Selenium has a role in preventing various forms of cancer (e.g., colon, prostate, lung and liver), heart disease and other cardiovascular and muscle disorders. It also serves as an antiviral agent and plays a role in delaying the aging process, in delaying the progression of AIDS in HIV positive patients, in immune function, in mammalian development and in male reproduction. Although the underlying metabolic effects of selenium are poorly understood, they are due, at least in part, to the presence of this element in selenoproteins as the amino acid, selenocysteine (Sec). Our program focuses on the means by which Sec is incorporated into protein and the role of specific selenoproteins in human health and development. In the last year, we discovered the gene for an important enzyme in Sec biosynthesis and characterized its product, phosphoseryl-tRNASerSec kinase (PSTK). In addition, we are using RNAi to knock down several selenoproteins and other proteins involved in Sec biosynthesis (e.g., SLA, PSTK, SPS1 and 2) to further elucidate their roles in selenium metabolism and determine how this element is incorporated into protein. Since Sec tRNA is the only known tRNA that governs the expression of an entire class of proteins, the selenoproteins, we have perturbed Sec tRNA synthesis to better understand the role of selenium and selenoproteins in health. Over the last few years, we have generated transgenic, knockout, conditional knockout and knockdown cells and mice involving the Sec tRNA gene to provide models for explicating the role of selenoproteins in health and development. We rescued standard Sec tRNA gene knockout mice that are embryonic lethal with transgenic mice carrying either a wild type or mutant Sec tRNA transgene and analyzed which selenoproteins are rescued. The mutant Sec tRNA is incapable of forming a methyl group at the 2'-O-hydroxy position on the ribose in position 34 which is the last step in the maturation of Sec tRNA. Mice rescued with the wild type transgene expressed all selenoproteins normally, but mice rescued with the mutant transgene did not express several stress-related selenoproteins, but expressed housekeeping selenoproteins in normal or reduced amounts. This novel regulation of protein expression occurred at the level of translation and manifested a tissue-specific pattern. The data indicate that Sec tRNA methylation at the 2'-O-hydroxylribosyl site plays a role in regulating the expression of various mammalian selenoproteins and is required for synthesis of non-essential, stress-related selenoproteins. Using loxP-Cre technology, we mated the conditional knockout mice with different Cre mice to selectively remove the Sec tRNA gene (and therefore alter selenoprotein expression) in various tissues including liver, prostate, breast, endothelial cells and skeletal and heart muscle. In this way, we can assess the role of selenoproteins in development, in tissue and organ function and in health. This past year we have knocked out selenoprotein expression in endothelial cells and heart and skeletal muscle. Animals lacking selenoprotein expression in endothelial cells died after 14.5 days dpc from multiple abnormalities, while those animals lacking selenoprotein expression in heart and skeletal muscle died abruptly at about 10 days after birth from heart disease. The latter studies clearly show that selenoproteins are involved in the development and normal function of endothelial cells and heart muscle. The conditional knockout of Sec tRNA in specifically targeted tissues and organs provides an excellent model for examining the role of selenoproteins in development and health.
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