Investigation of the SBP2-ribosome interaction in Sec incorporation

Sec 掺入中 SBP2-核糖体相互作用的研究

• 批准号: 7476328
• 负责人: Kelvin Caban
• 金额: $ 2.83万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7476328
• 关键词: 3' Untranslated Regions; Active Sites; Aging; Amino Acids; Antioxidants; Binding; Binding Proteins; Binding Sites; Biological; Biological Assay; Cardiovascular Diseases; Cells; Class; Codon Nucleotides; Communication; Complex; DNA; DNA Insertion Elements; Defect; Elongation Factor; Eukaryota; Eukaryotic Cell; Event; Family; Gelshift Analysis; Goals; IS Elements; In Vitro; Indium; Investigation; Lipids; Luciferases; Malignant Neoplasms; Micronutrients; Mutation; Numbers; Oxidative Stress; Process; Protein Biosynthesis; Proteins; RNA Binding; Reactive Oxygen Species; Reporter; Reporting; Research; Ribosomal RNA; Ribosomes; Role; Selenium; Selenocysteine; Signal Transduction; Specific qualifier value; Structure; Testing; Trace Elements; Trans-Activators; Translations; disorder prevention; glutathione peroxidase; human disease; in vivo; mutant; selenocysteine-tRNA; selenocysteinyl-tRNA; selenoprotein; stem; therapeutic target; transposon/insertion element

DESCRIPTION (provided by applicant): Selenium is a trace element whose biological activity is manifested in the form of the 21st amino acid selenocysteine (Sec), and its incorporation into an essential class of proteins called selenoproteins. Many selenoproteins are antioxidants that are intimately involved in the protection of DNA, lipids and proteins from oxidative damage. Oxidative damage arising from reactive oxygen species has been implicated in aging and a number of human diseases including cancer and cardiovascular disease. The ultimate goal of our research is to completely elucidate the mechanism for selenocysteine incorporation so that we could in turn manipulate the levels of these beneficial selenoproteins. Sec incorporation represents a unique process because unlike other amino acids, it is encoded by a UGA codon which canonically signals for translation termination. Several cis and trans-acting factors are essential for the "receding" of UGA; the known factors include a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region of all selenoprotein mRNAs, a SECIS binding protein (SBP2), and a Sec-specific elongation factor (eEFSec) that binds and delivers the Sec-tRNA[Ser]Sec. While many of the factors have been identified, the precise mechanism and the order of events have not been determined. In addition to binding the SECIS element, SBP2 has also been reported to bind stably and quantitatively to ribosomes in vivo. Our research focuses on SBP2 and its communication with the ribosome. Specifically, this proposal seeks to determine the significance of the SBP2-ribosome interaction and to characterize the SBP2 binding site on the ribosome. We will make use of an in vitro ribosome binding assay to test SBP2 mutants for defects in ribosome binding. The functional significance of these mutations will be investigated by assessing the effect on selenoprotein expression in cells and on Sec incorporation in a luciferase reporter construct in vitro. To characterize the binding site of SBP2 on the ribosome, mammalian rRNA fragments will be generated and tested for their ability to bind SBP2 by gel shift analysis and competition studies. The results obtained will aid in piecing together the mechanism for Sec incorporation and thus provide therapeutic targets to allow for the careful manipulation of selenoprotein expression in the treatment and prevention of disease.

描述（由申请人提供）：硒是一种微量元素，其生物活性以第21位氨基酸硒代半胱氨酸（Sec）的形式表现出来，并将其掺入称为硒蛋白的必需蛋白质类中。许多硒蛋白是抗氧化剂，密切参与保护DNA、脂质和蛋白质免受氧化损伤。由活性氧引起的氧化损伤与衰老和许多人类疾病包括癌症和心血管疾病有关。我们研究的最终目标是完全阐明硒代半胱氨酸掺入的机制，以便我们可以反过来操纵这些有益的硒蛋白的水平。Sec掺入代表了一个独特的过程，因为与其他氨基酸不同，它是由UGA密码子编码的，该密码子通常发出翻译终止的信号。几种顺式和反式作用因子对于UGA的“消退”是必不可少的;已知的因子包括所有硒蛋白mRNA的3'非翻译区中的硒代半胱氨酸插入序列（SECIS）元件、SECIS结合蛋白（SBP 2）和结合并递送Sec-tRNA[Ser]Sec的Sec特异性延伸因子（eEFSec）。虽然许多因素已经确定，但确切的机制和事件的顺序尚未确定。除了结合SECIS元件之外，还报道了SBP 2在体内稳定且定量地结合核糖体。我们的研究重点是SBP 2及其与核糖体的通讯。具体而言，该建议旨在确定SBP 2-核糖体相互作用的意义，并表征核糖体上的SBP 2结合位点。我们将利用体外核糖体结合试验来测试SBP 2突变体的核糖体结合缺陷。这些突变的功能意义将通过评估硒蛋白在细胞中的表达和Sec掺入荧光素酶报告构建体在体外的影响进行研究。为了表征核糖体上SBP 2的结合位点，将产生哺乳动物rRNA片段，并通过凝胶位移分析和竞争研究测试其结合SBP 2的能力。所获得的结果将有助于将Sec掺入的机制拼凑在一起，从而提供治疗靶点，以允许在疾病的治疗和预防中仔细操纵硒蛋白的表达。