Functional analysis of SBP2 and selenocysteine incorporation

硒是人体必需的微量元素，以硒半胱氨酸(Sec)的形式存在于人体的25种蛋白质中。含有Sec(硒蛋白)的蛋白质对许多细胞功能是必不可少的，包括对抗氧化应激、甲状腺激素的产生和蛋白质折叠。SEC被并入特定的UGA密码子，否则将发出翻译终止的信号。已知SEC掺入需要一组特殊的因子：将SEC-tRNASec运送到核糖体的特殊延伸因子，以及与Sec插入序列(SECI)结合的独特RNA结合蛋白。这种SECIS-蛋白质复合体向核糖体发出信号，使其结合SEC而不是翻译终止。我们之前的工作已经提供了每个必需因子的分子特征，但它们相互作用和其他细胞成分允许SEC掺入的机制仍不清楚。此外，我们提供了初步证据表明，将10秒残基连续掺入到硒转运蛋白硒蛋白P(SELENOP)中需要一个独特的机制和附加因素。这项提案的总体目标是确定SECIS结合蛋白促进单个和多个SEC掺入事件的机制。所有脊椎动物都有两个由不同基因编码的SECIS结合蛋白：SECISBP2(SBP2)和SECISBP2L。虽然SBP2的作用机制已经成为人们关注的焦点，但SECISBP2L在SEC合并中的作用尚未破译。我们的初步数据显示，SECISBP2L对于Sec在硒蛋白P中的持续整合是必不可少的。因此，我们建立了三个模型系统来研究SELENOP的合成：体外翻译、在转基因哺乳动物细胞中的表达和斑马鱼系统，该系统将允许前所未有地获得硒蛋白在发育过程中的作用。这些也被利用，并与结构生物学和转录组学相结合，以确定整个硒蛋白质组的合成如何受到SECIS结合蛋白的调节。在这项研究中，我们建议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来确定SECISBP2L的功能和体内SELENOP的合成机制；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，即开发能够选择性激活或抑制体内硒蛋白合成的试剂。

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.