title: Apollo-NADP(+): a spectrally tunable family of genetically encoded sensors for NADP(+).
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.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.|Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada.
authors.forename: William D
authors.initials: WD
authors.lastname: Cameron
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.|Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada.
authors.forename: Cindy V
authors.initials: CV
authors.lastname: Bui
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Ashley
authors.initials: A
authors.lastname: Hutchinson
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Peter
authors.initials: P
authors.lastname: Loppnau
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Susanne
authors.initials: S
authors.lastname: Gräslund
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.|Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada.|Banting and Best Diabetes Centre, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Jonathan V
authors.initials: JV
authors.lastname: Rocheleau
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002478:Cells, Cultured; D005454:Fluorescence Polarization / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism; D018384:Oxidative Stress; D011487:Protein Conformation
chemical_list: D016877:Oxidants; D009249:NADP; D006861:Hydrogen Peroxide; D005954:Glucosephosphate Dehydrogenase