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NADPH Oxidases pp 259-274 | Cite as

Visualization of Intracellular Hydrogen Peroxide with the Genetically Encoded Fluorescent Probe HyPer in NIH-3T3 Cells

  • Yulia G. Ermakova
  • Nataliya M. Mishina
  • Carsten Schultz
  • Vsevolod V. BelousovEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)

Abstract

Reactive oxygen species (ROS) are involved in regulating normal physiological cell functions as second messengers as well as nonspecific damage of biomolecules in a pathological process known as oxidative stress. The HyPer family of genetically encoded probes are a useful noninvasive tool for monitoring the real-time dynamics of ROS in individual cells or model organisms. HyPer, the first genetically encoded probe for detection of hydrogen peroxide (H2O2), is oxidized with high specificity and sensitivity by H2O2, leading to ratiometric changes in the fluorescence excitation spectrum of the probe. These changes can be detected with a wide range of commercial confocal and wide-field microscope systems. Here we describe a detailed protocol for ratiometric monitoring of H2O2 produced by D-amino acid oxidase (DAAO) or by NADPH oxidase (NOX) in NIH-3T3 cells using the HyPer probe.

Key words

ROS Hydrogen peroxide HyPer HyPerRed PDGF D-amino acid oxidase DAAO Ratiometric imaging Microscopy 

Notes

Acknowledgment

This work was supported by the Russian Science Foundation grant 17-14-01086 (experiments with D-amino acid oxidase), Russian Foundation for Basic Research grant 18-54-74003 (image processing and PDGF experiments), and DFG IRTG 1816. YGE received Sergey Shpiz fellowship. Experiments were partially carried out using the equipment provided by the IBCH сore facility (CKP IBCH, supported by the Russian Ministry of Education and Science, grant RFMEFI62117X0018).

