Person:

Chouchani, Edward

Nitrate (NO3−) and nitrite (NO2−) are known to be cardioprotective and to alter energy metabolism in vivo. NO3− action results from its conversion to NO2− by salivary bacteria, but the mechanism(s) by which NO2− affects metabolism remains obscure. NO2− may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO2−-dependent S-nitrosation of proteins thiols in vivo. Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO2− under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO2− in conjunction with ischemia led to extensive S-nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO2− on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2−, combined with the lack of S-nitrosation during anoxia alone or by NO2− during normoxia places constraints on how S-nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO2− exposure.