Functional Analysis of SBP2 and Selenocysteine Incorporation

描述(申请人提供)：翻译延伸周期是将信使核糖核酸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了极高的保真度，但有一些特殊的情况重新定义了 一个给定的信使核糖核酸的编码潜力。掺入第21种氨基酸--硒半胱氨酸(SEC)就是一个例子。将UGA终止密码子转化为SEC密码子需要利用一种新的翻译延伸因子(EEFSec)、位于硒蛋白mRNAs 3‘非翻译区的硒半胱氨酸插入序列(SECIS)元件以及一种名为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-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，其中许多形成了对抗氧化应激的基本防线。这些蛋白质还负责许多其他功能，包括甲状腺激素新陈代谢和维持适当的精子活力。我们最近已经证明，已知的对证券交易委员会成立至关重要的因素实际上是充分的。这些因子包括特化延伸因子(EEFSec)、SECIS结合蛋白(SBP2)、特化转移RNA(SEC-tRNASec)和哺乳动物核糖体。虽然关于哪些蛋白质结构域对SEC掺入活性很重要的一些信息，但这些因素允许SEC掺入的机制尚不清楚。需要完全了解这一过程，原因有三：1)许多硒蛋白是必不可少的；2)SBP2基因突变会导致人类疾病；3)调节硒蛋白的产生具有巨大的潜力，可以最大限度地发挥硒蛋白的有益抗氧化作用。我们建议在这个项目中解决三个核心问题：SEC在定义的UGA密码子上掺入的精致特异性的分子基础是什么？细胞动力学如何参与SEC掺入的特异性和有效性？3‘端非编码区是如何调控10个硒半胱氨酸残基高效、连续地掺入到血浆硒载体蛋白硒蛋白P(SEPP1)中的？这些目标的成功完成将对SEC掺入的分子机制产生重大影响，为确定在临床环境中调节这一过程的方法奠定基础。

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.