Biosynthesis of Selenocysteine and Its Incorporation into Protein

硒是人类和其他哺乳动物饮食中必需的微量营养素。许多健康益处都归功于这一元素。这些健康益处包括预防癌症、心脏病和其他心血管和肌肉疾病，抑制病毒表达，延缓艾滋病毒阳性患者艾滋病的进展，减缓衰老过程，并在哺乳动物发育、男性生殖和免疫功能中发挥作用。几年前，我们提出硒的健康益处是由于硒蛋白中硒半胱氨酸（Sec）的存在，硒半胱氨酸是一种含有硒的氨基酸。我们以前报道过真核生物和古细菌中遗传密码中第21个氨基酸Sec的生物合成途径。我们最近的注意力集中在两个Sec tRNA异构体上，我们已经证明它们负责硒蛋白的两个亚类的合成，即家政硒蛋白和应激相关硒蛋白。此外，我们之前研究了甲基化酶，称为Um34甲基化酶，它合成了Sec tRNA核苷酸34核糖基上2'- o位置的甲基。我们提供了强有力的证据，证明将Um34添加到5-甲基甲氧基甲基尿嘧啶（mcmU）上以形成5-甲基羧甲基，2'- o -甲基尿嘧啶（mcmUm）需要mcmU与Sec氨基酰化，即进行该反应的甲基化酶（指定为Um34甲基化酶）的底物是硒代半胱氨酸- trna。在过去的一年里，我们从牛肝脏中纯化了这两种异构体，并通过质谱分析对它们进行了表征。mcmU异构体是完全完整的，但mcmUm异构体既存在于完全完整的异构体中，也意外地存在于没有CCA末端的异构体中。我们正在研究这两种异构体是如何在HL60细胞中合成硒蛋白的过程中被合成和翻转的。HL60细胞高度富集Sec tRNA。在有硒存在的细胞中，应激相关的硒蛋白高度合成，mcmU异构体在这些细胞中高度富集，而在没有硒的情况下，家务活硒蛋白主要合成，mcmU异构体富集。当HL60细胞在存在和不存在硒的情况下生长时，对这两种异构体的质量分光光度分析应该能进一步了解这两种异构体是如何合成和转化的。此外，与Vadim Gladyshev博士和他的团队合作，通过生物信息学检查了克拉苏Euplotes和佛卡地Euplotes的基因组，发现很高比例的基因开放阅读框是通过核糖体移框翻译的。格拉迪舍夫博士的实验室此前已经对这两种生物体的基因组进行了测序。此外，在与Gladyshev博士及其团队的另一项合作中，研究了硒蛋白P的硒含量与饮食的关系。硒蛋白P中硒的含量随小鼠日粮硒含量的变化而变化，硒水平越低，硒蛋白P中硒半胱氨酸被半胱氨酸替代的程度越大。

Selenium is an essential micronutrient in the diet of humans and other mammals. Many health benefits have been ascribed to this element. These health benefits include 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. Several years ago, we proposed that the health benefits of selenium are due to the presence of selenium in selenoproteins as selenocysteine (Sec), which is the amino acid that contains selenium. We previously reported the biosynthetic pathway of Sec, the 21st amino acid in the genetic code, in eukaryotes and Archaea. Our more recent attention has focused on the two Sec tRNA isoforms that we have shown are responsible for the synthesis of the two subclasses of selenoproteins designated housekeeping and stress-related selenoproteins. In addition, we previously worked on the methylase, designated Um34 methylase, that synthesizes the methyl group at the 2'-O-postion on the ribosyl moiety at nucleotide 34 of Sec tRNA. We provided strong evidence that addition of Um34 to the isoform, 5-methylcarobxymethyl-uridine (mcmU), to form 5-methylcarboxymethyl, 2'-O-methyluridine (mcmUm) requires that mcmU is aminoacylated with Sec, i.e., that the substrate for the methylase (designated Um34 methylase) which carries out this reaction is selenocysteyl-tRNA. In the past year, we have purified these two isoforms from bovine liver and characterized them by mass spectrophometric analysis. The mcmU isoform was totally intact, but the mcmUm isoform existed in both the fully intact isoform and unexpectedly as an isoform without the CCA terminus. We are examining how these two isoforms are synthesized and are turned over during selenoprotein synthesis in HL60 cells. HL60 cells are highly enriched in Sec tRNA. In cells grown in the presence of selenium, stress-related selenoproteins are highly synthesized and the mcmUm isoform is highly enriched in these cells, while in the absence of selenium, housekeeping selenoproteins are primarily synthesized and the mcmU isoform is enriched. Mass spectrophotometric analysis of the two isoforms, when HL60 cells are grown in the presence and absence of selenium, should provide further insight into how the two isoforms are synthesized and turned over. In addition, and in collaboration with Dr. Vadim Gladyshev and his group, the genome of Euplotes crassus and Euplotes focardii were examined by bioinformatics and a high percentage of the open reading frames of genes were found to be translated by ribosomal frameshifting. Dr. Gladyshev's laboratory had previously sequenced the genomes of both of these species of organisms. Furthermore, and in an additional collaboration with Dr. Gladyshev and his group, the selenium content of selenoprotein P was examined in relation to diet. The amount of selenium in selenoprotein P varied with the amounts of selenium in the diet of mice and the lower the levels of dietary selenium, the greater the replacement of selenocysteine with cysteine in selenoprotein P.