Biosynthesis of Selenocysteine and Its Incorporation into Protein

正如我们在《硒在癌症与健康中的作用》项目中指出的那样，硒是人类饮食中一种必不可少的微量营养素，它对健康有许多好处。此外，正如在《硒在癌症与健康中的作用》项目中指出的那样，我们提出，硒对健康的益处在很大程度上是由于硒蛋白中的硒作为含硒氨基酸--硒半胱氨酸(Sec)的存在；因此，我们的计划侧重于1)SEC的生物合成和整合到蛋白质中的方法，2)建立小鼠模型，以评估硒和特定的硒蛋白在人类健康和癌症预防中的作用。在过去的一年中，我们在真核生物和古生菌中完成并发表了SEC生物合成的整个过程。进一步研究了两种硒磷酸合成酶SPS1和SPS2在SEC生物合成中的作用，这两种酶都被认为是SEC生物合成中的活性供体。我们发现SPS2确实是合成活性供体单硒磷酸的酶，而SPS1具有不同的作用，可能不参与硒的代谢。这些研究解决了硒生物学中的重要问题，即如何将硒插入SEC中，然后将其结合到蛋白质中。在过去的几年里，我们还发现，两种SEC tRNA亚型之一(命名为Um34)上34位核糖部分上的甲基负责合成与胁迫相关的硒蛋白(例如谷胱甘肽过氧化物酶1)，而另一种缺乏这种修饰的亚型参与了管家硒蛋白的合成(例如硫氧还蛋白还原酶1)。因此，我们正致力于鉴定、分离和鉴定导致Um34加入SEC tRNA的甲基酶。对甲基酶和潜在甲基酶的生物信息学分析为我们提供了几个候选基因，目前正在通过使用siRNA技术下调它们的表达来进行研究。此外，我们以前曾报道过一种名为SECp43的因子在Um34合成中发挥作用，在过去的一年里，我们产生了SECp43的条件基因敲除小鼠。这只鼠标目前正在进行角色化。

As noted in our project on the Role of Selenium in Cancer and Health, selenium is an essential micronutrient in the diet of humans and it has many health benefits. Furthermore, as also noted in the Role of Selenium in Cancer and Health project, we proposed that the health benefits are due in large part to the presence of selenium in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec); and therefore, our program focuses 1) on the means by which Sec is biosynthesized and incorporated into protein and 2) on developing mouse models to assess the role of selenium and specific selenoproteins in human health and cancer prevention. During the past year in our project on the biosynthesis of Sec, we completed and published the entire pathway of Sec biosynthesis in eukaryotes and archaea. The roles of the two selenophosphate synthetases, SPS1 and SPS2, which were both previously thought to provide the active selenium donor in the biosynthesis of Sec, were further characterized. We found that SPS2 is indeed the enzyme that synthesizes monoselenophosphate, the active donor, while SPS1 has a different role that may not be involved in selenium metabolism. These studies resolved important questions in selenium biology on how selenium is inserted into Sec to be subsequently incorporated into protein. We have also shown in the last few years that the methyl group attached to the ribosyl moiety at position 34 on one of the two Sec tRNA isoforms (designated Um34) is responsible for the synthesis of stress-related selenoproteins (e.g., glutathione peroxidase 1), while the other isoform lacking this modification is involved in the synthesis of housekeeping selenoproteins (e.g., thioredoxin reductase 1). We are therefore focusing on the identification, isolation and characterization of the methylase responsible for Um34 addition to Sec tRNA. Bioinformatic analysis of methylases and potential methylases have provided us with several candidates that are currently being examined by knocking down their expression using siRNA technology. In addition, we previously reported that a factor designated SECp43 had a role in Um34 synthesis, and in the past year, we have generated the conditional knockout mouse of SECp43. This mouse is currently being characterized.