Functional analysis of SBP2 and selenocysteine incorporation

描述（由申请人提供）：硒是人类饮食的重要组成部分，因为它被纳入至少25种人类蛋白质中，这些蛋白质的功能对人类健康的关键要素是必需的，包括调节炎症、男性生育能力和细胞抗氧化活性。虽然已知硒以“21”氨基酸硒半胱氨酸（Sec）的形式结合，但Sec结合发生的机制尚不清楚。迄今为止，已知在真核生物中将UGA终止密码子转化为编码Sec需要四个因子：硒蛋白mRNA中称为Sec插入序列（SECIS）元件的顺式序列，携带Sec- trnasec的Sec特异性延伸因子（eEFSec），以及SECIS结合蛋白SBP2。虽然这四个因素对Sec的成立至关重要，但它们的作用机制尚未确定。这项工作的总体目标是破译Sec合并的机制，重点关注延伸因子特异性，以及识别促进高效和进程Sec合并的新因素。我们的指导假设是，SBP2/SECIS复合物向eEFSec三元复合物（eEFSec/Sec-tRNASec/GTP）提供信号，使其能够结合核糖体a位点，并且该机制被尚未确定的因素修改以产生有效的过程性反应。为了验证这一假设，提出了三个具体目标。首先，我们将确定sec特异性延伸因子（eEFSec）特异性的决定因素。由于eEFSec的功能与典型翻译延伸相似且平行，我们建议在典型同源物eEF1A的已知功能背景下分析eEFSec的功能，包括开发新的eEFSec检测系统和确定GTP水解在Sec结合中的作用。二是确定eEFSec的职能和监管机制。SBP2/SECIS复合物对eEFSec的招募可能是Sec结合反应的组成部分，因此我们建议阐明SBP2/SECIS /eEFSec复合物形成的机制、构象后果和所需的氨基酸序列。最后，我们提出了识别和表征过程和有效的Sec整合所需的新因素。SBP2和eEFSec可能足以满足Sec的基础掺入，但这两个因素无法在体外或转染细胞中支持Sec的有效掺入。由于效率和加工能力都是体内硒蛋白生产的基本特征，我们建议确定所需的因素和顺式元件，从而扩大领域，包括基础硒结合机制的特定调节因子。这三个目标共同代表了一种综合的分子方法来确定Sec结合的机制，以实现增强硒蛋白在体内功能的长期目标。

DESCRIPTION (provided by applicant): Selenium is an essential component of the human diet because it is incorporated into at least 25 human proteins whose functions are required for key elements in human health, including the regulation of inflammation, male fertility and cellular antioxidant activity. Although it is known that selenium is incorporated in the form of the "21st" amino acid, selenocysteine (Sec), the mechanism by which Sec incorporation occurs is unknown. To date, four factors are known to be required for the conversion of a UGA stop codon into one encoding Sec in eukaryotes: a cis-sequence in the selenoprotein mRNA termed a Sec insertion sequence (SECIS) element, the Sec-specific elongation factor (eEFSec) that carries the Sec-tRNASec, and a SECIS binding protein, SBP2. While these four factors are essential for Sec incorporation, their mechanism of action has not been determined. The overall goal of this work is to decipher the mechanism by which Sec incorporation is achieved with a focus on elongation factor specificity as well as the identification of novel factors that promote efficient and processive Sec incorporation. Our guiding hypothesis is that an SBP2/SECIS complex provides a signal to the eEFSec ternary complex (eEFSec/Sec-tRNASec/GTP) that allows it to bind the ribosomal A site, and that this mechanism is modified by as-yet unidentified factors to yield an efficient and processive reaction. To test this hypothesis, three specific aims are proposed. First, we will identify the determinants for specificity in the Sec-specific elongation factor, eEFSec. Since eEFSec functions analogously and in parallel with canonical translation elongation, we propose to analyze eEFSec function in the context of the known functions of the canonical homolog eEF1A, including the development of a novel eEFSec assay system and a determination of the role GTP hydrolysis in Sec incorporation. Second, we will determine the mechanism of eEFSec function and regulation. The recruitment of eEFSec by the SBP2/SECIS complex is likely an integral part of the Sec incorporation reaction, thus we propose to elucidate the mechanism, conformational consequences and amino acid sequences required for SBP2/ SECIS/eEFSec complex formation. Finally, we propose the identification and characterization of the novel factors required for processive and efficient Sec incorporation. SBP2 and eEFSec may be sufficient for basal Sec incorporation, but these two factors are unable to support efficient Sec incorporation in vitro or in transfected cells. Since both efficiency and processivity are essential features of selenoprotein production in vivo, we propose to identify the factors and cis-elements required, thus expanding the field to include specific regulators of the basal Sec incorporation machinery. Together these three aims represent an integrated molecular approach to identifying the mechanism of Sec incorporation in order to fulfill the long term goal of enhancing selenoprotein function in vivo. PUBLIC HEALTH RELEVANCE: This proposal is designed to provide essential information regarding the processes required for the utilization of dietary selenium. Selenium is incorporated into proteins as selenocysteine by means of a unique modification of standard protein synthesis. The completion of this project will reveal the molecular mechanism involved in Sec incorporation so that the system may be used as a target for regulating selenoprotein expression so as to maximize the beneficial properties of this group of antioxidative enzymes.