CRISPR/Cas9-mediated knockout of p22phox leads to loss of Nox1 and Nox4, but not Nox5 activity

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• 作者：Kim-Kristin Prior^a ; ^b ; Matthias S. Leisegang^a ; ^b ; Ivana Josipovic^a ; ^b ; Oliver Lö ; we^a ; ^b ; Ajay M. Shah^d ; Norbert Weissmann^c ; Katrin Schrö ; der^a ; ^b ; Ralf P. Brandes^a ; ^b ; ^{brandes@vrc.uni-frankfurt.de" class="auth_mail" title="E-mail the corresponding author}
• 关键词：CRISPR ; Clustered regulatory interspaced short palindromic repeats ; Cas9 ; CRISPR-associated protein 9 ; HEK293 ; human embryonic kidney 293 cells ; HRP ; horseradish peroxidase ; Nox4-HEK293 ; HEK293 cells stably expressing Nox4 ; PMA ; phorbol 12-myristate 13-acetate ; tetNox4-HEK293 ; HEK293 cells expressing Nox4 in tetracycline-inducible tet-on vector
• 刊名：Redox Biology
• 出版年：2016
• 出版时间：October 2016
• 年：2016
• 卷：9
• 期：Complete
• 页码：287-295
• 全文大小：1106 K

文摘

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.