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: 7167444
• 负责人: PAUL R COPELAND
• 金额: $ 25.9万
• 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 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7167444
• 关键词: 3' Untranslated Regions; Active Sites; Affinity Chromatography; Amino Acids; Binding; Binding Proteins; Biological Assay; Biological Process; C-terminal; Cells; Chromatography; Code; Codon Nucleotides; Complex; Condition; DNA Insertion Elements; Dietary Selenium; Elongation Factor; Event; Family; Foundations; Glutathione S-Transferase; Goals; Human; IS Elements; In Vitro; Indium; MS2 coat protein; Mammalian Cell; Measures; Messenger RNA; Methods; Modification; Molecular; Mutagenesis; N-terminal; Oxidoreductase; Process; Property; Protein Binding; Proteins; RNA Binding; RNA Interference; RNA-Binding Proteins; Research Personnel; Ribosomal Proteins; Ribosomes; Role; Selenium; Selenocysteine; Solid; Specific qualifier value; Standards of Weights and Measures; Structure; Structure-Activity Relationship; System; Terminator Codon; Tertiary Protein Structure; Trace Elements; Trans-Activators; Translations; Triplet Multiple Birth; Work; base; cis acting element; design; in vivo; insight; mutant; novel; polypeptide; programs; research study; selenocysteine-tRNA; selenocysteinyl-tRNA; selenoprotein; stem; synthetic protein; transposon/insertion element

DESCRIPTION (provided by applicant): Dietary selenium is incorporated into at least 25 human proteins as the amino acid selenocysteine (Sec). Sec incorporation in an elongating polypeptide represents a modification of the standard protein synthetic machinery in that it requires the utilization of a novel translation elongation factor (eEFSec), a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region of selenoprotein mRNAs, and a novel SECIS binding protein termed SBP2. These factors act in concert to alter the coding potential of specific UGA codons by specifying the insertion of the Sec-specific tRNA, Sec-tRNA [Ser] Sec. The focus of this proposal is on SBP2 and its mechanism of action. To date, functional analyses have established that SBP2 is required for Sec incorporation, possesses specific SECIS element binding activity and also physically interacts with the ribosome. Structure/function analysis of SBP2 has shown that it is comprised of three distinct domain: a dispensable N-terminal domain with no known function, a central "functional domain" that is required for Sec incorporation but not SECIS element binding, and a C-terminal SECIS element binding domain containing an RNA binding motif found in the family of kink-turn binding proteins (e.g. ribosomal protein L7Ae). Using a combination of in vitro studies and cell-based assays, the experiments proposed are designed to decipher the structure/function relationships within the SBP2 subdomains and identify novel components of the Sec incorporation machinery using a three-tiered approach. First, we propose to precisely define the amino acids required for Sec incorporation in order to lay a solid foundation for structural studies. Second, we will develop assays to study the function of the SBP2 N-terminal domain in order to gain insight into its potential regulatory role in Sec incorporation. Third, we will identify components of the Sec incorporation complex (SIC) by assembling SBP2-centered and selenoprotein mRNA-centered complexes in mammalian cells followed by complex purification and identification. As a whole, this work will provide fundamental and essential information regarding the mechanism of Sec incorporation - an essential process that will be an important target for strategies designed to maximize the beneficial properties of selenoprotein function.

描述（由申请人提供）：膳食硒以氨基酸硒代半胱氨酸（Sec）的形式掺入至少 25 种人类蛋白质中。延伸多肽中的 Sec 掺入代表了对标准蛋白质合成机制的修改，因为它需要利用新型翻译延伸因子 (eEFSec)、硒蛋白 mRNA 3' 非翻译区中的硒代半胱氨酸插入序列 (SECIS) 元件以及称为 SBP2 的新型 SECIS 结合蛋白。这些因素协同作用，通过指定 Sec 特异性 tRNA、Sec-tRNA [Ser] Sec 的插入来改变特定 UGA 密码子的编码潜力。该提案的重点是 SBP2 及其作用机制。迄今为止，功能分析已确定 SBP2 是 Sec 掺入所必需的，具有特定的 SECIS 元件结合活性，并且还与核糖体发生物理相互作用。 SBP2 的结构/功能分析表明，它由三个不同的结构域组成：一个功能未知的可有可无的 N 端结构域，一个 Sec 掺入所需的中央“功能结构域”，但不需要 SECIS 元件结合，以及一个包含扭结转角结合蛋白家族（例如核糖体蛋白 L7Ae）中发现的 RNA 结合基序的 C 端 SECIS 元件结合结构域。结合体外研究和基于细胞的测定，所提出的实验旨在破译 SBP2 子域内的结构/功能关系，并使用三层方法识别 Sec 掺入机制的新组件。首先，我们建议精确定义Sec掺入所需的氨基酸，以便为结构研究奠定坚实的基础。其次，我们将开发分析方法来研究 SBP2 N 末端结构域的功能，以便深入了解其在 Sec 掺入中的潜在调节作用。第三，我们将通过在哺乳动物细胞中组装以 SBP2 为中心和以硒蛋白 mRNA 为中心的复合物，然后进行复杂的纯化和鉴定来鉴定 Sec 掺入复合物 (SIC) 的成分。 总的来说，这项工作将提供有关 Sec 掺入机制的基本和重要信息，这是一个重要的过程，将成为旨在最大限度地发挥硒蛋白功能的有益特性的策略的重要目标。