Functional analysis of SBP2 and selenocysteine incorporation

说明(申请人提供)：硒是人类饮食中的基本成分，因为它包含在至少25种人类蛋白质中，这些蛋白质的功能是人类健康所需的关键元素，包括调节炎症、男性生育和细胞抗氧化活性。虽然已知硒以“第21”氨基酸--硒半胱氨酸(SEC)的形式被掺入，但SEC掺入的机制尚不清楚。到目前为止，在真核生物中，将UGA终止密码子转换为编码SEC的终止密码子需要四个因素：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。虽然这四个因素对于证券交易委员会的成立是必不可少的，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并入的机制，重点是延长因子的特异性以及识别促进有效和过程SEC并入的新因素。我们的指导假设是，SBP2/SECIS复合体向eEFSec三元复合体(eEFSec/SEC-tRNASec/GTP)提供信号，使其能够与核糖体A位点结合，并且这一机制可被迄今未知的因素改变，以产生有效的过程反应。为了验证这一假设，本文提出了三个具体目标。首先，我们将确定SEC特异性伸长因子eEFSec的决定因素。由于eEFSec的功能与典型的翻译延伸相似且平行，我们建议在规范同系物eEF1A的已知功能的背景下分析eEFSec的功能，包括开发一种新的eEFSec分析系统，以及确定GTP水解物在SEC掺入中的作用。第二，我们将确定eEFSec的功能和调控机制。SBP2/SECIS复合体对eEFSec的募集可能是SEC掺入反应的一个组成部分，因此我们建议阐明SBP2/SECIS/eEFSec复合体形成的机制、构象结果和所需的氨基酸序列。最后，我们建议识别和表征过程中和有效的证券交易委员会成立所需的新因素。SBP2和eEFSec可能足以支持基本的SEC掺入，但这两个因素不能支持在体外或在转基因细胞中有效的SEC掺入。由于效率和可加工性都是体内生产硒蛋白的基本特征，我们建议确定所需的因子和顺式元件，从而扩大研究领域，包括基本SEC掺入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的掺入机制，以实现在体内增强硒蛋白功能的长期目标。 公共卫生相关性：本提案旨在提供有关膳食硒利用所需过程的基本信息。通过对标准蛋白质合成的独特修饰，硒以硒半胱氨酸的形式被结合到蛋白质中。该项目的完成将揭示SEC掺入的分子机制，从而可以将该系统作为调控硒蛋白表达的靶点，从而最大限度地发挥这类抗氧化酶的有益性质。

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.