CRISPR/Cas9-mediated knockout of p22phox leads to loss of Nox1 and Nox4, but not Nox5 activity
| Autor: | Matthias S. Leisegang, Norbert Weissmann, Oliver Löwe, Ivana Josipovic, Ralf P. Brandes, Ajay M. Shah, Kim-Kristin Prior, Katrin Schröder |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
PMA
phorbol 12-myristate 13-acetate 0301 basic medicine Clinical Biochemistry CRISPR Clustered regulatory interspaced short palindromic repeats HEK293 human embryonic kidney 293 cells Biochemistry Gene Knockout Techniques 03 medical and health sciences Cell Line Tumor Humans CRISPR ddc:610 lcsh:QH301-705.5 CRISPR/Cas9 chemistry.chemical_classification lcsh:R5-920 Reactive oxygen species Cas9 CRISPR-associated protein 9 tetNox4-HEK293 HEK293 cells expressing Nox4 in tetracycline-inducible tet-on vector NADPH oxidase 030102 biochemistry & molecular biology biology Cas9 Organic Chemistry Membrane Proteins NADPH Oxidases HRP horseradish peroxidase NOX4 Hydrogen Peroxide Nox4-HEK293 HEK293 cells stably expressing Nox4 Transmembrane protein 030104 developmental biology lcsh:Biology (General) NADPH Oxidase 5 chemistry NADPH Oxidase 4 NOX1 NADPH Oxidase 1 biology.protein cardiovascular system CYBA/p22phox P22phox CRISPR-Cas Systems lcsh:Medicine (General) Reactive Oxygen Species Research Paper |
| Zdroj: | Prior, K-K, Leisegang, M S, Josipovic, I, Löwe, O, Shah, A M, Weissmann, N, Schröder, K & Brandes, R P 2016, ' CRISPR/Cas9-mediated knockout of p22phox leads to loss of Nox1 and Nox4, but not Nox5 activity ', Redox Biology, vol. 9, pp. 287–295 . https://doi.org/10.1016/j.redox.2016.08.013 Redox Biology Redox Biology, Vol 9, Iss C, Pp 287-295 (2016) |
| Popis: | 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 H2O2 production. 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. Graphical abstract fx1 |
| Databáze: | OpenAIRE |
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