Biosynthesis of Selenocysteine and Its Incorporation into Protein

硒代半胱氨酸的生物合成及其与蛋白质的结合

• 批准号: 8937830
• 负责人: Dolph Hatfield
• 金额: $ 45.38万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8937830
• 关键词: 2' -O-methyluridine; Acquired Immunodeficiency Syndrome; Aging-Related Process; Amino Acids; Anabolism; Archaea; Attention; Bioinformatics; Cardiovascular Diseases; Cattle; Cells; Collaborations; Cysteine; Development; Diet; Dietary Selenium; Disease; Elements; Eukaryota; Euplotes; Euplotes crassus; Genes; Genetic Code; Genome; Glycine decarboxylase; Goals; HIV Seropositivity; HL-60 Cells; Health Benefit; Heart Diseases; Housekeeping; Human; Laboratories; Liver; Malignant Neoplasms; Mammals; Methyltransferase; Micronutrients; Mus; Myopathy; Nucleotides; Open Reading Frames; Organism; Pathway interactions; Patients; Play; Protein Biosynthesis; Protein Isoforms; Proteins; Reaction; Reporting; Reproduction; Ribosomal Frameshifting; Role; Selenium; Selenocysteine; Stress; Transfer RNA; Translating; Uridine; Viral; Work; genome sequencing; immune function; insight; male; methyl group; prevent; selenocysteine-tRNA

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.

硒是人类和其他哺乳动物饮食中必不可少的微量营养素。这一因素已归因于许多健康益处。这些健康益处包括预防癌症，心脏病和其他心血管疾病和肌肉疾病，抑制病毒表达，延迟艾滋病毒阳性患者的艾滋病进展，减慢衰老过程，并在哺乳动物发育，男性繁殖和免疫功能中起作用。几年前，我们提出硒的健康益处是由于硒蛋白中硒作为硒代半胱氨酸（SEC）的存在，这是含有硒的氨基酸。我们以前报道了真核生物和古细菌中SEC的生物合成途径，SEC是遗传密码中的第21个氨基酸。我们最近的关注集中在我们所展示的两个SEC tRNA同工型上，是造成硒蛋白指定管家和与压力相关的硒蛋白的两个亚类的合成。此外，我们以前曾在甲基酶的甲基化合物中合成甲基在2'-O-postion上的甲基基团在SEC tRNA的核苷34上合成甲基。我们提供了有力的证据表明，将UM34添加到同工型，5-甲基羟基甲基 - 尿尿酸尿素（MCMU）中形成5-甲基羧甲基，2'-O-O-甲基尿疗（MCMUM），要求MCMU用SEC氨基酰基氨基酰基，即以Sec氨基糖化，以使其属于IS甲基酶（即甲基酶），以甲基酶（甲基酶）为甲基酶（甲基酶），以甲基酶（甲基酶）的反应。 Selenocysteyl-tRNA。在过去的一年中，我们从牛肝脏中纯化了这两种同工型，并通过质量分光光度计分析对其进行了表征。 MCMU同工型完全完整，但是MCMUM同工型在完全完整的同工型中都存在，并且是没有CCA末端的同工型的。我们正在研究如何合成这两种同工型，并在HL60细胞中的硒蛋白合成过程中翻转。 HL60细胞高度富集在SEC tRNA中。在硒存在下生长的细胞中，与应激相关的硒蛋白高度合成，MCMUM同工型高度富集在这些细胞中，而在缺乏硒的情况下，家政硒蛋白主要合成，MCMU同工型富集。当HL60细胞在存在和不存在硒的情况下生长时，两种同工型的质量分光光度法分析应进一步了解两种同工型如何合成并翻转。此外，通过生物信息学研究了euplotes crassus和crassus和Euplotes focardi的基因组和他的小组的合作，并通过生物信息学研究了基因的开放式读数框架的很大比例，这些基因的开放式读数框架被发现由核糖体帧片剂翻译。 Gladyshev博士的实验室先前已经对这两种生物种的基因组进行了测序。此外，在与Gladyshev博士及其小组的额外合作中，硒蛋白P的硒含量与饮食有关。硒蛋白P中的硒量随小鼠饮食中的硒量而变化，饮食中的硒水平越低，硒代蛋白在硒蛋白P中用半胱氨酸的替代量就越大。