Investigation of the SBP2-ribosome interaction in Sec incorporation

说明(申请人提供)：硒是一种微量元素，其生物活性以第21位氨基酸硒半胱氨酸(SEC)的形式表现出来，并将其结合到一种称为硒蛋白的基本蛋白质类别中。许多硒蛋白是抗氧化剂，与保护DNA、脂质和蛋白质免受氧化损伤密切相关。由活性氧引起的氧化损伤与衰老和包括癌症和心血管疾病在内的许多人类疾病有关。我们研究的最终目标是彻底阐明硒半胱氨酸掺入的机制，以便我们能够反过来操纵这些有益的硒蛋白的水平。SEC的掺入代表了一个独特的过程，因为与其他氨基酸不同的是，它由一个UGA密码子编码，该密码子规范地发出翻译终止的信号。一些顺式和反式作用因子是UGA“退缩”所必需的；已知的因素包括所有硒蛋白mRNA3‘非翻译区的硒半胱氨酸插入序列(SECIS)元件，SECIS结合蛋白(SBP2)，以及结合和传递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。除了与SECIS元件结合外，SBP2还被报道在体内稳定和定量地与核糖体结合。我们的研究重点是SBP2及其与核糖体的通讯。具体地说，这一建议旨在确定SBP2与核糖体相互作用的意义，并表征核糖体上的SBP2结合部位。我们将利用体外核糖体结合试验来测试SBP2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对细胞中硒蛋白表达的影响和对体外荧光素酶报告结构中SEC掺入的影响来研究。为了确定SBP2在核糖体上的结合位置，我们将制备哺乳动物rRNA片段，并通过凝胶位移分析和竞争研究来测试它们与SBP2结合的能力。所获得的结果将有助于拼凑SEC掺入的机制，从而为在疾病的治疗和预防中仔细操纵硒蛋白的表达提供治疗靶点。

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.