Genetically encoded FRET-based nanosensor for in vivo measurement of leucine.

Besides fundamental role in protein synthesis, leucine has metabolic roles as energy substrates, precursors for synthesis of other amino acids and as a modulator of muscle protein synthesis via the insulin-signaling pathway. Leucine concentration in cell and tissue is temporally dynamic as the metabolism of leucine is regulated through multiple enzymes and transporters. Assessment of cell-type specific activities of transporters and enzymes by physical fractionation is extremely challenging. Therefore, a method of reporting leucine dynamics at the cellular level is highly desirable. Given this, we developed a series of genetically encoded nanosensors for real-time in vivo measurement of leucine at cellular level. A leucine binding periplasmic binding protein (LivK) of Escherichia coli K12 was flanked with CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) at N-terminus and C-terminus, respectively. The constructed nanosensors allowed in vitro determination of fluorescence resonance energy transfer (FRET) changes in a concentration-dependent manner. These sensors were found to be specific to leucine, and stable to pH-changes within a physiological range. Genetically encoded sensors can be targeted to a specific cell type, and allow dynamic measurement of leucine concentration in bacterial and yeast cells.

[1]  S. Kimball,et al.  Regulation of protein synthesis by branched-chain amino acids , 2001, Current opinion in clinical nutrition and metabolic care.

[2]  A. Burkovski,et al.  Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applications , 2002, Applied Microbiology and Biotechnology.

[3]  W. Frommer,et al.  Optical sensors for measuring dynamic changes of cytosolic metabolite levels in yeast , 2011, Nature Protocols.

[4]  A. Lesk,et al.  Structural mechanisms for domain movements in proteins. , 1994, Biochemistry.

[5]  S. Brul,et al.  In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth. , 2009, Microbiology.

[6]  D. Evanko,et al.  Elimination of environmental sensitivity in a cameleon FRET-based calcium sensor via replacement of the acceptor with Venus. , 2005, Cell calcium.

[7]  L. Looger,et al.  Fluorescence resonance energy transfer sensors for quantitative monitoring of pentose and disaccharide accumulation in bacteria , 2008, Biotechnology for biofuels.

[8]  M. Buse,et al.  Leucine. A possible regulator of protein turnover in muscle. , 1975, The Journal of clinical investigation.

[9]  U. Magnusson,et al.  X-ray Structures of the Leucine-binding Protein Illustrate Conformational Changes and the Basis of Ligand Specificity* , 2004, Journal of Biological Chemistry.

[10]  M. Buse,et al.  In vitro effect of branched chain amino acids on the ribosomal cycle in muscles of fasted rats. , 1979, Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme.

[11]  D. Piston,et al.  Fluorescent protein FRET: the good, the bad and the ugly. , 2007, Trends in biochemical sciences.

[12]  L. Jefferson,et al.  Influence of amino acid availability on protein turnover in perfused skeletal muscle. , 1978, Biochimica et biophysica acta.

[13]  Alexander M. Jones,et al.  In vivo biochemistry: quantifying ion and metabolite levels in individual cells or cultures of yeast. , 2011, The Biochemical journal.

[14]  R. Tsien,et al.  Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin , 1997, Nature.

[15]  Marcus Fehr,et al.  In Vivo Imaging of the Dynamics of Glucose Uptake in the Cytosol of COS-7 Cells by Fluorescent Nanosensors* , 2003, Journal of Biological Chemistry.

[16]  L. Deldicque,et al.  ER Stress Induces Anabolic Resistance in Muscle Cells through PKB-Induced Blockade of mTORC1 , 2011, PloS one.

[17]  D. Sabatini,et al.  Defective regulation of autophagy upon leucine deprivation reveals a targetable liability of human melanoma cells in vitro and in vivo. , 2011, Cancer cell.

[18]  Elizabeth A Jares-Erijman,et al.  Imaging molecular interactions in living cells by FRET microscopy. , 2006, Current opinion in chemical biology.

[19]  S. Rapoport,et al.  Kinetics of Neutral Amino Acid Transport Across the Blood‐Brain Barrier , 1987, Journal of neurochemistry.

[20]  R. Boileau,et al.  Oat, wheat or corn cereal ingestion before exercise alters metabolism in humans. , 1996, The Journal of nutrition.

[21]  Igor L. Medintz,et al.  Maltose-binding protein: a versatile platform for prototyping biosensing. , 2006, Current opinion in biotechnology.

[22]  U. Ludewig,et al.  Visualization of Arginine Influx into Plant Cells Using a Specific FRET-sensor , 2007, Journal of Fluorescence.

[23]  S. Kimball,et al.  Orally administered leucine stimulates protein synthesis in skeletal muscle of postabsorptive rats in association with increased eIF4F formation. , 2000, The Journal of nutrition.

[24]  W. Frommer,et al.  Minimally invasive dynamic imaging of ions and metabolites in living cells. , 2004, Current opinion in plant biology.

[25]  L. Looger,et al.  Nanosensor Detection of an Immunoregulatory Tryptophan Influx/Kynurenine Efflux Cycle , 2007, PLoS biology.

[26]  L. Looger,et al.  A novel analytical method for in vivo phosphate tracking , 2006, FEBS letters.

[27]  Roger Y. Tsien,et al.  Spatiotemporal dynamics of guanosine 3′,5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[28]  L. Looger,et al.  Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[29]  W. Frommer,et al.  Development of a fluorescent nanosensor for ribose , 2003, FEBS letters.

[30]  M. Bixel,et al.  Generation of Ketone Bodies from Leucine by Cultured Astroglial Cells , 1995, Journal of neurochemistry.

[31]  Daniel Raftery,et al.  Quantitative Metabolomics by 1H-NMR and LC-MS/MS Confirms Altered Metabolic Pathways in Diabetes , 2010, PloS one.

[32]  L L Looger,et al.  Development and use of fluorescent nanosensors for metabolite imaging in living cells. , 2005, Biochemical Society transactions.

[33]  V. Grill,et al.  Brain uptake and release of amino acids in nondiabetic and insulin-dependent diabetic subjects: important role of glutamine release for nitrogen balance. , 1992, Metabolism: clinical and experimental.

[34]  Marcus Fehr,et al.  Visualization of maltose uptake in living yeast cells by fluorescent nanosensors , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[35]  L. Looger,et al.  Construction of a fluorescent biosensor family , 2002, Protein science : a publication of the Protein Society.

[36]  Igor L. Medintz,et al.  A Reagentless Biosensing Assembly Based on Quantum Dot–Donor Förster Resonance Energy Transfer , 2005 .

[37]  R. Tsien,et al.  Creating new fluorescent probes for cell biology , 2002, Nature Reviews Molecular Cell Biology.

[38]  S. Okumoto,et al.  Visualization of Glutamine Transporter Activities in Living Cells Using Genetically Encoded Glutamine Sensors , 2012, PloS one.