Oxidation of HRas cysteine thiols by metabolic stress prevents palmitoylation in vivo and contributes to endothelial cell apoptosis

Here we demonstrate a new paradigm in redox signaling, whereby oxidants resulting from metabolic stress directly alter protein palmitoylation by oxidizing reactive cysteine thiolates. In mice fed a high-fat, high-sucrose diet and in cultured endothelial cells (ECs) treated with high palmitate and high glucose (HPHG), there was decreased HRas palmitoylation on Cys181/184 (61 +/- 24% decrease for cardiac tissue and 38 +/- 7.0% in ECs). This was due to oxidation of Cys181/184, detected using matrix-assisted laser desorption/ionization time of flight (MALDI TOF)-TOF. Decrease in HRas palmitoylation affected its compartmentalization and Ras binding domain binding activity, with a shift from plasma membrane tethering to Golgi localization. Loss of plasma membrane-bound HRas decreased growth factor-stimulated ERK phosphorylation (84 +/- 8.6% decrease) and increased apoptotic signaling (24 +/- 6.5-fold increase) after HPHG treatment that was prevented by overexpressing wild-type but not C181/184S HRas. The essential role of HRas in metabolic stress was made evident by the similar effects of expressing an inactive dominant negative N17-HRas or a MEK inhibitor. Furthermore, the relevance of thiol oxidation was demonstrated by overexpressing manganese superoxide dismutase, which improved HRas palmitoylation and ERK phosphorylation, while lessening apoptosis in HPHG treated ECs.-Burgoyne, J. R., Haeussler, D. J., Kumar, V., Ji, Y., Pimental, D. R., Zee, R. S., Costello, C. E., Lin, C., McComb, M. E., Cohen, R. A., Bachschmid, M. M. Oxidation of HRas cysteine thiols by metabolic stress prevents palmitoylation in vivo and contributes to endothelial cell apoptosis. FASEB J. 26, 832-841 (2012). www.fasebj.org