This proposal seeks to address the fundamental mechanism governing the insertion of selenocysteine into the enzyme phospholipid hydroperoxide gluathione peroxidase (PhGPx). The long term objective of this research is to understand the points of regulation in selenoprotein synthesis. Most selenoproteins are involved in maintaining the proper redox potential both intra- and extracellularly. Specifically, PhGPx acts to reduce oxidized lipids which are known to be toxic to cells are potentially pathogenic. A basic understanding of PhGPx synthesis will allow a manipulation of expression levels with either pharmacological agents or gene therapy. It is the insertion of the active-site selenocysteine (Sec) residue at an in-frame UGA codon that is the rate-limiting step in PhGPx synthesis, and as the activity of the enzyme is dependent on the presence of selenocysteine, the study of this prices is essential to the eventual manipulation of expression levels. Sec insertion requires the presence of a stable stem-loop structure in the 3' UTR of selenoprotein mRNAs which are believed to interact with translation elongation machinery to allow recognition and utilization of the selenocysteyl-tRNA/sec. WE have used UV crosslinking assay to identify a 120kDA protein, termed SBP2 binding also prevent Sec insertion. We hypothesize that SBP2 plays a critical role in the long-range interactions necessary for the eukaryotic ribosome to recognize specific UGA condons as those encoding selenocysteine. Specifically the work described in this proposal will aim to complete the following objectives: 1) Isolation of SBP2 protein and identification of its cognate cDNA; 2) Characterization of the function and regulation of SBP2; 3) Analysis of PhGPx translation in vitro.
| Copeland, P R; Stepanik, V A; Driscoll, D M (2001) Insight into mammalian selenocysteine insertion: domain structure and ribosome binding properties of Sec insertion sequence binding protein 2. Mol Cell Biol 21:1491-8 |
| Copeland, P R; Fletcher, J E; Carlson, B A et al. (2000) A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs. EMBO J 19:306-14 |
| Fletcher, J E; Copeland, P R; Driscoll, D M (2000) Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon. RNA 6:1573-84 |
| Copeland, P R; Driscoll, D M (1999) Purification, redox sensitivity, and RNA binding properties of SECIS-binding protein 2, a protein involved in selenoprotein biosynthesis. J Biol Chem 274:25447-54 |
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