Functional analysis of SBP2 and selenocysteine incorporation

硒是一种必需的微量元素，以氨基酸硒半胱氨酸（Sec）的形式存在于25种人体蛋白质中。含有硒蛋白的蛋白质对许多细胞功能至关重要，包括对抗氧化应激、甲状腺激素的产生和蛋白质折叠。Sec结合在特定的UGA密码子上，否则将表示翻译终止。已知Sec整合需要一组特殊的因子：将Sec- trnasec传递到核糖体的特殊延伸因子和与硒蛋白mRNA 3' utr中的Sec插入序列（SECIS）结合的独特RNA结合蛋白。这种secis蛋白复合物向核糖体发出信号，使其加入Sec而不是翻译终止。我们之前的工作已经提供了每个所需因子的分子特征，但它们相互作用的机制以及其他细胞成分允许Sec掺入的机制仍然未知。此外，我们提供了初步证据，表明10 Sec残基在硒转运蛋白硒蛋白P （SELENOP）中的结合需要一个独特的机制和其他因素。本提案的总体目标是确定SECIS结合蛋白促进单个和多个Sec结合事件的机制。所有脊椎动物都有两种SECIS结合蛋白，分别由不同的基因编码：SECISBP2 （SBP2）和SECISBP2L。虽然SBP2的作用机制正在成为焦点，但SECISBP2L在Sec合并中的作用尚未被破译。我们的初步数据表明，SECISBP2L对于硒蛋白p的合成至关重要。因此，我们建立了三个模型系统来研究硒蛋白的合成：体外翻译、转染的哺乳动物细胞中的表达和斑马鱼系统，这将使我们能够前所未有地了解硒蛋白在发育过程中的作用。这些还与结构生物学和转录组学相结合，以确定整个硒蛋白组的合成是如何由SECIS结合蛋白调节的。在本研究中，我们提出：1)破译SECIS元件和SECIS结合蛋白使Sec进入硒转运蛋白SELENOP的机制；2)利用斑马鱼模型系统确定SECISBP2L在体内的功能和SELENOP合成的机制；3)确定硒蛋白差异表达的分子基础。这些目标的成功完成将使我们更接近我们的长期目标，即开发能够在体内选择性激活或抑制硒蛋白合成的试剂。

Selenium is an essential trace element that is incorporated into 25 human proteins as the amino acid selenocysteine (Sec). The proteins that contain Sec (selenoproteins) are essential for many cellular functions including combatting oxidative stress, thyroid hormone production and protein folding. Sec is incorporated at specific UGA codons that would otherwise signal translation termination. A specialized set of factors are known to be required for Sec incorporation: a specialized elongation factor that delivers the Sec-tRNASec to the ribosome and unique RNA binding proteins that bind to a Sec insertion sequence (SECIS) in selenoprotein mRNA 3' UTRs. This SECIS-protein complex signals the ribosome to incorporate Sec instead of translation termination. Our prior work has provided molecular characterization of each of the required factors, but the mechanism by which they interact with each other and other cellular components to allow Sec incorporation remains unknown. In addition, we provide preliminary evidence that the processive incorporation of 10 Sec residues into the selenium transport protein Selenoprotein P (SELENOP) requires a unique mechanism and additional factors. The overall goals for this proposal are to determine the mechanism by which SECIS binding proteins promote single and multiple Sec incorporation events. All vertebrates possess two SECIS binding proteins encoded by separate genes: SECISBP2 (SBP2) and SECISBP2L. While the mechanism of action for SBP2 is coming into focus, the role for SECISBP2L in Sec incorporation has not been deciphered. Our preliminary data shows that SECISBP2L is essential for the processive incorporation of Sec into Selenoprotein P. As such, we have established three model systems to study the synthesis of SELENOP: in vitro translation, expression in transfected mammalian cells and a zebrafish system that will allow unprecedented access to the role of selenoprotein function during development. These are also leveraged and combined with structural biology and transcriptomics to determine how synthesis of the entire selenoproteome is regulated by SECIS binding proteins. In this proposal we propose to 1) Decipher the mechanism by which SECIS elements and SECIS binding proteins enable processive Sec incorporation into the selenium transport protein, SELENOP; 2) Utilize a zebrafish model system to determine the function of SECISBP2L and the mechanism of SELENOP synthesis in vivo; 3) Determine the molecular basis for differential selenoprotein expression. The successful completion of these aims will bring us significantly closer to our long term goal of developing reagents that will permit selective activation or inhibition of selenoprotein synthesis in vivo.