| Oxidized LDL (oxLDL) is thought to be a main contributor to the pathogenesis of atherosclerosis and has been shown to initiate a variety of contrasting responses within the endothelium, including apoptosis, monocyte adhesion, and induction of cellular antioxidants. Developments in analytical techniques have led to the identification of a variety of classes of lipids within oxLDL that have distinct properties, including those lipids with electrophilic carbon centers. Electrophilic lipids include the compounds 15-deoxy-Delta 12,14-Prostaglandin J2 (15d-PGJ2) and 4-hydroxy-2-nonenal both which have been detected in atherosclerotic lesions. Recent studies have provided mechanistic insights into the modulation of cellular signaling by electrophilic lipids, which includes modification of critical protein thiols. Although individual target proteins modified by electrophilic lipids have been identified, the effect of these species on protein modifications from a global perspective has not been examined. It is hypothesized that electrophilic lipids such as 15d-PGJ2 can react with a specific subset of proteins and that this process is governed by the reactivity of the protein thiol. In this study, a proteomics approach was used to identify and define the electrophile responsive proteome in endothelial cells. Using (biotinoyl)-N-(iodoacetyl) ethylenediamine, which reacts with free protein thiols, it was found that both pH and the concentration of glutathione can affect the number of reduced thiols available. It was determined using biotinylated 15d-PGJ2 that 15d-PGJ2 can form adducts with a specific subset of cellular proteins in a time- and concentration-dependent manner. Fluorescent microscopy showed that 15d-PGJ2 can enter the mitochondria. The effects of oxidized lipids on mitochondrial function is of great interest because of the emerging role of these lipids in the pathogenesis of atherosclerosis. Studies into the functional changes associated with exposure to electrophilic lipids showed that noncytotoxic concentrations of 15d-PGJ2 can induce mitochondrial complex I activity, which is thought to be part of an adaptive response that includes the induction of glutathione, heme oxygenase, and catalase. Taken together, these data support the hypothesis that there is a subproteome within endothelial cells that contains reactive thiols that are modified by electrophilic lipids and that modification of protein thiols represents a potential mechanism of signaling by electrophilic lipids. |
