Functional analysis of SBP2 and selenocysteine incorporation

描述（由申请人提供）：膳食硒作为氨基酸硒代半胱氨酸（Sec）掺入至少25种人类蛋白质中。在延伸多肽中的Sec掺入代表了标准蛋白质合成机制的修饰，因为它需要利用新的翻译延伸因子（eEFSec）、硒蛋白mRNA的3'非翻译区中的硒代半胱氨酸插入序列（SECIS）元件和称为SBP 2的新SECIS结合蛋白。这些因子协同作用，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。本提案的重点是SBP 2及其作用机制。到目前为止，功能分析已经确定，SBP 2是所需的Sec纳入，具有特定的SECIS元件结合活性，也与核糖体的物理相互作用。SBP 2的结构/功能分析表明，它由三个不同的结构域组成：一个没有已知功能的JNK-末端结构域，一个中心“功能结构域”，它是Sec掺入所需的，但不是SECIS元件结合，和一个C-末端SECIS元件结合结构域，含有在扭结-转角结合蛋白家族中发现的RNA结合基序（例如核糖体蛋白L7 Ae）。使用体外研究和基于细胞的测定的组合，提出的实验旨在破译SBP 2亚结构域内的结构/功能关系，并使用三层方法鉴定Sec掺入机制的新组分。首先，我们建议精确地定义所需的氨基酸Sec的掺入，以奠定坚实的基础，结构研究。其次，我们将开发检测方法来研究SBP 2 N-末端结构域的功能，以深入了解其在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SBP 2为中心的复合物和以硒蛋白mRNA为中心的复合物，然后进行复合物纯化和鉴定来鉴定Sec掺入复合物（SIC）的组分。 作为一个整体，这项工作将提供基本的和必要的信息有关的机制，证券交易委员会纳入-一个重要的过程，将是一个重要的目标，旨在最大限度地发挥硒蛋白功能的有益特性的战略。

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.