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种元素。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，延缓艾滋病病毒阳性患者的艾滋病进展，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存在硒，如硒代半胱氨酸（Sec），这是含有硒的氨基酸。我们以前报道了Sec（遗传密码中的第21个氨基酸）在真核生物和真核生物中的生物合成途径。我们最近的注意力集中在两个Sec tRNA异构体，我们已经表明是负责的两个亚类的硒蛋白指定管家和应力相关的硒蛋白的合成。此外，我们先前研究了甲基化酶，命名为Um 34甲基化酶，其在Sec tRNA的核苷酸34处的核糖基部分上的2 '-O-位置合成甲基。我们提供了强有力的证据，证明将Um 34添加到同种型5-甲基羧甲基尿苷（mcmU）中以形成5-甲基羧甲基，2 '-O-甲基尿苷（mcmUm）需要mcmU被Sec氨酰化，即，进行该反应的甲基化酶（命名为Um 34甲基化酶）的底物是硒代半胱氨酸-tRNA。在过去的一年中，我们已经从牛肝脏中纯化了这两种异构体，并通过质谱分析对其进行了表征。mcmU同种型是完全完整的，但mcmUm同种型以完全完整的同种型和意外地作为没有CCA末端的同种型存在。我们正在研究这两种异构体是如何合成的，并在HL 60细胞的硒蛋白合成过程中被翻转。HL 60细胞高度富集Sec tRNA。在硒存在下生长的细胞中，应激相关的硒蛋白高度合成，并且mcmU同种型在这些细胞中高度富集，而在硒不存在下，管家硒蛋白主要合成，并且mcmU同种型富集。当HL 60细胞在硒存在和不存在的情况下生长时，对这两种异构体的质谱分光光度分析应该提供对这两种异构体如何合成和转化的进一步了解。此外，与Vadim Gladyshev博士及其团队合作，通过生物信息学检查了厚游仆虫和Euplotes focardii的基因组，发现高比例的基因开放阅读框架通过核糖体移码翻译。Gladyshev博士的实验室之前已经对这两种生物的基因组进行了测序。此外，在与Gladyshev博士及其小组的另一次合作中，研究了硒蛋白P的硒含量与饮食的关系。硒蛋白P中硒的含量随饲料硒含量的变化而变化，饲料硒含量越低，硒蛋白P中半胱氨酸对硒代半胱氨酸的替代作用越大。

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.