Mitochondria are the central organelle for cellular metabolism while being an important source of ROS production.<sup>1</sup> 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 regards to sensitivity and selectivity of detecting protein glutathionylation induced by endogenous production of mROS. We recently developed a novel chemical method 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. Our GS mutant catalyzes azido-Ala in place of Gly, forming azido-glutathione (γGlu-Cys-azido-Ala) efficiently in cells. In response to ROS, this clickable glutathione allows for selective and sensitive detection of glutathionylation after click reaction with biotin-alkyne or fluorophore-alkyne. With this approach, we have analyzed mROS-induced global and individual protein glutathionylation during glucose starvation, which occurs in a pathophysiological ischemic condition. Proteomic and biochemical analysis with our clickable glutathione approach led to identifying multiple target proteins of glutathionylation and determining their functional changes upon glutathionylation in response to mROS.

Chemoselective identification of protein glutathionylation in response to mitochondrial ROS

251st National Meeting (2016)

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