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Smoking increases the risk of hypertension and contributes to premature mortality from cardiovascular disease, the leading cause of death in the United States. Dysfunction of the endothelium is one pathological response to smoking that contributes to cardiovascular disease. The endothelium is a layer of specialized cells that line the inside of blood vessels and is in constant contact with circulating blood. The endothelial cells regulate the passage of materials in the blood to the surrounding tissue. Endothelial cell damage induced by smoking has been linked to decreased production of the anti-coagulant protease, activated protein C (APC). As a consequence, there is enhanced blood clotting (thrombosis) that can contribute to an obstruction of blood flow within blood vessels, increasing the risk for heart attacks and strokes. Thrombin is a protease that is generated at sites of vascular injury and regulates bleeding (hemostasis) and thrombosis. Interestingly, thrombin and APC mediate their effects through protease-activated receptor-1 (PAR1), a receptor that is present on platelets and endothelial cells. The goal of this study is to better understand how PAR1 signaling is regulated and its effects on the endothelial barrier.
The focus of my work is to assess the role of palmitoylation on PAR1 signaling and trafficking. Palmitoylation is a posttranslational modification that results in the addition of palmitate, a 16-carbon fatty acid, to the amino acid cysteine. I have determined that palmitoylation occurs on specific cysteines within PAR1. Palmitoylation at these sites has the potential to influence how PAR1 signaling is regulated. To test the role of palmitoylation, I generated a mutation that prevents the addition of palmitate to PAR1, and examined its effect on signaling and trafficking. As a consequence of palmitoylation-deficiency, the cell internalizes PAR1 more rapidly when the receptor is inactive. This result suggests that palmitoylation controls PAR1 accessibility to the cell’s internalization machinery.
Palmitoylation of PAR1 also has the potential to influence its ability to be activated by APC versus thrombin. We recently found that localization of PAR1 into cholesterol rich domains on the cell surface (caveolar microdomains) is critical for APC, but not for thrombin, mediated PAR1 signaling. We hypothesize that palmitoylation plays a role in localizing a population of receptors into these microdomains to allow for PAR1 to elicit anti-inflammatory, barrier protective signaling in endothelial cells. The results from the proposed study will have the potential to reveal a new mechanism by which PAR1 elicits distinct endothelial cellular responses, and may provide new therapeutic targets for the prevention and treatment of vascular endothelial cell dysfunction associated with smoking and cardiovascular disease. |
