BIAM switch assay coupled to mass spectrometry identifies novel redox targets of NADPH oxidase 4

  • Aim: NADPH oxidase (Nox) -derived reactive oxygen species have been implicated in redox signaling via cysteine oxidation in target proteins. Although the importance of oxidation of target proteins is well known, the specificity of such events is often debated. Only a limited number of Nox-oxidized proteins have been identified thus far; especially little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4. In this study, HEK293 cells with tetracycline-inducible Nox4 overexpression (HEK-tet-Nox4), as well as podocytes of WT and Nox4-/- mice, were utilized to identify Nox4-dependent redox-modified proteins. Results: TGFβ1 induced an elevation in Nox4 expression in podocytes from WT but not Nox4-/- mice. Using BIAM based redox switch assay in combination with mass spectrometry and western blot analysis, 142 proteins were identified as differentially oxidized in podocytes from wild type vs. Nox4-/- mice and 131 proteins were differentially oxidized in HEK-tet-Nox4 cells upon Nox4 overexpression. A predominant overlap was found for peroxiredoxins and thioredoxins, as expected. More interestingly, the GRB2-associated-binding protein 1 (Gab1) was identified as being differentially oxidized in both approaches. Further analysis using mass spectrometry-coupled BIAM switch assay and site directed mutagenesis, revealed Cys374 and Cys405 as the major Nox4 targeted oxidation sites in Gab1. Innovation & conclusion: BIAM switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins in a global untargeted way. Nox4, as a source of hydrogen peroxide, changes the redox-state of numerous proteins. Of those, we identified Gab1 as a novel redox target of Nox4.
Author:Oliver Löwe, Flávia Figueiredo de Rezende FelipeORCiDGND, Juliana HeidlerORCiD, Ilka WittigORCiD, Valeska HelfingerORCiDGND, Ralf BrandesORCiDGND, Katrin SchröderORCiDGND
Pubmed Id:
Parent Title (English):Redox Biology
Place of publication:Amsterdam [u. a.]
Document Type:Article
Year of Completion:2019
Date of first Publication:2019/01/29
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2019/04/23
Tag:BIAM switch assay; Mass spectrometry; NADPH oxidase; Nox4; Reactive oxygen species
Issue:Art. 101125
Page Number:11
First Page:1
Last Page:11
© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (
Institutes:Medizin / Medizin
Wissenschaftliche Zentren und koordinierte Programme / Sonderforschungsbereiche / Forschungskollegs
Dewey Decimal Classification:6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Licence (German):License LogoCreative Commons - Namensnennung-Nicht kommerziell - Keine Bearbeitung 4.0