MECHANISM OF SELENOPEROXIDASE BIOSYNTHESIS

项目2的长期目标是了解硒蛋白的合成和调节，以保护血管细胞免受氧化脂蛋白引起的脂氢过氧化损伤。硒是人体必需的微量元素，以第21位氨基酸--硒半胱氨酸(Sec)的形式存在于含硒蛋白质中。许多已知的硒蛋白是催化氧化还原反应的酶，其活性部位含有SEC。磷脂氢谷胱甘肽过氧化物酶(PHGPx)通过减少磷脂、胆固醇和胆固醇酯氢过氧化来保护细胞免受膜脂过氧化。 PHGPx对血管细胞凋亡和动脉病变发展具有保护作用的假设将在项目1中得到验证。项目2的目标是了解如何使用PHGPx作为模型来合成硒蛋白。Sec由硒蛋白基因中的UGA密码子编码。将UGA解码为SEC需要重新编程翻译，因为UGA通常被读取为终止密码子。哺乳动物硒蛋白mRNAs的翻译需要3‘非翻译区(3’UTR)，它包含一个SEC插入序列(SECIS)元件，对于将UGA解码为SEC是必不可少的。在之前的资助期间，我们纯化、克隆并鉴定了一种新的RNA结合蛋白--SECIS结合蛋白2(SBP2 蛋白质，它与SECIS元件结合。我们开发了第一个体外翻译硒蛋白mRNAs的系统，并表明SBP2是这一途径中一个必不可少的限制因子。SBP2蛋白在不同组织中的表达水平差异很大，从睾丸的高表达到在主动脉平滑肌细胞(SMC)中检测不到的水平不等。在初步研究中，我们发现了一种新的SECIS结合蛋白SBP3，它在SMC和其他组织中高表达。我们假设SBP2和SBP3对硒蛋白的合成都是必不可少的，并且它们在SEC插入中执行不同的功能。在本项目中，我们提出：1)分析SBP2的结构和功能；2)研究SEC的机制 使用我们的体外翻译系统插入；以及3)表征和鉴定SBP3。对这些目标的成功追求将有助于更好地了解哺乳动物细胞中硒蛋白的合成，并可能识别限制因素和调控途径，这些限制因素和调控途径可以通过调节体内硒蛋白的表达来用于治疗预防动脉粥样硬化的发展。

The long-term goal of Project 2 is to understand the synthesis and regulation of selenoproteins that protect vascular cells from lipid-hydroperoxide-mediated injury caused by oxidized lipoproteins. Selenium is an essential trace element, which is incorporated into selenoproteins as selenocysteine (Sec), the 21st amino acid. Many of the known selenoproteins are enzymes that catalyze oxidation-reduction reactions and contain Sec at their active site. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) protects cells against membrane lipid peroxidation by reducing phospholipid, cholesterol, and cholesterol ester hydroperoxides. The hypothesis that PHGPx is protective against vascular cell apoptosis and arterial lesion development will be tested in Project 1. The goal of Project 2 is to understand how selenoproteins are synthesized using PHGPx as a model. Sec is encoded by a UGA codon in the selenoprotein mRNA. The decoding of UGA as Sec requires the reprogramming of translation since UGA is normally read as a stop codon. The translation of mammalian selenoprotein mRNAs requires the 3' untranslated region (3' UTR), which contains a Sec Insertion Sequence (SECIS) element that is essential for the decoding of UGA as Sec. During the previous funding period, we purified, cloned, and characterized SECIS-binding Protein 2 (SBP2), a novel RNA-binding protein, which binds to the SECIS element. We developed the first system for translating selenoprotein mRNAs in vitro and showed that SBP2 is an essential and limiting factor in this pathway. The levels of SBP2 protein vary dramatically between tissues, ranging from high expression in testis to undetectable levels in aortic smooth muscle cells (SMC). In preliminary studies, we identified a new SECIS-binding protein, SBP3, which is highly expressed in SMC and other tissues. We hypothesize that SBP2 and SBP3 are both essential for selenoprotein synthesis and that they perform separate functions in Sec insertion. In this project, we propose to: 1) analyze the structure and function of SBP2; 2) investigate the mechanism of Sec insertion using our in vitro translation system; and 3) characterize and identify SBP3. The successful pursuit of these aims will lead to a better understanding of selenoprotein synthesis in mammalian cells and may identify limiting factors and regulatory pathways that could be used therapeutically to prevent the development of atherosclerosis by modulating selenoprotein expression in vivo.