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975867 
Journal Article 
Abstract 
Ras activity is modulated by glutathione and nitric oxide via one electron but not two electron mediated mechanisms 
Hobbs, A; Bonini, M 
2008 
Yes 
Free Radical Biology and Medicine
ISSN: 0891-5849
EISSN: 1873-4596 
45 
Suppl. 
S30-S31 
English 
WOS:000260867900076 
is part of a larger document 3452652 SFRBM's 15th Annual Meeting: Program and Abstracts
The Ras protein cycles between active GTP-bound and inactive GDP-bound states to regulate a host of cellular processes, including cell growth, differentiation and apoptosis. Single point mutations in Ras that increase Ras-GTP levels in cells result in deregulation of Ras activity, leading to cancer and developmental disorders. Ras activity can be regulated by protein modulatory agents and redox molecules. We uncovered a novel mechanism of Ras regulation by free radical-oxidants, and have demonstrated that superoxide and nitric oxide (NO) can react with C118, resulting in Ras activation via a radical mediated electron transfer mechanism. Intriguingly, stable nitrosation of Ras does not alter its activity. Although we anticipated that this radical-mediated mechanism could be generalized to other redox active molecules capable of reacting with Ras to generate a thiyl radical, recent reports indicate that glutathionylation of Ras at C118, alters its structure and activity. We conducted biochemical, NMR and EPR studies to characterize the effects of one-electron (NO) and two-electron (GSSG) oxidation on Ras activity and structure. Our results indicate that NO is able to modify Ras and cause guanine nucleotide dissociation (GND) via a radical-based mechanism. In contrast, NMR and kinetic data indicate that modification with glutathione via 2-electron oxidation does not alter Ras structure or activity. Using EPR spin trapping and kinetic measurements, we show that reaction of Ras with DEANO (an NO donor) promotes for mation of a radical at C118 in Ras, resulting in radical mediated transfer to the guanine nucleotide base and GND. The rate of GND is unaltered by treatment of Ras with oxidized glutathione under conditions that specifically modify C118 by a non-radical mechanism. in summary, our data suggest that free radicals induce GND through the intermediacy of a thiyl radical centered at Ras C118 what constitutes an alternative route of Ras activation in the cell. 
Society for Free Radical Biology and Medicine 15th Annual Meeting 
Indianapolis, IN 
November 19-23, 2008