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，因为它被掺入至少25种人类蛋白质中，这些蛋白质的功能是人类健康的关键要素所需的，包括调节炎症、男性生育力和细胞抗氧化活性。虽然已知硒以“第21”氨基酸硒代半胱氨酸（Sec）的形式掺入，但Sec掺入发生的机制尚不清楚。到目前为止，已知在真核生物中将UGA终止密码子转化为编码Sec的密码子需要四个因子：硒蛋白mRNA中的顺式序列，称为Sec插入序列（SECIS）元件，携带Sec-tRNASec的Sec特异性延伸因子（eEFSec），以及SECIS结合蛋白SBP 2。虽然这四个因素是必不可少的SEC纳入，其作用机制尚未确定。这项工作的总体目标是破译的机制，通过该机制，SEC的掺入实现了延伸因子的特异性，以及识别新的因素，促进有效的和进行性的SEC的掺入。我们的指导假设是，SBP 2/SECIS复合物提供了一个信号的eEFSec三元复合物（eEFSec/Sec-tRNASec/GTP），使其结合核糖体A位点，这一机制是修改的，迄今尚未确定的因素，以产生一个有效的和进行性的反应。为了检验这一假设，提出了三个具体目标。首先，我们将确定的决定因素的特异性，在秒特异性延长因子，eEFSec。由于eEFSec功能类似，并与典型的翻译延伸平行，我们建议分析eEFSec功能的背景下，已知的功能的典型同源物eEF 1A，包括开发一种新的eEFSec检测系统和确定的作用GTP水解Sec的掺入。其次，我们将确定eEFSec的功能和调节机制。通过SBP 2/SECIS复合物的eEFSec的招募可能是Sec掺入反应的一个组成部分，因此我们建议阐明SBP 2/ 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的新的因素所需的过程和有效的证券交易委员会成立。SBP 2和eEFSec可能足以用于基础Sec掺入，但这两个因子不能支持体外或转染细胞中的有效Sec掺入。由于效率和持续合成能力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，从而扩大该领域，包括特定的监管机构的基础证券公司的掺入机制。这三个目标共同代表了一个完整的分子方法来确定Sec掺入的机制，以实现增强硒蛋白在体内功能的长期目标。 公共卫生相关性：该建议旨在提供有关利用膳食硒所需过程的基本信息。硒通过标准蛋白质合成的独特修饰以硒代半胱氨酸的形式掺入蛋白质中。该项目的完成将揭示参与Sec掺入的分子机制，以便该系统可以用作调控硒蛋白表达的靶点，从而最大限度地发挥这组抗氧化酶的有益特性。

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.