Biosynthesis of Selenocysteine and Its Incorporation into Protein

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Selenium is an essential micronutrient in the diet of humans and other mammals and it has many health benefits have been ascribed to this element including preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process, and having roles in mammalian development, male reproduction and immune function. We proposed previously that the health benefits of selenium are due in large part to the presence of selenium in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec). Since little was known about how Sec was biosynthesized, we undertook a project to elucidate how this amino acid, which is the 21st amino acid in the genetic code, was synthesized and to identify and characterize each of the components involved in the pathway. We established the biosynthetic pathway of Sec in eukaryotes and archeae (PLoS Biology 5: 96-105, 2007) and are continuing our characterization of the components responsible for Sec synthesis. During the past year, we have identified that the enzymes involved in the biosynthetic pathway of Sec can be used to synthesize Cys on the tRNA[Ser]Sec using sulfide as a substrate. The Cys-tRNA[Ser]Sec can be used to insert Cys upon decoding the codeword, UGA, in selenoprotein translation. This pathway of misincorporating Cys into protein is likely used under dietary conditions of selenium deficiency in mammalian cells. We have also generated two conditional knock-out mouse models, selenophosphate synthetase 1 (SPS1) and SECp43 knockout mice. Both mouse lines have shown that the total knockout of the corresponding gene is embryonic lethal. We have found that the SPS1 liver knockout did not affect selenoprotein metabolism while the SECp43 liver knockout is still being investigated. We are conducting embryo analysis of SPS1 knockout mice to elucidate the function of SPS1 in mammalian embryo development. We also found a new factor in mammalian cells which may be involved in tRNA[Ser]Sec modification, and this modification can dramatically increase seryl-tRNA synthetase attachment of serine to tRNA[Ser]Sec. The activity of this factor was not found to be present in yeast seryl-tRNA synthetase, and interestingly, yeast do not encode the selenocysteine biosynthesis machinery. We are purifying this factor to characterize its role in modifying tRNA[Ser]Sec.