The long-term goal of Project 2 is to understand the synthesis and regulation of selenoproteins that protect vascular cells from lipid-hydroperoxide-mediated injury caused by oxidized lipoproteins. Selenium is an essential trace element, which is incorporated into selenoproteins as selenocysteine (Sec), the 21st amino acid. Many of the known selenoproteins are enzymes that catalyze oxidation-reduction reactions and contain Sec at their active site. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) protects cells against membrane lipid peroxidation by reducing phospholipid, cholesterol, and cholesterol ester hydroperoxides. The hypothesis that PHGPx is protective against vascular cell apoptosis and arterial lesion development will be tested in Project 1. The goal of Project 2 is to understand how selenoproteins are synthesized using PHGPx as a model. Sec is encoded by a UGA codon in the selenoprotein mRNA. The decoding of UGA as Sec requires the reprogramming of translation since UGA is normally read as a stop codon. The translation of mammalian selenoprotein mRNAs requires the 3' untranslated region (3' UTR), which contains a Sec Insertion Sequence (SECIS) element that is essential for the decoding of UGA as Sec. During the previous funding period, we purified, cloned, and characterized SECIS-binding Protein 2 (SBP2), a novel RNA-binding protein, which binds to the SECIS element. We developed the first system for translating selenoprotein mRNAs in vitro and showed that SBP2 is an essential and limiting factor in this pathway. The levels of SBP2 protein vary dramatically between tissues, ranging from high expression in testis to undetectable levels in aortic smooth muscle cells (SMC). In preliminary studies, we identified a new SECIS-binding protein, SBP3, which is highly expressed in SMC and other tissues. We hypothesize that SBP2 and SBP3 are both essential for selenoprotein synthesis and that they perform separate functions in Sec insertion. In this project, we propose to: 1) analyze the structure and function of SBP2; 2) investigate the mechanism of Sec insertion using our in vitro translation system; and 3) characterize and identify SBP3. The successful pursuit of these aims will lead to a better understanding of selenoprotein synthesis in mammalian cells and may identify limiting factors and regulatory pathways that could be used therapeutically to prevent the development of atherosclerosis by modulating selenoprotein expression in vivo.
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