Characterizing Protein Glutathionylation in Response to Mitochondrial ROS

Mitochondria are the central organelle for cellular metabolism while being an important source of ROS production.1 Importantly, mitochondrial ROS (mROS) are emerging as key pathophysiological molecules in cardiac cells as well as a broad cardiovascular system. The complex role of mROS is largely mediated by protein oxidative modifications, including glutathionylation that is a disulfide bond formation of protein cysteine residue with intracellular glutathione. Although several biochemical methods for identifying glutathionylation are available, individual approach has its own limitations. In order to identify and characterize protein glutathionylation, we recently developed a novel chemical method, namely clickable glutathione, that selectively detects glutathionylation in response to ROS. The central idea was to tag a ‘clickable’ functionality to glutathione by engineering glutathione synthetase (GS), a non-rate limiting enzyme in glutathione (γGlu-Cys-Gly) biosynthesis. With this approach, we analyzed glutathionylation of global and individual proteins in response to metabolic alteration that increases mROS production. Proteomic and biochemical analysis with our clickable glutathione approach led to identifying multiple target proteins of glutathionylation, including SMYD2 implicated in cardiac and skeletal muscle. In this talk, I will present our clickable glutathione approach, characterization of glutathionylation in metabolic alteration, and functional significance of SMYD2 glutathionylation in muscle.
Young-Hoon Ahn (Wayne State University)