Apollo-NADP(+): A Spectrally Tunable Family of Genetically Encoded Sensors for NADP(+)

Nat Methods. 2016 Apr;13(4):352-8. doi: 10.1038/nmeth.3764. Epub 2016 Feb 15.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biosensing Techniques / methods*
  • Cells, Cultured
  • Fluorescence Polarization / methods
  • Fluorescence Resonance Energy Transfer
  • Glucosephosphate Dehydrogenase / chemistry
  • Glucosephosphate Dehydrogenase / genetics
  • Glucosephosphate Dehydrogenase / metabolism*
  • Humans
  • Hydrogen Peroxide / metabolism
  • Image Processing, Computer-Assisted
  • Insulin-Secreting Cells / metabolism*
  • NADP / chemistry*
  • NADP / metabolism
  • Oxidants / metabolism
  • Oxidative Stress
  • Protein Conformation

Substances

  • Oxidants
  • NADP
  • Hydrogen Peroxide
  • Glucosephosphate Dehydrogenase