GYF domain mediated protein: protein interactions

含有GYF结构域的蛋白质在细胞质中的mRNA监视（Smy 2，PERQ 2）或在细胞核中的剪接体组装（CD 2BP 2）中起作用。因此，GYF结构域赋予与富含脯氨酸的序列（PRS）的结合，并且这种相互作用已经在Smy 2和CD 2BP 2型亚家族的原子水平上被定义。结构知识作为进一步将肽模拟物和非蛋白质氨基酸类似物掺入肽支架的基础，与其他PRS识别结构域相比，所述肽支架特异性地针对GYF。此外，我们希望鉴定捕获CD 2BP 2-GYF结构域的第二个结合位点的抑制剂，该结构域介导与snRNP蛋白U 5 - 15 K的相互作用。固定化GYF结构域抑制剂的蛋白质组学分析将通过结合质谱和细胞培养中的稳定同位素标记（SILAC）来实现。此外，我们将通过所选候选mRNA的RT-PCR和深度测序来表征共沉淀的RNA。功能测定包括报告mRNA的剪接和mRNA稳定性的测量及其3 '-非翻译区的修饰。最后，我们将使用CD 2BP 2敲除小鼠直接比较化学和遗传干扰与这种范式蛋白的功能。

Proteins containing GYF domains act in the context of mRNA surveillance in the cytoplasm (Smy2, PERQ2) or in spliceosome assembly in the nucleus (CD2BP2). The GYF domain thereby confers binding to proline-rich sequence (PRS) and this interaction has been defined at the atomic level for Smy2 and CD2BP2-type subfamilies. Structural knowledge serves as a basis to further incorporate peptide-mimetic and non-proteinogenic amino acid analogs into peptide scaffolds that specifically address GYF in comparison to other PRS recognition domains. In addition, we want to identify inhibitors that capture the second binding site of the CD2BP2-GYF domain that mediates interactions with the snRNP protein U5-15K. Proteomic profiling of immobilized GYF domain inhibitors will be achieved by combining mass spectrometry and stable isotope labeling in cell culture (SILAC). Furthermore, we will characterize co-precipitated RNA by RT-PCR of selected candidate mRNAs and deep sequencing. Functional assays comprise splicing of reporter mRNAs and the measurement of mRNA stability and modification of their 3’-untranslated regions. Finally, we will use CD2BP2 knockout mice to directly compare chemical and genetic interference with the function of this paradigmatic protein.