利用擴充基因密碼方法合成泛素-SUMO2異元二聚體與其功能分析之研究

Abstract

泛素化和類泛素化為細胞內重要之蛋白質轉譯後修飾作用，廣泛參與並影響生物體作用。USP7近期發現亦為泛素-類泛素降解酶，能將鍵結在類泛素2上的泛素移除，暗示泛素-類泛素的動態鍵結之間有著調控受質蛋白功能的生物機制。然而，調控類泛素-泛素異元聚合物轉譯後修飾的其餘調控因子，尚未進一步發現及探討。本研究利用化學生物學方法及非典型胺基酸體內嵌入的技術，合成特定鍵結的泛素-類泛素異元二聚體，做為深入探討相關蛋白間作用的基體。本研究中，類泛素2中的八個賴胺酸基因位點，分別以TAG基因密碼突變並以生物正交配對PylRS•tRNA_CUA^Pyl體內嵌入非典型胺基酸SeCbzK；類泛素2進一步經過硒亞碸脫去反應轉換成類泛素2-Dha，進而衍生化的泛素-ethylthio進行硫醇麥可加成反應，得到泛素-類泛素2異元二聚體產物。 此論文研究亦針對以雜環類非典型胺基酸中對環境敏感而有螢光強弱變化的特性，發展辨認特定鍵結泛素二聚體的分子探針。利用演化後的PylRS•tRNA_CUA^Pyl正交配對，在超折疊綠螢光蛋白的發色團中酪胺酸66及苯丙胺酸27分別取代為7種不同雜環胺基酸。在螢光光譜的測定實驗中，顯示放射光譜和控制組實驗相比，有著顯著發射光譜位移、斯托克斯位移、分子內多重FRET光學現象、增加螢光發光團，相關結果可做為非典型胺基酸分子探針分辨特定泛素二聚體的工具。

Protein Ubiquitination and SUMOylation are unique protein post-translational modifications involved in a myriad biological processes. The discovery of USP7 as exclusive SUMO deubiquitinase has manifested the cross-talk between Ubiquitin (Ub) and SUMO. However, the corresponding effectors and biological language of such hetero-linkage are still indecipherable. Here, we report an unbiased novel chemical method in synthesizing Ub-SUMO2 dimers. Se-alkylselenocysteine was incorporated into SUMO2 protein at eight different lysine positions individually and further converted to SUMO2-dehydroalanine (Dha) through selenoxide-elimination chemistry. Subsequently, SUMO2-Dha was covalently tethered with Ub-ethylthio by thiol-Michael addition yielding Ub-SUMO2 heterodimer. This study also centers on the development of sensitive heterocyclic fluorescent probe which can sense Di-Ub or Ub-SUMO linkage in response to environmental changes. With the evolved pyrrolysyl-tRNA synthetase • pyrrolysyl-tRNA (PylRS•tRNA_CUA^Pyl) pairs, the tyrosine 66 and phenylalanine 27 positions of superfolder green fluorescent protein were efficiently substituted with seven heterocyclic noncanonical amino acids. The photophysical characterization of these proteins have unraveled the significant differences at the emission wavelengths, stoke shift, intramolecular FRET, and additional chromophore. These findings have demonstrated that ncAA-based tool was employed to establish molecular probe which can specifically recognize Ubiquitin dimers.