MECHANISM OF SELENOPEROXIDASE BIOSYNTHESIS

脂蛋白的自由基氧化修饰被认为是 成为……发起和发展中的一件大事 动脉硬化。氧化型脂蛋白的致动脉粥样硬化作用 可能部分与它们的细胞毒性有关。这个项目的目标是 了解可能保护血管的硒蛋白的生物合成 过氧化脂质介导的细胞损伤 脂蛋白。这个项目的重点是过氧化氢磷脂。 谷胱甘肽过氧化物酶(PHGPx)，一种硒过氧化物酶 磷脂胆固醇和胆固醇酯氢氧化物。PHGPx和 其他感光蛋白是通过一种新的途径合成的，该途径涉及 硒半胱氨酸(SEC)残基的共翻译掺入 对信使核糖核酸中UGA密码子的反应。这一机制涉及 重新编制翻译程序，因为UGA通常作为翻译程序进行 停止密码子。在真核生物中，UGA的识别是SEC密码子所要求的 该基因的3‘非翻译区含有一个稳定的茎-- 循环结构。在初步研究中，我们开发了一种翻译阅读 利用报告基因检测硒蛋白的合成 荧光素酶。这个系统的发展已经让我们开始 确定PHGPx mRNA中阅读所需的序列 活动。我们还鉴定了一个120 kDa的蛋白(SBP2)，它能特异性地 与PHGPx 3‘非编码区结合。我们的突变研究表明SBP2在 在硒蛋白生物合成中起着重要作用。在这个项目中，我们将 测试SEC并入PHGPx涉及的假设 基因3‘端非编码区序列与SBP2的相互作用(目标1) 将使用定点突变和二级结构分析来 确定PHGPx mRNA中需要的序列和结构 将UGA解码为SEC。目标2)SBP2将通过以下方式提纯至均一 生化方法，包括RNA亲和层析。一个 与纯化的多肽序列相对应的寡核苷酸 蛋白质将用于聚合酶链式反应来克隆SBP CDNA.或者，我们将配基筛选细菌表达文库 使用32/P标记的PHGPx 3‘非编码区。SBP2是否受硒调控 或者，氧化应激也将被调查。(目标3)我们会进行调查 PHGPx生物合成的翻译调控机制 硒。这些研究的结果将为我们提供对 硒蛋白生物合成的机制和调控，并可能识别 可用于治疗的调控途径可用于预防 硒蛋白表达调控动脉粥样硬化的研究进展 活着。

The modification of lipoproteins by free radical oxidation is proposed to be an important event in the initiation and development of atherosclerosis. The pro-atherogenic properties of oxidized lipoproteins may in part be related to their cytotoxicity. The goal of this project is to understand the biosynthesis of selenoproteins that may protect vascular cells from lipid hydroperoxide-mediated injury caused by oxidized lipoproteins. This project will focus on phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenoperoxidase that reduces phospholipid cholesterol, and cholesterol ester hydroperoxides. PHGPx and other seneloproteins are synthesized by a novel pathway that involves the co-translational incorporation of a selenocysteine (Sec) residue in response to a UGA codon in the mRNA. This mechanism involves the reprogramming of translation since UGA is normally lead as a translational stop codon. In eukaryotes, the recognition of UGA is a Sec codon requires the 3' untranslated region (UTR) of the mRNA which contains a stable stem- loop structure. In preliminary studies, we developed a translational read through assay for selenoprotein synthesis using the reporter gene luciferase. The development of this system has allowed us to began to identify the sequences in PHGPx mRNA that are required for read through activity. We also identified a 120 kDa protein (SBP2) that specifically binds to the PHGPx 3' UTR. Our mutagenesis studies suggest that SBP2 plays an important role in selenoprotein biosynthesis. In this project, we will test the hypothesis that the incorporation of Sec into PHGPx involves interactions between sequences in the 3' UTR of the mRNA and SBP2. (Aim 1) Site-directed mutagenesis and secondary structure analyses will be used to identify the sequence and structures in PHGPx mRNA that are required to decode UGA as Sec. Aim 2) SBP2 will be purified to homogeneity by biochemical approaches, including RNA affinity chromatography. An oligonucleotide corresponding to the peptide sequence of the purified protein will be used in the polymerase chain reaction to clone the SBP cDNA. Alternatively, we will ligand screen a bacterial expression library using the 32/P-labeled PHGPx 3' UTR. Whether SBP2 is regulated by selenium or oxidative stress will also be investigated. (Aim 3) We will investigate the mechanism of translational regulation of PHGPx biosynthesis by selenium. The results from these studies will provide insight into the mechanism and regulation of selenoprotein biosynthesis, and may identify regulatory pathways that could be used therapeutically to prevent the development of atherosclerosis by modulating selenoprotein expression in vivo.