GYF domain mediated protein: protein interactions

含有GYF结构域的蛋白质在细胞质（Smy2, PERQ2）或细胞核剪接体组装（CD2BP2）的mRNA监视背景下起作用。因此GYF结构域与富含脯氨酸序列（PRS）结合，这种相互作用已经在Smy2和cd2bp2型亚家族的原子水平上被定义。与其他PRS识别结构域相比，结构知识可以作为进一步将肽模拟和非蛋白质原性氨基酸类似物纳入特异性处理GYF的肽支架的基础。此外，我们希望确定能够捕获介导与snRNP蛋白U5-15K相互作用的CD2BP2-GYF结构域的第二个结合位点的抑制剂。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法和细胞培养（SILAC）中的稳定同位素标记来实现。此外，我们将通过选择候选mrna的RT-PCR和深度测序来表征共沉淀RNA。功能分析包括报告mRNA的剪接和mRNA稳定性的测量及其3 ' -非翻译区域的修饰。最后，我们将使用CD2BP2敲除小鼠来直接比较化学和遗传干扰对该范例蛋白功能的影响。

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.