Incorporation of selenocysteine into proteins

在哺乳动物细胞中，第21个氨基酸硒半胱氨酸(Sec)的掺入是由硒小体引导的。这种蛋白质复合体的功能需要大量的蛋白质-蛋白质和蛋白质-RNA相互作用，导致硒半胱氨酸在UGA密码子上掺入，由硒蛋白编码基因3‘-非翻译区的茎环结构引导。我们研究计划的长期目标是了解SEC整合到蛋白质中的机制。这一过程对哺乳动物细胞和许多细菌的新陈代谢至关重要，例如对于细菌发酵。在我们的初步工作中，我们使用基于生物发光共振能量转移(BRET)的体内实验研究了硒小体所有已知成分之间的相互作用。我们还使用了生化方法来验证这些相互作用，并使用生化和遗传方法进一步表征这些相互作用，从而发现了SEC生物合成机制的组件之间的新的相互作用。这些结果导致了我们下一个资助期工作的指导性假设：SPS1、SPS2、SECS和SECp43之间的一系列蛋白质-蛋白质相互作用指导硒进入其特定的tRNA。这些反应之后，SEC转移到延伸因子上，并加入到不断增长的多肽链中。我们建议使用生物化学、细胞生物学、分子生物学和结构生物学的前沿方法，以高分辨率研究SEC掺入途径的这些早期步骤。 具体目的1.用噬菌体展示和生物化学相结合的方法研究SPS1、SPS2、SECS和SECp43之间的相互作用。 具体目标2.用X射线结晶学和单粒子低温电子显微镜分析SPS1-SPS2-SECS-SECp43相互作用的原子分辨率水平。 特定目的3.哺乳动物细胞内SPS1、SPS2、SECS、SECp43与其他硒小体成分动态相互作用的体内分析。 我们的长期计划将揭示SEC精确结合到蛋白质中的分子基础，我们将获得硒小体的原子分辨结构。此外，我们还将使用最高分辨率成像技术显示活细胞中SEC的掺入情况。更详细地了解这一过程的分子基础将使我们能够在未来对其进行修饰，以将非天然氨基酸引入蛋白质中，这在蛋白质工程和合成生物学中具有非常有趣的应用。

The incorporation of the 21st amino acid selenocysteine (Sec) into proteins in mammalian cells is guided by the selenosome. The function of this protein complex requires a multitude of protein-protein and protein-RNA interactions leading to the incorporation of selenocysteine at UGA codons, guided by stem-loop structures in the 3'-non translated regions of the selenoprotein-encoding genes. The long-term objective of our research program is to understand the mechanism of Sec incorporation into proteins. This process is of vital importance for mammalian cells and for the metabolism of many bacteria, e.g. for bacterial fermentations. In our preliminary work we have studied the interactions between all the known components of the selenosome using an in vivo assay that is based on bioluminescence resonance energy transfer (BRET). We have also used biochemical approaches to validate and to further characterize these interactions using biochemical and genetic approaches leading to the discovery of novel interactions among components of the Sec biosynthesis machinery. These results led to the guiding hypothesis of our work for the next funding period: A sequence of protein-protein interactions between SPS1, SPS2, SecS and SECp43 guides the incorporation of selenium into its specific tRNA. These reactions are followed by transfer of Sec to the elongation factor and incorporation into the growing polypeptide chain. We here propose to study these early steps of the Sec incorporation pathway at high resolution using cutting edge approaches of biochemistry, cell biology, molecular biology and structural biology. Specific aim 1. Characterizing the interaction between SPS1, SPS2, SecS and SECp43 using a combination of phage display and biochemical approaches. Specific aim 2. Atomic resolution-level analysis of the SPS1-SPS2-SecS-SECp43 interaction using X-ray crystallography and single-particle cryo-electron microscopy. Specific aim 3. In vivo analysis of the dynamic interactions between SPS1, SPS2, SecS, SECp43 and other selenosome components inside mammalian cells. Our long-term program will reveal the molecular basis for the precise incorporation of Sec into proteins and we will obtain atomic resolution structures of the selenosome. Also, we will visualize Sec incorporation in living cells using highest resolution imaging techniques. More detailed understanding of the molecular basis of this process will enable us to modify it in future to incorporate unnatural amino acids intro proteins, which has very interesting applications in protein engineering and synthetic biology.