Thermal proteome profiling reveals glutathione peroxidase 4 as the target of the autophagy inducer conophylline

Abstract

Conophylline (CNP) is a vinca alkaloid extracted from the Tabernaemontana divaricata plant. It has been reported that CNP induces autophagy in a mammalian target of rapamycin-independent manner, and thereby inhibits protein aggregation. However, the mode of action of CNP in inducing autophagy remains unknown. In this study, we identified glutathione peroxidase 4 (GPX4) as a CNP-binding protein by using thermal proteome profiling. The technique exploits changes in the thermal stability of proteins resulting from ligand interaction, which is capable of identifying compound-binding proteins without chemical modification. GPX4, an antioxidant protein that uses reduced glutathione as a cofactor, directly catalyzes the reduction of hydrogen peroxide, organic hydroperoxides, and lipid peroxides. GPX4 suppresses lipid peroxide accumulation, and thus plays a key role in protecting cells from oxidative damage. We found that treatment with CNP caused accumulation of lipid reactive oxygen species (ROS) in cultured cells. Furthermore, similarly with CNP treatment, GPX4 deficiency caused accumulation of lipid ROS and induced autophagy. These findings indicate that GPX4 is a direct target of CNP involved in autophagy induction. SIGNIFICANCE STATEMENT The present study identified glutathione peroxidase 4 (GPX4) as a binding protein of conophylline (CNP) by using thermal proteome profiling (TPP). This study showed that CNP treatment, similarly with the inhibition of GPX4, induced lipid reactive oxygen species accumulation and autophagy. The present findings suggest that GPX4 is the CNP target protein involved in autophagy induction. Furthermore, these results indicate that TPP is a useful technique for determining the mechanism of natural compounds.