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CRISPR/Cas9-mediated modification of the extreme C-terminus impairs PDGF-stimulated activity of Duox2

  • Pyotr A. Tyurin-Kuzmin EMAIL logo , Maxim N. Karagyaur , Yury P. Rubtsov , Daniyar T. Dyikanov , Pyotr A. Vasiliev and Alexander V. Vorotnikov
Published/Copyright: January 26, 2018
Biological Chemistry
From the journal Biological Chemistry Volume 399 Issue 5

Abstract

Duox2 belongs to the large family of NADPH-oxidase enzymes that are implicated in immune response, vasoregulation, hormone synthesis, cell growth and differentiation via the regulated synthesis of H2O2 and reactive oxygen species. We and others have shown that Duox2 and H2O2 are involved in platelet-derived growth factor (PDGF) induced migration of fibroblasts. Now, using the CRISPR/Cas9-mediated genome editing we demonstrate that the extreme C-terminal region of Duox2 is required for PDGF-stimulated activity of Duox2 and H2O2 production. We generated the fibroblast cells that stably co-express the wild-type or C-terminally modified Duox2 and fluorescent H2O2 probe Hyper. We found that nonsense substitution of the last 23 amino acids in Duox2 results in complete loss of PDGF stimulation of intracellular H2O2 and fibroblast migration, yet these mutations have no effects on the expression of Duox2 and other NADPH-oxidases in cells. These findings illustrate for the first time that the extreme C-terminus of Duox2 is required for the functional activity of the enzyme. Furthermore, the conservative nature of the C-terminus suggests its role for activity in other NADPH-oxidases.

Keywords: CRISPR-Cas9; Duox2; migration; NADPH oxidases; PDGF

Acknowledgments

We thank Dr. K.Y. Kulebyakin for fruitful discussions on this manuscript. This work was supported by the Russian Science Foundation (RSF), grant 14-24-00086. Partial support from M.V. Lomonosov Moscow State University Program of Development for the use of microscope equipment is also acknowledged.

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Received: 2017-08-29
Accepted: 2018-01-11
Published Online: 2018-01-26
Published in Print: 2018-04-25

©2018 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

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https://doi.org/10.1515/hsz-2017-0229
Keywords for this article
CRISPR-Cas9; Duox2; migration; NADPH oxidases; PDGF

Articles in the same Issue

  1. Frontmatter
  2. Reviews
  3. Generation of superoxide and hydrogen peroxide by side reactions of mitochondrial 2-oxoacid dehydrogenase complexes in isolation and in cells
  4. Update on mitochondria and muscle aging: all wrong roads lead to sarcopenia
  5. Research Articles/Short Communications
  6. Protein Structure and Function
  7. CRISPR/Cas9-mediated modification of the extreme C-terminus impairs PDGF-stimulated activity of Duox2
  8. Membranes, Lipids, Glycobiology
  9. Changes of the peripheral blood mononuclear cells membrane fluidity from type 1 Gaucher disease patients: an electron paramagnetic resonance study
  10. Molecular Medicine
  11. Aβ42 oligomers impair the bioenergetic activity in hippocampal synaptosomes derived from APP-KO mice
  12. Cell Biology and Signaling
  13. Molecular determinants of Drosophila immunophilin FKBP39 nuclear localization
  14. The effect of lncRNA HOTAIR on chemoresistance of ovarian cancer through regulation of HOXA7
  15. Novel Techniques
  16. Determination of selenium during pathogenesis of hepatic fibrosis employing hydride generation and inductively coupled plasma mass spectrometry
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