References

  1. 1.
    Droge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82(1):47–95.  https://doi.org/10.1152/physrev.00018.2001 CrossRefPubMedGoogle Scholar
  2. 2.
    Winterbourn CC, Hampton MB (2008) Thiol chemistry and specificity in redox signaling. Free Radical Bio Med 45(5):549–561.  https://doi.org/10.1016/j.freeradbiomed.2008.05.004 CrossRefGoogle Scholar
  3. 3.
    D'Autreaux B, Toledano MB (2007) ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol 8(10):813–824.  https://doi.org/10.1038/nrm2256 CrossRefPubMedGoogle Scholar
  4. 4.
    Bedard K, Krause KH (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87(1):245–313.  https://doi.org/10.1152/physrev.00044.2005 CrossRefPubMedGoogle Scholar
  5. 5.
    Folkes LK, Christlieb M, Madej E, Stratford MR, Wardman P (2007) Oxidative metabolism of combretastatin A-1 produces quinone intermediates with the potential to bind to nucleophiles and to enhance oxidative stress via free radicals. Chem Res Toxicol 20(12):1885–1894.  https://doi.org/10.1021/tx7002195 CrossRefPubMedGoogle Scholar
  6. 6.
    Serrander L, Jaquet V, Bedard K, Plastre O, Hartley O, Arnaudeau S, Demaurex N, Schlegel W, Krause KH (2007) NOX5 is expressed at the plasma membrane and generates superoxide in response to protein kinase C activation. Biochimie 89(9):1159–1167.  https://doi.org/10.1016/j.biochi.2007.05.004 CrossRefPubMedGoogle Scholar
  7. 7.
    Stocker S, Maurer M, Ruppert T, Dick TP (2018) A role for 2-Cys peroxiredoxins in facilitating cytosolic protein thiol oxidation. Nat Chem Biol 14(2):148–155.  https://doi.org/10.1038/nchembio.2536 CrossRefPubMedGoogle Scholar
  8. 8.
    Mendez I, Vazquez-Martinez O, Hernandez-Munoz R, Valente-Godinez H, Diaz-Munoz M (2016) Redox regulation and pro-oxidant reactions in the physiology of circadian systems. Biochimie 124:178–186.  https://doi.org/10.1016/j.biochi.2015.04.014 CrossRefPubMedGoogle Scholar
  9. 9.
    Rhee SG, Kil IS (2017) Multiple functions and regulation of mammalian peroxiredoxins. Annu Rev Biochem 86:749–775.  https://doi.org/10.1146/annurev-biochem-060815-014431 CrossRefPubMedGoogle Scholar
  10. 10.
    Sies H (2015) Oxidative stress: a concept in redox biology and medicine. Redox Biol 4:180–183.  https://doi.org/10.1016/j.redox.2015.01.002 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Zhu Y, Sun J, Wang L, Qi B (2015) Endogenic oxidative stress response contributes to glutathione over-accumulation in mutant Saccharomyces cerevisiae Y518. Appl Microbiol Biotechnol 99(17):7069–7078.  https://doi.org/10.1007/s00253-015-6629-7 CrossRefPubMedGoogle Scholar
  12. 12.
    Gomes A, Fernandes E, Lima JL (2005) Fluorescence probes used for detection of reactive oxygen species. J Biochem Biophys Methods 65(2–3):45–80.  https://doi.org/10.1016/j.jbbm.2005.10.003 CrossRefPubMedGoogle Scholar
  13. 13.
    Yang G, de Castro Reis F, Sundukova M, Pimpinella S, Asaro A, Castaldi L, Batti L, Bilbao D, Reymond L, Johnsson K, Heppenstall PA (2015) Genetic targeting of chemical indicators in vivo. Nat Methods 12(2):137–139.  https://doi.org/10.1038/nmeth.3207 CrossRefPubMedGoogle Scholar
  14. 14.
    Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, Lukyanov S (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3(4):281–286.  https://doi.org/10.1038/nmeth866 CrossRefPubMedGoogle Scholar
  15. 15.
    Markvicheva KN, Bilan DS, Mishina NM, Gorokhovatsky AY, Vinokurov LM, Lukyanov S, Belousov VV (2011) A genetically encoded sensor for H2O2 with expanded dynamic range. Bioorg Med Chem 19(3):1079–1084.  https://doi.org/10.1016/j.bmc.2010.07.014 CrossRefPubMedGoogle Scholar
  16. 16.
    Bilan DS, Pase L, Joosen L, Gorokhovatsky AY, Ermakova YG, Gadella TW, Grabher C, Schultz C, Lukyanov S, Belousov VV (2013) HyPer-3: a genetically encoded H(2)O(2) probe with improved performance for ratiometric and fluorescence lifetime imaging. ACS Chem Biol 8(3):535–542.  https://doi.org/10.1021/cb300625g CrossRefPubMedGoogle Scholar
  17. 17.
    Ermakova YG, Bilan DS, Matlashov ME, Mishina NM, Markvicheva KN, Subach OM, Subach FV, Bogeski I, Hoth M, Enikolopov G, Belousov VV (2014) Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide. Nat Comm 5:5222.  https://doi.org/10.1038/ncomms6222 CrossRefGoogle Scholar
  18. 18.
