Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA151

Poster Communications

Novel redox-targets of NADPH oxidase 4 identified by the BIAM switch assay

O. Löwe1, J. Heidler2, I. Wittig2, F. Rezende1, K. Schröder1, R. Brandes1

1. Cardiovascular Physiology, Goethe University Frankfurt, Frankfurt, Germany. 2. Functional Proteomics, Goethe University Frankfurt, Frankfurt, Germany.

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, therefore podocytes of WT and Nox4-/- were used. Redox-modified proteins were identified by the BIAM switch assay in combination with mass spectrometry. Increased Nox4 expression in response to TGFβ-1 was detected 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 and show up some interesting novel redox-targets of Nox4. The BIAM-Switch assay coupled to Mass spec 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.

Where applicable, experiments conform with Society ethical requirements