The modification of lipoproteins by free radical oxidation is proposed to be an important event in the initiation and development of atherosclerosis. The pro-atherogenic properties of oxidized lipoproteins may in part be related to their cytotoxicity. The goal of this project is to understand the biosynthesis of selenoproteins that may protect vascular cells from lipid hydroperoxide-mediated injury caused by oxidized lipoproteins. This project will focus on phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenoperoxidase that reduces phospholipid cholesterol, and cholesterol ester hydroperoxides. PHGPx and other seneloproteins are synthesized by a novel pathway that involves the co-translational incorporation of a selenocysteine (Sec) residue in response to a UGA codon in the mRNA. This mechanism involves the reprogramming of translation since UGA is normally lead as a translational stop codon. In eukaryotes, the recognition of UGA is a Sec codon requires the 3' untranslated region (UTR) of the mRNA which contains a stable stem- loop structure. In preliminary studies, we developed a translational read through assay for selenoprotein synthesis using the reporter gene luciferase. The development of this system has allowed us to began to identify the sequences in PHGPx mRNA that are required for read through activity. We also identified a 120 kDa protein (SBP2) that specifically binds to the PHGPx 3' UTR. Our mutagenesis studies suggest that SBP2 plays an important role in selenoprotein biosynthesis. In this project, we will test the hypothesis that the incorporation of Sec into PHGPx involves interactions between sequences in the 3' UTR of the mRNA and SBP2.
(Aim 1) Site-directed mutagenesis and secondary structure analyses will be used to identify the sequence and structures in PHGPx mRNA that are required to decode UGA as Sec.
Aim 2) SBP2 will be purified to homogeneity by biochemical approaches, including RNA affinity chromatography. An oligonucleotide corresponding to the peptide sequence of the purified protein will be used in the polymerase chain reaction to clone the SBP cDNA. Alternatively, we will ligand screen a bacterial expression library using the 32/P-labeled PHGPx 3' UTR. Whether SBP2 is regulated by selenium or oxidative stress will also be investigated.
(Aim 3) We will investigate the mechanism of translational regulation of PHGPx biosynthesis by selenium. The results from these studies will provide insight into the mechanism and regulation of selenoprotein biosynthesis, and may identify regulatory pathways that could be used therapeutically to prevent the development of atherosclerosis by modulating selenoprotein expression in vivo.
Showing the most recent 10 out of 276 publications
