ability to counteract oxidative challenges. In zebrafish, iNap was used to monitor kinetic changes of the NADP+/NADPH redox state in response to a tail fin cut4. Previous work from the same group showed pyruvate-induced changes of NAD+/NADH in nude mice carrying tumor cells expressing SoNar7. Therefore, iNap and SoNar can be applied successfully for kinetic studies of redox changes in vivo. With these new tools, many unresolved questions regarding the regulation and physiological role of different redox couples can be directly addressed. Highlighting the potential of the new GSH/thiol sensors roGFP and rxYFP to study glutathione and thiol redox dynamics, Schwarzländer et al.8 commented in a recent review, “the best we have been able to achieve is the measurement of defined redox couples in whole cell lysates. By analogy, this situation is comparable to trying to understand calcium and kinase signaling by measuring the total calcium and phosphate level of the cell.” Although this statement may be provocative, biochemical studies of NAD and NADP likely have similar limitations. The opportunities that lie ahead of us with the newly developed
NAD+/NADH and NADP+/NADPH sensors may be equally promising. The recently developed, genetically encoded sensors for NAD+/ NADH (Frex, Peredox, RexYFP and SoNar) as well as for NADP+/NADPH (Apollo-NADP+ and iNap) should lead to a much better understanding of the kinetic changes of these parameters in situ2,4,5,7. With these new probes, we will enter a new era where the monitoring of transient perturbations of the redox network in response to physiologically relevant nutrient changes, bioactives and stressors becomes possible. We expect that these sensors will revolutionize the study of redox biology as much as fluorescent probes revolutionized the study of calcium signaling. Which of these probes will stand the test of time is hard to foresee. iNAP has good chances of doing so, as its selectivity and spectral properties make it an optimal sensor reporting on the intracellular NADP+/NADPH redox balance. Despite the name iNap, this probe will likely wake up scientists and illuminate NADP+/NADPH redox biology. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Ghosh, D., Levault, K.R. & Brewer, G.J. Aging Cell 13, 631–640 (2014). 2. Cameron, W.D. et al. Nat. Methods 13, 352–358 (2016). 3. Wu, J., Jin, Z. & Yan, L.J. Redox Biol. 11, 51–59 (2017). 4. Tao, R. et al. Nat. Methods 14, 720–728 (2017). 5. Bilan, D.S. & Belousov, V.V. Free Radic. Biol. Med. 100, 32–42 (2016). 6. Pollak, N., Niere, M. & Ziegler, M. J. Biol. Chem. 282, 33562–33571 (2007). 7. Zhao, Y. et al. Cell Metab. 21, 777–789 (2015). 8. Schwarzländer, M., Dick, T.P., Meyer, A.J. & Morgan, B. Antioxid. Redox Signal. 24, 680–712 (2016).
Andreas Wiederkehr & Nicolas Demaurex
Genetically encoded NAD and NADP sensors will revolutionize the study of redox biology. Most metabolic pathways depend on either of the two important cofactors, nicotinamide adenine dinucleotide (NAD+) or nicotinamide
a
adenine dinucleotide phosphate (NADP+), that exist as redox couples with their corresponding reduced forms, NADH and NADPH.
NAD+
NADP+
O
O NH2
NH2
+ N
O
+ N
O
NADH
NADPH NH2
O NH2 N
O
P
N
O–
O– O
O HO
NH2 O
N
O
P
NH2
N
O
O
N
N
O
P
P
O
O HO
OH
HO
N
O–
O– O
O
OH
N
O
O
O
OH
HO
O
O P O–
482 nm
Rex + + +
NADP+
+ + Rex cpY FP
407 nm
+
FP
Rex + 407 nm 482 nm
cp
YF
NADPH
P
528 nm
iNap
iNap
O–
+ + NADPH + Rex
+ +
528 nm
Debbie Maizels/Springer Nature
b
cpY
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
Illuminating redox biology using NADH- and NADPH-specific sensors
P
YF
cp
Figure 1 | Sensing changes in the NADP+/NADPH ratio using the genetically encoded sensor iNap. (a) Chemical structure of the cofactors NAD and NADP in their reduced and oxidized forms. Blue circles indicate the functional group of the cofactor in its reduced and oxidized form; green circle indicates the phosphate group that distinguishes NAD from NADP. (b) Conformational changes in iNap following NADPH binding change the spectral properties of cpYFP (alterations in cpYFP indicated by shape changes). Andreas Wiederkehr is in the Mitochondrial Function group at the Nestlé Institute of Health Sciences, Lausanne, Switzerland, and Molecular Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. Nicolas Demaurex is in the Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland. e-mail: [email protected]
Alterations in the NAD+/NADH or NADP+/ NADPH ratios reflect changes in the redox status of cells or tissues. Such changes have also been found to be associated with pathological conditions and aging1–3. In this issue, Tao et al.4 describe the development and applications of a novel fluorescent indicator protein for NADPH, iNAP, to selectively follow kinetic changes of the NADP+/NADPH ratio in situ. Glycolysis and mitochondrial oxidative metabolism reduce NAD+ to NADH, which is used as an electron donor by the mitochondrial respiratory chain to initiate mitochondrial ATP synthesis. NADPH is regenerated from NADP+ by the pentose phosphate pathway2,4 and used, for example, in the reduction of glutathione, thioredoxin and peroxiredoxin as part of the cellular antioxidant system. Biochemical analytical methods do not capture the transient intracellular redox changes associated with metabolic activation or dysfunction. Therefore, cell biologists rely on the autofluorescence of NADH and NADPH to obtain kinetic information on the intracellular redox state of these cofactors. Unfortunately, the fluorescence spectra of NADH and NADPH are identical, and autofluorescence imaging fails to discriminate the two cofactors. Structurally, NAD and NADP differ only in a phosphate group at position 2′ of the ribose carrying the adenine group in NADP (Fig. 1). Yet most enzymes show high selectivity for either NAD or