    Zhao Y, Araki S, Wu J, Teramoto T, Chang YF, Nakano M, Abdelfattah AS, Fujiwara M, Ishihara T, Nagai T, Campbell RE (2011) An expanded palette of genetically encoded Ca(2)(+) indicators. Science 333(6051):1888–1891.  https://doi.org/10.1126/science.1208592 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA, Tsien RY, Remington SJ (2004) Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 279(13):13044–13053.  https://doi.org/10.1074/jbc.M312846200 CrossRefPubMedGoogle Scholar
  20. 20.
    Berg J, Hung YP, Yellen G (2009) A genetically encoded fluorescent reporter of ATP: ADP ratio. Nat Methods 6(2):161–166.  https://doi.org/10.1038/nmeth.1288 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Poburko D, Santo-Domingo J, Demaurex N (2011) Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations. J Biol Chem 286(13):11672–11684.  https://doi.org/10.1074/jbc.M110.159962 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Whitaker M (2010) Genetically encoded probes for measurement of intracellular calcium. Methods Cell Biol 99:153–182.  https://doi.org/10.1016/B978-0-12-374841-6.00006-2 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Boselli A, Piubelli L, Molla G, Pilone MS, Pollegioni L, Sacchi S (2007) Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity. Biochimie 89(3):360–368.  https://doi.org/10.1016/j.biochi.2006.10.017 CrossRefPubMedGoogle Scholar
  24. 24.
    Pollegioni L, Diederichs K, Molla G, Umhau S, Welte W, Ghisla S, Pilone MS (2002) Yeast D-amino acid oxidase: structural basis of its catalytic properties. J Mol Biol 324(3):535–546CrossRefGoogle Scholar
  25. 25.
    Matlashov ME, Belousov VV, Enikolopov G (2014) How much H(2)O(2) is produced by recombinant D-amino acid oxidase in mammalian cells? Antioxid Redox Signal 20(7):1039–1044.  https://doi.org/10.1089/ars.2013.5618 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Pollegioni L, Falbo A, Pilone MS (1992) Specificity and kinetics of Rhodotorula gracilis D-amino acid oxidase. Biochim Biophys Acta 1120(1):11–16CrossRefGoogle Scholar
  27. 27.
    Bogdanova YA, Schultz C, Belousov VV (2017) Local generation and imaging of hydrogen peroxide in living cells. Curr Protocols Chem Biol 9(2):117–127.  https://doi.org/10.1002/cpch.20 CrossRefGoogle Scholar
  28. 28.
    Chen KC, Zhou Y, Zhang W, Lou MF (2007) Control of PDGF-induced reactive oxygen species (ROS) generation and signal transduction in human lens epithelial cells. Mol Vis 13:374–387PubMedPubMedCentralGoogle Scholar
  29. 29.
    Matlashov ME, Bogdanova YA, Ermakova GV, Mishina NM, Ermakova YG, Nikitin ES, Balaban PM, Okabe S, Lukyanov S, Enikolopov G, Zaraisky AG, Belousov VV (2015) Fluorescent ratiometric pH indicator SypHer2: applications in neuroscience and regenerative biology. Biochim Biophys Acta 1850(11):2318–2328.  https://doi.org/10.1016/j.bbagen.2015.08.002 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Ermakova YG, Pak VV, Bogdanova YA, Kotlobay AA, Yampolsky IV, Shokhina AG, Panova AS, Marygin RA, Staroverov DB, Bilan DS, Sies H, Belousov VV (2018) SypHer3s: a genetically encoded fluorescent ratiometric probe with enhanced brightness and an improved dynamic range. Chem Commun (Camb) 54(23):2898–2901.  https://doi.org/10.1039/c7cc08740c CrossRefGoogle Scholar
  31. 31.
    Mishina NM, Markvicheva KN, Fradkov AF, Zagaynova EV, Schultz C, Lukyanov S, Belousov VV (2013) Imaging H2O2 microdomains in receptor tyrosine kinases signaling. Methods Enzymol 526:175–187.  https://doi.org/10.1016/B978-0-12-405883-5.00011-9 CrossRefPubMedGoogle Scholar
  32. 32.
    Stein F, Kress M, Reither S, Piljic A, Schultz C (2013) FluoQ: a tool for rapid analysis of multiparameter fluorescence imaging data applied to oscillatory events. ACS Chem Biol 8(9):1862–1868.  https://doi.org/10.1021/cb4003442 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Yulia G. Ermakova
    • 1
    • 2
  • Nataliya M. Mishina
    • 1
  • Carsten Schultz
    • 2
    • 3
  • Vsevolod V. Belousov
    • 1
    • 4
    • 5
    Email author
  1. 1.Shemyakin-Ovchinnikov Institute of Bioorganic ChemistryMoscowRussia
  2. 2.European Molecular Biology LaboratoryHeidelbergGermany
  3. 3.Oregon Health and Science UniversityPortlandUSA
  4. 4.Pirogov Russian National Research Medical UniversityMoscowRussia
  5. 5.Georg August University of GöttingenGöttingenGermany

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