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Date of Award
Elevated intracellular levels of reactive oxygen species (ROS) are associated with induction of oxidative stress. Increased levels of ROS participate in development of cardiovascular diseases by modifying proteins reversibly or irreversibly. Especially, the cysteine residues can undergo oxidative post-translational modifications such as glutathionylation. Glutathionylation is an important regulatory reversible thiol oxidation. Identification and quantification of glutathionylated proteins is important for the understanding of the molecular mechanisms behind initiation and progression of many diseases. Our lab previously developed a click chemistry-based method to detect and identify glutathionylation in cells by synthesizing clickable-glutathione.We established a mass spectrometry-based strategy coupled with clickable glutathione in which two azido-Ala derivatives with isotopic mass difference were used to metabolically synthesize isotopically labelled clickable glutathione for identification and quantification of glutathionylated proteins. We applied this approach on HL-1 cardiomyocyte cells to identify and quantify 1398 glutathionylated peptides upon the addition of H2O2. During ischemia and reperfusion, levels of ROS can be elevated causing oxidative modifications in cardiac proteins. By applying clickable glutathione approach on HL-1 cells under different metabolic alterations, we found that glucose is important for maintaining redox homeostasis, while fatty acid oxidation induces a high level of glutathionylation during reoxygenation. We applied our isotopically labeled clickable glutathione approach upon addition of fatty acids during reoxygenation, identifying and quantifying 248 glutathionylated proteins. We developed a proteomic approach for the direct quantification of glutathionylated cysteines compared to the unmodified cysteines using a cysteine blocking clickable glutathione reagent (L-N3-GS-SPy) that mimics the structure of clickable glutathione. We applied this quantification approach on C2C12 mouse skeletal muscle cell line and identified 1518 glutathionylated cysteines upon addition of H2O2. Desmin is an intermediate filament that plays a key role in maintaining structural and mechanical integrity of sarcomere. From our proteomic analyses, we identified that desmin is undergoing glutathionylation under stress. Using rat cardiomyocytes, we investigate the effect of glutathionylation on myofibril integrity of desmin. We found that desmin lose its myofibril integrity upon oxidative stress. However, cysteine mutant of desmin recovers myofibril integrity, suggesting that glutathionylation may interfere with the desmin filament assembly.
Yapa Abeywardana, Maheeshi, "Chemical Proteomic Identification And Functional Studies Of Cardiac Protein Glutathionylation" (2022). Wayne State University Dissertations. 3585.