Biosynthesis of Selenocysteine and Its Incorporation into Protein

硒是人类和其他哺乳动物饮食中的必需微量营养素，并且许多健康益处已归因于该元素，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HIV阳性患者中的AIDS进展，减缓衰老过程，以及在哺乳动物发育、男性生殖和免疫功能中发挥作用。我们以前提出，硒的健康益处在很大程度上是由于硒蛋白中存在硒作为含硒氨基酸，硒代半胱氨酸（Sec），由于Sec生物合成的途径尚未在古细菌和真核生物中确定，我们进行了一个项目，以阐明这种氨基酸，这是遗传密码中的第21个氨基酸，合成并鉴定和表征所涉及的每种组分。我们以前建立了真核生物和古生菌中Sec的生物合成途径，现在将我们的注意力集中在两种Sec tRNA异构体上，我们已经证明这两种异构体负责合成两个亚类的硒蛋白，命名为管家蛋白和应激相关硒蛋白;以及甲基化酶，命名为Um 34甲基化酶，其在2？在Sec tRNA的核苷酸34处的核糖基部分上的O-位置。我们先前证实，将Um 34添加到同种型5-甲基羧甲基尿苷（mcmU）中，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，氨酰化的研究完成并发表，它提供了重要的见解，我们了解如何亚类硒蛋白，指定的压力相关硒蛋白，是生物合成的mcmUm异构体特异性。我们确定了甲基化酶，指定Um 34甲基化酶，这是负责添加的2？在位置34处将0-甲基基团连接至核糖基部分。Um 34甲基化酶基因已被克隆到表达载体中，在细菌和哺乳动物细胞中表达，分离出蛋白质产物，目前正在对其进行表征。Um 34甲基化酶在其氨基末端具有线粒体信号。我们已经表明，甲基化酶位于线粒体中，其中约20个氨基酸的线粒体信号被切割时，蛋白质进入这个细胞器。我们目前正在开发一种检测方法，以进一步表征Um 34甲基化酶使用mcmU Sec tRNA异构体作为底物和S-腺苷甲硫氨酸作为甲基donor.In的，我们以前证明，半胱氨酸可以取代硫氧还蛋白还原酶1（TR 1）中的Sec，并在过去的一年中广泛扩展了这些研究。我们正在研究在癌细胞和用抗生素处理的细胞中，半胱氨酸取代Sec和其他氨基酸误译Sec UGA密码子。我们发现抗生素强力霉素（Dox）、氯霉素（Cp）和遗传霉素（G418）通过抑制真核细胞中的Sec插入步骤来阻断硒蛋白的合成。在这项研究中，我们专注于三个硒蛋白，TR 1，谷胱甘肽过氧化物酶1（GPx 1）和谷胱甘肽过氧化物酶4（GPx 4），因为Sec密码子被发现在不同的位置在每个蛋白质和蛋白质显示差异使用的Sec tRNA异构体，mcmU和mcmUm。所有三种抗生素诱导TR 1和GPx 4的蛋白质表达，如通过蛋白质印迹分析测量的，而GPx 1表达在治疗后降低。然而，硒-75标记和酶的测定表明，所有三个硒蛋白的表达和活性下降。质谱分析纯化的，His-标记的重组TR 1，GPx 1和GPx 4显示，Sec残基被替换为其他氨基酸，如半胱氨酸，精氨酸和色氨酸，在一个依赖性的方式，因此误读发生，而不是Cys/Sec替换。在人类肿瘤组织中，我们发现TR 1蛋白在肝和肺肿瘤组织中的表达与周围正常组织相比增加，而TR 1在肿瘤中的活性与周围正常组织相比略有下降。从肿瘤和周围正常组织中纯化的TR 1的质谱分析显示，在肿瘤组织中插入半胱氨酸代替Sec或蛋白质在Sec密码子处被截短。

Selenium is an essential micronutrient in the diet of humans and other mammals and 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), and since the pathway for Sec biosynthesis had not been determined in archaea and eukaryotes, 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. We previously established the biosynthetic pathway of Sec in eukaryotes and archaea and are now focusing our attention 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; and 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 previously confirmed that addition of Um34 to the isoform, 5-methylcarobxymethyluridine (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, the aminoacylation study was completed and published and it provided important insights into our understanding of how the subclass of selenoproteins, designated stress-related selenoproteins, is biosynthesized specifically by the mcmUm isoform.We identified the methylase, designated Um34 methylase, that is responsible for the addition of the 2?-O-methyl group to the ribosyl moiety at position 34. The Um34 methylase gene has been cloned into expression vectors, expressed in bacterial and mammalian cells, the protein product isolated and it is currently being characterized. Um34 methylase has a mitochondrial signal at its amino terminus. We have shown that the methylase is localized in the mitochondria, wherein the approximately 20 amino acid mitochondrial signal is cleaved when the protein enters this organelle. We are currently developing an assay to further characterize Um34 methylase using the mcmU Sec tRNA isoform as substrate and S-adenosylmethionine as the methyl donor.In addition, we previously demonstrated that cysteine could replace Sec in thioredoxin reductase 1 (TR1) and have expanded these studies extensively in the past year. We are examining the replacement of Sec by cysteine and the mistranslation of the Sec UGA codon by other amino acids in cancer cells and in cells treated with antibiotics. We found that the antibiotics doxycycline (Dox), chloramphenicol (Cp) and Geneticin (G418) block selenoprotein synthesis by inhibiting the Sec insertion step in eukaryotic cells. In this study, we focused on three selenoproteins, TR1, glutathione peroxidase 1 (GPx1) and glutathione peroxidase 4 (GPx4), because the Sec codon is found at a different position in each protein and the proteins show differential use of the Sec tRNA isoforms, mcmU and mcmUm. All three antibiotics induced the protein expression of TR1 and GPx4 as measured by western blot analysis, while GPx1 expression decreased upon treatment. However, selenium-75 labeling and enzymatic assays showed a decrease in expression and activity of all three selenoproteins. Mass spec analysis of purified, His-tagged recombinant TR1, GPx1 and GPx4 revealed that Sec residues were replaced by other amino acids, such as cysteine, arginine and tryptophan, in an antibiotic-dependent manner and thus misreading was occurring and not Cys/Sec replacement. In human cancer tissues, we found that TR1 protein expression was increased in both liver and lung tumor tissues compared with surrounding normal tissues, whereas the activities of TR1 in tumors were slightly decreased compared with surrounding normal tissue. Mass spec analysis of purified TR1 from tumors and surrounding normal tissue revealed that cysteine is inserted in place of Sec in tumor tissues or the protein was truncated at the Sec codon.