Identification and Quantification of Protein S-nitrosation by Nitrite in the Mouse Heart during Ischemia [Methods and Resources]

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 Snitrosation sites across the mammalian proteome remains 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-labelling technique, ICAT (Isotope-Coded Affinity Tag), to quantify the extent of NO2- -dependent S-nitrosation of proteins thiols by in vivo. Using this approach, called SNOxICAT, 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 Snitrosation 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 place 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 for determining modified cysteines across the proteome and for identifying those most likely responsible for the functional consequences of NO2- exposure.

from # All Medicine by Alexandros G. Sfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2vmYewV
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