Understanding the genetic regulation and function of Selenoprotein P

Selenium is an essential trace element that has been linked to beneficial health effects in multiple disease states. These effects have been attributed to antioxidant activity of selenoproteins; proteins containing selenium incorporated as the amino acid selenocysteine during translation of the protein. Selenoprotein P is an extracellular glycoprotein containing multiple selenocysteine residues. It is the primary selenium distribution protein of the body as well as the major selenium containing protein in serum. An antioxidant function has been observed for this protein. The experiments presented in this dissertation were designed to further characterize the mechanisms of selenoprotein P regulation and function and test the hypothesis that mechanisms regulating the expression of selenoprotein P provide for modulation of this protein so it may function to provide antioxidant protection in extrahepatic tissues. When stimulated with ecdysone analogs, selenoprotein P expression was increased with the use of a fusion transcription factor that contains the glucocorticoid receptor DNA binding domain, an ecdysone ligand-binding domain, and a strong transactivation domain as well as the retinoid X receptor. In silico analysis of the selenoprotein P promoter identified putative glucocorticoid and retinoid responsive binding sites. Luciferase reporter assays and quantitative PCR were used to measure selenoprotein P transcription in engineered HEK-293 cells. The native glucocorticoid receptor inhibited selenoprotein P transactivation, and selenoprotein P was further attenuated in the presence of dexamethasone. These studies also aimed to determine if selenoprotein P possessed hydroperoxidase activity against lipid hydroperoxides generated from the metabolism of arachidonic acid by 15-lipoxygenase-1. Enzymatic reduction of 15- hydroperoxyeicosatetraenoic acid (15-HpETE) by selenoprotein P was observed in a NADPH-coupled biochemical assay. Diphenylpyrenylphosphin was used to measure lipid hydroperoxides in human embryonic kidney cells treated with selenoprotein P following exposure to 15-HpETE. Cellular oxidation increased with 15-HpETE treatment and selenoprotein P reduced this effect. These results suggest that selenoprotein P can function as an antioxidant enzyme during inflammation. An increased understanding of the mechanisms regulating selenoprotein P expression and activity could provide insight into the way in which selenium exerts its physiological effects.