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ScienceDirect Profiling metabolic states with genetically encoded fluorescent biosensors for NADH Yuzheng Zhao1,2 and Yi Yang1,2 NADH and its oxidized form, NAD+, play central roles in energy metabolism and are ideal indicators of cellular metabolic states. In this review, we will introduce recent progress made in the developing of a series of genetically encoded NADH sensors, which offer the potential to fill the gap in currently used techniques of endogenous NAD(P)H fluorescence imaging. These sensors are bright, specific and organelles targetable, allowing real-time tracking and quantification of intracellular NADH levels in different subcellular compartments. The individual strengths and weaknesses of these sensors when applied to the study of metabolic states profiling will be also discussed. Addresses 1 Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai, China 2 Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China Corresponding author: Yang, Yi ([email protected], [email protected])
Current Opinion in Biotechnology 2015, 31:86–92 This review comes from a themed issue on Analytical biotechnology Edited by Hadley D Sikes & Nicola Zamboni
surrogate marker for human IDH1 and IDH2-mutated glioma and acute myeloid leukemia [5,6]. The spatiotemporal patterns of metabolite levels in living cells are pivotal in understanding signal transduction and metabolite flux in physiological and pathological conditions. Usually, measurement of metabolites is based on enzymatic cycling assay, chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. Recently, metabolomic analyses became popular for quantitating the entire spectrum of small molecules in biological samples in order to assess global metabolic state or perturbations [7], however, such analyses provide only static information of a population of cells and need sample destruction. In this respect, fluorescent imaging with metabolite sensors may be used to profile metabolic states of living cells in real-time and with single-cell or even subcellular resolution. Compare to chemical probes for metabolite, genetically encoded metabolite sensors based on fluorescent proteins have significant advantages. First, they can be genetically introduced into any cell or organism, and targeted to any subcellular localization. Second, they can be conveniently engineered to fluoresce of different colors, or different sensitivities. In this review, we focused mainly on sensors for nicotinamide adenine dinucleotide (NADH), the most important metabolite in redox metabolism.
http://dx.doi.org/10.1016/j.copbio.2014.08.007
Genetically encoded metabolite sensors
0958-1669/# 2014 Elsevier Ltd. All right reserved.
In nature, members of the bacterial transcription regulatory protein and periplasmic binding protein families bind their substrates with high affinity and specificity, exhibiting significant conformational changes [8–13]. Genetically encoded sensors [14,15] were created by fusing these proteins with fluorescent protein (FP), which transforms conformational changes caused by ligand binding into changes of fluorescence. A varieties of genetically encoded sensors were created to sense key metabolites involved in energy and redox metabolic processes, including NADH [16,17,18], ATP [10,19,20], glucose [21–24], glutamine [25,26], glutamate [12,21,27,28], leucine [29], lactate [30], pyruvate [31], and 2-oxogluatarate [32,33] (Table 1). These sensors are categorized into two categories, namely, Foster resonance energy transfer (FRET) based sensors and single FP sensors. FRETbased sensors contain donor and acceptor fluorescent proteins, and the FRET efficiency between these proteins changes upon ligand binding [34]. In single FP sensors, ligand-binding results in a change local environment of its chromophore, shifting the fluorescence intensity and/or
Introduction Many studies have demonstrated evidence that metabolic disorders are closely related to the occurrence of many diseases, such as cancer, diabetes, obesity, dyslipidemia, and hypercholesterolemia. Thus, determination of metabolite levels is relevant to pathogenesis, diagnosis, monitoring, and possible treatment of such diseases. For example, cancer cells shift energy metabolism from mitochondrial oxidative phosphorylation to aerobic glycolysis, characterized by increased uptake of glucose and accumulation of large amounts of lactate [1–3]. These have become the basis for the clinical detection of tumors by 18F-fluorodeoxyglucose positron emission tomography imaging [4]. Another key metabolite, 2-hydroxyglutarate, could be used as a suitable Current Opinion in Biotechnology 2015, 31:86–92
www.sciencedirect.com
Metabolite sensing, NADH Zhao and Yang 87
Table 1 Fluorescent biosensors for key metabolite in living cells Sensors
Kd for substrate
Dynamic range (%)
NADH and NAD+/NADH ratio sensors Frex-Cyt a 3.7 mM 11 mM c FrexH a 0.04 mM C3L194K a 50 mM c P0 b
