All ETDs from UAB

Advisory Committee Chair

Aimee Landar

Advisory Committee Members

Scott W Ballinger

Victor M Darley-Usmar

Rakesh P Patel

Matthew B Renfrow

Victor J Thannickal

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The GTPase Rac1 is a protein which has been shown to be dysregulated in the context of a number of inflammatory diseases including atherosclerosis. Rac1 responds to extracellular signals and is involved in cellular cytoskeletal rearrangement, reactive oxygen species generation and cell cycle progression. When activated Rac1 interacts with effector proteins on the plasma membrane. This activation has recently been shown to be modulated by redox signaling via an active site cysteine. However, it is not known whether other redox signaling compounds can modulate Rac1 activity. An important redox signaling mediator is the electrophilic lipid, 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2). This lipid is endogenously produced downstream of cyclooxygenase during inflammatory conditions such as atherosclerosis. Biological responses of 15d-PGJ2 are produced in a pleiotropic manner via the formation of covalent adducts with cysteine residues on target proteins; however, not all protein members of the 15d-PGJ2 specific proteome are currently known. In this study, we demonstrate that 15d-PGJ2 forms adducts with Rac1 in vitro at residues, C157 and C178. In cultured primary bovine aortic endothelial (BAECs) cells, treatment with 15d-PGJ2 resulted in increased activity which coincided with the modification of Rac1 by the lipid. Furthermore, this modification was associated with decreased BAEC migration and cell spreading. These results demonstrate for the first time that Rac1 is a target for 15d-PGJ2 in the context of vascular cells, such as endothelial cells, and suggest that Rac1 modification by electrophilic lipids may alter redox signaling and cellular function. This study is significant because work done identifies a new member of the electrophile responsive proteome, Rac1. Additionally, other methods used to study protein thiol modification in cell culture are discussed. There is a lack of understanding about reactive species and the functional implications of protein thiol modification in the context of cells and in disease. In this thesis, multiple methods are used to assess protein thiol modification in vascular cells.

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