Session: Current Topics in Biological Chemistry:

Elevated intracellular levels of reactive oxygen species (ROS) or reactive nitrogen species (RNS) are associated with induction of oxidative stress. Even though ROS/RNS can act as signaling molecules for maintaining cardiovascular function when they are present in suboptimal levels, increased levels of ROS/RNS participate in pathological roles by damaging proteins. There is evidence that development of cardiovascular diseases (CVD) can be caused by post-translational modifications of cardiac proteins by ROS/RNS. Ischemia and ischemic reperfusion are known to cause cardiovascular diseases by altering the flux of oxygen and nutrient rich blood to the cardiomyocytes. Altered levels of ROS participate in pathological roles by causing oxidative modifications on proteins. One such modification is protein glutathionylation. Identification and quantification of glutathionylated proteins is important for understanding the molecular mechanisms behind initiation and progression of cardiovascular diseases. Despite previous extensive studies, proteomic identification of glutathionylated proteins in cardiomyocytes under altered levels of nutrients and oxygen has been relatively limited. Recently we have developed a new mass spectrometry-based method to identify glutathionylation in cells by synthesizing clickable-glutathione, a glutathione derivative containing an azide group. A mutant of glutathione synthetase enzyme was used for the in-situ biosynthesis of clickable glutathione upon the addition of azido-alanine in cells. Two azido-Ala derivatives with isotopic mass difference were used to metabolically synthesize isotopically labelled clickable glutathione, which were effectively used for the identification of glutathionylated proteins by proteomic mass spectrometry after the bio-orthogonal click reaction with a biotin alkyne. We have applied our clickable glutathione approach to HL-1 cardiomyocyte cell line under metabolic alterations of glucose, oxygen, and fatty acids to detect and identify 249 peptides undergoing glutathionylation. Also, we did a bioinformatic analysis to find glutathionylated cysteines whose mutations are associated with cardiac diseases, including cardiomyopathy, thus building a list of cysteines that may contribute to cardiac diseases upon glutathionylation.

Young-Hoon Ahn

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