Functional Analysis of SBP2 and Selenocysteine Incorporation

描述（由申请人提供）：翻译延伸循环是mRNA正确解码为蛋白质所需的中心过程。尽管在这种反应中观察到的保真度极高，但仍有特殊情况重新定义了 一个给定的mRNA的编码潜力。第21个氨基酸硒代半胱氨酸（Sec）的掺入就是一个例子。将UGA终止密码子转化为Sec密码子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的新的SECIS结合蛋白。这些因子协同作用，通过指定Sec特异性tRNA（Sec-tRNASec）的插入来改变特定UGA密码子的编码潜力。这一过程是生产25种人类硒蛋白所必需的，其中许多硒蛋白是抵御氧化应激的重要防线。这些蛋白质还负责无数其他功能，包括甲状腺激素代谢和维持正常的精子活力。我们最近已经证明，SEC成立所必需的已知因素实际上是足够的。这些因子包括特化延伸因子（eEFSec）、SECIS结合蛋白（SBP 2）、特化转移RNA（Sec-tRNASec）和哺乳动物核糖体。虽然有一些关于哪些蛋白质结构域对Sec掺入活性很重要的信息，但这些因素允许Sec掺入的机制尚不清楚。有三个关键原因需要对这一过程进行全面了解：1）许多硒蛋白是必不可少的; 2）SBP 2基因突变导致人类疾病; 3）硒蛋白产生的调节具有最大限度地发挥硒蛋白有益的抗氧化作用的巨大潜力。我们建议在这个项目中解决三个核心问题：什么是精细的特异性SEC纳入在定义的UGA密码子的分子基础？细胞动力学如何参与Sec掺入的特异性和效率？3'非翻译区如何调节10个硒代半胱氨酸残基有效地和进行性地掺入血浆硒载体蛋白硒蛋白P（SEPP 1）？这些目标的成功完成，将揭示显着的Sec纳入的分子机制，为确定该过程可以在临床环境中进行调节的手段奠定了基础。

DESCRIPTION (provided by applicant): The translation elongation cycle is the central process required for the correct decoding of mRNA into protein. Despite the extremely high level of fidelity that has been observed in this reaction, there are special circumstances that redefine the coding potential of a given mRNA. The incorporation of the 21st amino acid, selenocysteine (Sec), is one example. The transformation of a UGA stop codon into a Sec codon 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-tRNASec. This process is required for the production of 25 human selenoproteins, many of which form an essential line of defense against oxidative stress. The proteins are also responsible for a myriad of other functions including thyroid hormone metabolism and maintaining proper sperm motility. We have recently demonstrated that the factors known to be essential for Sec incorporation are, in fact, sufficient. These factors include a specialized elongation factor (eEFSec), a SECIS binding protein (SBP2), a specialized transfer RNA (Sec-tRNASec) and mammalian ribosomes. Although some information about which protein domains are important for Sec incorporation activity, the mechanism by which these factors allow Sec incorporation is not known. A complete understanding of this process is required for three key reasons: 1) many selenoproteins are essential; 2) mutations in the SBP2 gene cause human disease; 3) modulation of selenoprotein production has tremendous potential to maximize the beneficial antioxidant effects of selenoproteins. We propose to address three central questions in this project: What is the molecular basis for the exquisite specificity of Sec incorporation at defined UGA codons? How are cellular dynamics involved in the specificity and efficiency of Sec incorporation? How does the 3' UTR regulate the efficient and processive incorporation of 10 selenocysteine residues into the plasma selenium carrier protein selenoprotein P (SEPP1)? Successful completion of these Aims will shed significant light on the molecular mechanism of Sec incorporation, setting the stage for identifying the means by which the process can be regulated in a clinical setting.