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Honors Thesis Archive

Author Amanda Jane Hicks
Title Is Rac1 Glutathionylated in Intact Cells?
Department Biochemistry/Molecular Biology
Advisor Margaret Goodman
Year 2006
Honors Departmental Honors
Full Text View Thesis (133 KB)
Abstract Rac1 is a member of the Ras superfamily and Rho subfamily of small GTP-binding proteins or GTPases. It functions as a molecular switch, cycling between an active (GTP-bound) and inactive (GDP-bound) conformation, to regulate diverse physiological processes within mammalian cells. Active Rac1 signals to downstream effectors to control cellular processes such as membrane ruffling. While the downstream signaling pathways that lead to Rac1 mediated events are reasonably well understood, the upstream events that regulate Rac1 are not well characterized.

The goal of this study is to determine if Rac1 is post-translationally glutathionylated. Glutathione (GSH) is a tripeptide consisting of glutamate, cysteine, and glycine with a free thiol. Glutathionylation is the addition of glutathione to a target protein, via a disulfide bond. Recent studies have shown that Angiotensin II induces the glutathionylation of Ras, which affects its activity. These studies are designed to determine if the Ras-related GTPase, Rac1, is glutathionylated.

NIH 3T3 cells were metabolically labeled with [35S]-cysteine to determine if Rac1 is glutathionylated in intact cells. Western Blot and fluorograph analysis revealed that diamide, a chemical that forces the formation of disulfide bonds, can increase the amount of glutathionylated FLAG-epitope tagged Rac1. Preliminary studies indicate that under such conditions, Rac1 is modified by [35S]-cysteine in a DTT reversible manner. Future studies will focus on utilizing physiological stimuli to promote and assess reversible glutathionylation of Rac1 in intact cells.

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