CRISPR/Cas9-mediated knockout of p22phox leads to loss of Nox1 and Nox4, but not Nox5 activity.
The NADPH oxidases are important transmembrane proteins producing reactive oxygen species (ROS). Within the Nox family, different modes of activation can be discriminated. Nox1-3 are dependent on different cytosolic subunits, Nox4 seems to be constitutively active and Nox5 is directly activated by calcium. With the exception of Nox5, all Nox family members are thought to depend on the small transmembrane protein p22phox. With the discovery of the CRISPR/Cas9-system, a tool to alter genomic DNA sequences has become available. So far, this method has not been widely used in the redox community. On such basis, we decided to study the requirement of p22phox in the Nox complex using CRISPR/Cas9-mediated knockout. Knockout of the gene of p22phox, CYBA, led to an ablation of activity of Nox4 and Nox1 but not of Nox5. Production of hydrogen peroxide or superoxide after knockout could be rescued with either human or rat p22phox, but not with the DUOX-maturation factors DUOXA1/A2. Furthermore, different mutations of p22phox were studied regarding the influence on Nox4-dependent H2O2production. P22phox Q130* and Y121H affected maturation and activity of Nox4. Hence, Nox5-dependent O2•-production is independent of p22phox, but native p22phox is needed for maturation of Nox4 and production of H2O2.
Institut für Kardiovaskuläre Physiologie, Goethe-Universität, 60590 Frankfurt am Main, Germany; German Center for Cardiovascular Research (DZHK), Partner site RheinMain, 60590 Frankfurt am Main, Germany.
Kim-Kristin
KK
Prior
Institut für Kardiovaskuläre Physiologie, Goethe-Universität, 60590 Frankfurt am Main, Germany; German Center for Cardiovascular Research (DZHK), Partner site RheinMain, 60590 Frankfurt am Main, Germany.
Matthias S
MS
Leisegang
Institut für Kardiovaskuläre Physiologie, Goethe-Universität, 60590 Frankfurt am Main, Germany; German Center for Cardiovascular Research (DZHK), Partner site RheinMain, 60590 Frankfurt am Main, Germany.
Ivana
I
Josipovic
Institut für Kardiovaskuläre Physiologie, Goethe-Universität, 60590 Frankfurt am Main, Germany; German Center for Cardiovascular Research (DZHK), Partner site RheinMain, 60590 Frankfurt am Main, Germany.
Oliver
O
Löwe
King's College London British Heart Foundation Centre, Cardiovascular Division, London, UK.
Ajay M
AM
Shah
Excellence Cluster Cardio-Pulmonary System, Member of the German Center for Lung Research (DZL), Justus-Liebig-University, 35390 Giessen, Germany.
Norbert
N
Weissmann
Institut für Kardiovaskuläre Physiologie, Goethe-Universität, 60590 Frankfurt am Main, Germany; German Center for Cardiovascular Research (DZHK), Partner site RheinMain, 60590 Frankfurt am Main, Germany.
Katrin
K
Schröder
Institut für Kardiovaskuläre Physiologie, Goethe-Universität, 60590 Frankfurt am Main, Germany; German Center for Cardiovascular Research (DZHK), Partner site RheinMain, 60590 Frankfurt am Main, Germany. Electronic address: brandes@vrc.uni-frankfurt.de.
Ralf P
RP
Brandes
D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D055786:Gene Knockout Techniques; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics; D000074624:NADPH Oxidase 1 / Q000378:metabolism*; D000074663:NADPH Oxidase 4 / Q000378:metabolism*; D000074664:NADPH Oxidase 5 / Q000378:metabolism*; D019255:NADPH Oxidases / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism
C513411:DUOXA2 protein, human; D008565:Membrane Proteins; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases; C086986:CYBA protein, human
CRISPR/Cas9; CYBA/p22phox; NADPH oxidase