Functional analysis of SBP2 and selenocysteine incorporation

SBP2 和硒代半胱氨酸掺入的功能分析

• 批准号: 8248704
• 负责人: PAUL R COPELAND
• 金额: $ 29.19万
• 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: 2006
• 资助国家: 美国
• 起止时间: 2006-01-06 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8248704
• 关键词: 3' Untranslated Regions; Address; Amino Acid Sequence; Amino Acids; Antioxidants; Binding; Binding Proteins; Biological Assay; Biological Models; Cells; Codon Nucleotides; Complex; DNA Insertion Elements; Development; Diet; Dietary Selenium; Elements; Elongation Factor; Enzymes; Eukaryota; Fertility; Goals; Guanosine Triphosphate; Health; Homologous Gene; Human; Hydrolysis; IS Elements; In Vitro; Indium; Inflammation; Insecta; Light; Mammalian Cell; Messenger RNA; Modification; Molecular; Oxidative Stress; Phase; Process; Production; Property; Protein Biosynthesis; Proteins; RNA-Binding Proteins; Reaction; Regulation; Regulatory Element; Role; Selenium; Selenocysteine; Signal Transduction; Site; Specificity; Sum; System; Terminator Codon; Testing; Translations; Work; base; cofactor; design; in vivo; insight; male; novel; public health relevance; selenoprotein

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.

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，这些蛋白质的功能是人类健康关键要素所必需的，包括调节炎症、男性生育力和细胞抗氧化活性。尽管已知硒以“第21个”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。迄今为止，已知真核生物中将 UGA 终止密码子转换为编码 Sec 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、Sec 特异性延伸因子（ eEFSec）携带 Sec-tRNASec 和 SECIS 结合蛋白 SBP2。虽然这四个因素对于 Sec 合并至关重要，但其作用机制尚未确定。这项工作的总体目标是破译实现 Sec 合并的机制，重点关注延伸因子的特异性以及促进高效和持续 Sec 合并的新因素的识别。我们的指导性假设是，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为产生有效且持续的反应。为了检验这一假设，提出了三个具体目标。首先，我们将确定 Sec 特异性延伸因子 eEFSec 特异性的决定因素。由于 eEFSec 的功能与规范翻译延伸类似且平行，因此我们建议在规范同系物 eEF1A 的已知功能的背景下分析 eEFSec 功能，包括开发新型 eEFSec 测定系统以及确定 GTP 水解在 Sec 中的作用合并。其次，我们将确定eEFSec的功能和监管机制。 SBP2/SECIS 复合物招募 eEFSec 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 复合物形成所需的机制、构象结果和氨基酸序列。最后，我们提出了持续有效的 Sec 合并所需的新因素的识别和表征。 SBP2 和 eEFSec 可能足以用于基础 Sec 掺入，但这两个因素无法支持体外或转染细胞中有效的 Sec 掺入。由于效率和持续合成能力都是体内硒蛋白生产的基本特征，因此我们建议确定所需的因子和顺式元件，从而扩大该领域以包括基础 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分子方法，用于识别 Sec 掺入机制，以实现增强体内硒蛋白功能的长期目标。 公共健康相关性：该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，硒以硒代半胱氨酸的形式掺入蛋白质中。该项目的完成将揭示Sec掺入的分子机制，从而使该系统可以作为调节硒蛋白表达的靶标，从而最大限度地发挥这组抗氧化酶的有益特性。