ScienceDirect Profiling metabolic states with genetically encoded fluorescent biosensors for NADH Yuzheng Zhao1,2 and Yi Yang1,2 NADH and its oxidized form, NAD+, play central roles in energy metabolism and are ideal indicators of cellular metabolic states. In this review, we will introduce recent progress made in the developing of a series of genetically encoded NADH sensors, which offer the potential to fill the gap in currently used techniques of endogenous NAD(P)H fluorescence imaging. These sensors are bright, specific and organelles targetable, allowing real-time tracking and quantification of intracellular NADH levels in different subcellular compartments. The individual strengths and weaknesses of these sensors when applied to the study of metabolic states profiling will be also discussed. Addresses 1 Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai, China 2 Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China Corresponding author: Yang, Yi ([email protected], [email protected])
Current Opinion in Biotechnology 2015, 31:86–92 This review comes from a themed issue on Analytical biotechnology Edited by Hadley D Sikes & Nicola Zamboni
surrogate marker for human IDH1 and IDH2-mutated glioma and acute myeloid leukemia [5,6]. The spatiotemporal patterns of metabolite levels in living cells are pivotal in understanding signal transduction and metabolite flux in physiological and pathological conditions. Usually, measurement of metabolites is based on enzymatic cycling assay, chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. Recently, metabolomic analyses became popular for quantitating the entire spectrum of small molecules in biological samples in order to assess global metabolic state or perturbations [7], however, such analyses provide only static information of a population of cells and need sample destruction. In this respect, fluorescent imaging with metabolite sensors may be used to profile metabolic states of living cells in real-time and with single-cell or even subcellular resolution. Compare to chemical probes for metabolite, genetically encoded metabolite sensors based on fluorescent proteins have significant advantages. First, they can be genetically introduced into any cell or organism, and targeted to any subcellular localization. Second, they can be conveniently engineered to fluoresce of different colors, or different sensitivities. In this review, we focused mainly on sensors for nicotinamide adenine dinucleotide (NADH), the most important metabolite in redox metabolism.
http://dx.doi.org/10.1016/j.copbio.2014.08.007
Genetically encoded metabolite sensors
0958-1669/# 2014 Elsevier Ltd. All right reserved.
In nature, members of the bacterial transcription regulatory protein and periplasmic binding protein families bind their substrates with high affinity and specificity, exhibiting significant conformational changes [8–13]. Genetically encoded sensors [14,15] were created by fusing these proteins with fluorescent protein (FP), which transforms conformational changes caused by ligand binding into changes of fluorescence. A varieties of genetically encoded sensors were created to sense key metabolites involved in energy and redox metabolic processes, including NADH [16,17,18], ATP [10,19,20], glucose [21–24], glutamine [25,26], glutamate [12,21,27,28], leucine [29], lactate [30], pyruvate [31], and 2-oxogluatarate [32,33] (Table 1). These sensors are categorized into two categories, namely, Foster resonance energy transfer (FRET) based sensors and single FP sensors. FRETbased sensors contain donor and acceptor fluorescent proteins, and the FRET efficiency between these proteins changes upon ligand binding [34]. In single FP sensors, ligand-binding results in a change local environment of its chromophore, shifting the fluorescence intensity and/or
Introduction Many studies have demonstrated evidence that metabolic disorders are closely related to the occurrence of many diseases, such as cancer, diabetes, obesity, dyslipidemia, and hypercholesterolemia. Thus, determination of metabolite levels is relevant to pathogenesis, diagnosis, monitoring, and possible treatment of such diseases. For example, cancer cells shift energy metabolism from mitochondrial oxidative phosphorylation to aerobic glycolysis, characterized by increased uptake of glucose and accumulation of large amounts of lactate [1–3]. These have become the basis for the clinical detection of tumors by 18F-fluorodeoxyglucose positron emission tomography imaging [4]. Another key metabolite, 2-hydroxyglutarate, could be used as a suitable Current Opinion in Biotechnology 2015, 31:86–92
www.sciencedirect.com
Metabolite sensing, NADH Zhao and Yang 87
Table 1 Fluorescent biosensors for key metabolite in living cells Sensors
Kd for substrate
Dynamic range (%)
NADH and NAD+/NADH ratio sensors Frex-Cyt a 3.7 mM 11 mM c FrexH a 0.04 mM C3L194K a 50 mM c P0 b