NADP, enabling these two cofactors to regulate different biochemical reactions. Specific probes for NAD and NADP are highly desirable, as these cofactors are involved in distinct biological processes and catalyze different enzymatic reactions. Today, only one fluorescent sensor designed to follow intracellular NADP+ concentrations has been described2. The Apollo-NADP+ sensor measures a reduction of fluorescence aniso tropy polarization due to NADP+-dependent homodimerization of a fluorescent glucose-6phosphate dehydrogenase fusion protein2. However, a number of genetically encoded sensors measuring NADH or the related NAD+/NADH ratio have been developed5. These probes are derived from Rex, a bacterial transcriptional repressor and natural NAD+/ NADH sensor. Rex alternatively binds either one molecule of NAD+ or two molecules of nature methods | VOL.14 NO.7 | JULY 2017 | 671
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
news and views NADH. Binding of NADH causes a conformational change that results in the release of Rex from its specific promoter regions. In the NAD+/NADH sensor SoNar, the nucleotidebinding domain of Rex from Thermus aquaticus (T-Rex) is fused to a circularly permutated YFP (cpYFP). Conformational changes of the T-Rex domain alter the chromophore microenvironment of cpYFP and thereby the spectral properties of SoNar. Interestingly, binding of NADH and NAD+ have opposite effects on SoNar fluorescence emission; as a result large ratiometric responses to changes in the NAD+/ NADH redox state are recorded. To develop iNap, Tao et al.4 converted the bright ratiometric NAD+/NADH-selective probe SoNar into a specific NADP+/NADPH sensor (Fig. 1). Applying the structural informatics tool CPASS (comparison of protein active site structures) to compare a large collection of proteins binding either NAD or NADP, the researchers established structural rules for proteins to accommodate either NAD or NADP. Based on these rules, SoNar mutants with switched cofactor selectivity were engineered. Several iNap variants with different affinities for NADPH that retained the spectral properties of SoNar were created. NADPH binding to iNAP sensors caused a nine-fold ratiometric fluorescence change when the probe was excited with 420 and 485 nm light. Importantly, the iNaps are NADP+/NADPH specific, insensitive to NADH and other nucleotides. The authors demonstrate the usefulness of iNap to study redox changes in situ. They confirm earlier results, which showed that NAD+ kinase is a key enzyme for modulating cytosolic
672 | VOL.14 NO.7| JULY 2017 | nature methods
NADPH levels4,6 and, interestingly, also the mitochondrial NADPH pool4. Such clear demonstration of organelle-specific NADPH responses has only become possible through the development of genetically encoded, compartment-specific, fluorescent-based probes. In vitro, rapid and transient increases of the NADP+/NADPH ratio in response to oxidants were observed. These time-resolved recordings highlight the robustness of the cellular NADP+/NADPH redox system in its ability to counteract oxidative challenges. In zebrafish, iNap was used to monitor kinetic changes of the NADP+/NADPH redox state in response to a tail fin cut4. Previous work from the same group showed pyruvate-induced changes of NAD+/NADH in nude mice carrying tumor cells expressing SoNar7. Therefore, iNap and SoNar can be applied successfully for kinetic studies of redox changes in vivo. With these new tools, many unresolved questions regarding the regulation and physiological role of different redox couples can be directly addressed. Highlighting the potential of the new GSH/thiol sensors roGFP and rxYFP to study glutathione and thiol redox dynamics, Schwarzländer et al.8 commented in a recent review, “the best we have been able to achieve is the measurement of defined redox couples in whole cell lysates. By analogy, this situation is comparable to trying to understand calcium and kinase signaling by measuring the total calcium and phosphate level of the cell.” Although this statement may be provocative, biochemical studies of NAD and NADP likely have similar limitations. The opportunities that lie ahead of us with the newly developed
NAD+/NADH and NADP+/NADPH sensors may be equally promising. The recently developed, genetically encoded sensors for NAD+/ NADH (Frex, Peredox, RexYFP and SoNar) as well as for NADP+/NADPH (Apollo-NADP+ and iNap) should lead to a much better understanding of the kinetic changes of these parameters in situ2,4,5,7. With these new probes, we will enter a new era where the monitoring of transient perturbations of the redox network in response to physiologically relevant nutrient changes, bioactives and stressors becomes possible. We expect that these sensors will revolutionize the study of redox biology as much as fluorescent probes revolutionized the study of calcium signaling. Which of these probes will stand the test of time is hard to foresee. iNAP has good chances of doing so, as its selectivity and spectral properties make it an optimal sensor reporting on the intracellular NADP+/NADPH redox balance. Despite the name iNap, this probe will likely wake up scientists and illuminate NADP+/NADPH redox biology. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Ghosh, D., Levault, K.R. & Brewer, G.J. Aging Cell 13, 631–640 (2014). 2. Cameron, W.D. et al. Nat. Methods 13, 352–358 (2016). 3. Wu, J., Jin, Z. & Yan, L.J. Redox Biol. 11, 51–59 (2017). 4. Tao, R. et al. Nat. Methods 14, 720–728 (2017). 5. Bilan, D.S. & Belousov, V.V. Free Radic. Biol. Med. 100, 32–42 (2016). 6. Pollak, N., Niere, M. & Ziegler, M. J. Biol. Chem. 282, 33562–33571 (2007). 7. Zhao, Y. et al. Cell Metab. 21, 777–789 (2015). 8. Schwarzländer, M., Dick, T.P., Meyer, A.J. & Morgan, B. Antioxid. Redox Signal. 24, 680–712 (2016).
