198 - Novel Redox-Targets of NADPH Oxidase 4 Identified by Untargeted BIAM Switch Assay

Abstract

NADPH oxidases of the Nox family are important sources of reactive oxygen species. Nox-derived ROS have been implicated in redox-signaling as they can elicit cysteine oxidation in target proteins. Only a limited number of Nox-differentially oxidized proteins have been identified so far and particularly little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4. We set out to identify redox-targets of Nox4 by redox-proteomics. Tetracycline-inducible HEK293 cells overexpressing Nox4 (HEK-TET-Nox4) and podocytes of WT and Nox4-/- mice were used. Redox-modified proteins were identified by the BIAM switch assay in combination with mass spectrometry and western blot analysis. Increased Nox4 expression in response to TGFβ-1 was detected by western blot analysis in podocytes of WT mice. In response to TGFβ-1, oxidation of 138 proteins increased in podocytes of wildtype but not in podocytes of Nox4-/- mice. Identified proteins clustered in different groups of cellular processes like “cellular oxidant detoxification” and “activation of protein kinase activity”. The difference in oxidation of identified proteins could be validated for selected proteins by the BIAM-switch assay and western blot analysis. As a second approach, HEK-TET-Nox4 cells were used in which Nox4 overexpression and ROS production can be stimulated by tetracycline. In this protocol, an overlap in the oxidized proteins to the WT/KO system was found for proteins with antioxidant capacity as well for some interesting novel redox-targets of Nox4. The BIAM-Switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins. Nox4 is a source of ROS which changes the redox-state of numerous proteins.