建構新穎胜肽辨識分子以應用於直線型泛素鏈修飾蛋白質分析

Abstract

在不同的細胞訊號傳遞路徑中，藉由泛素結合域(UBDs)與泛素鏈間特異性親和作用可觸發許多細胞內生理機制之調控，包含蛋白質降解、蛋白質運輸、轉錄、DNA修復、細胞生長與凋亡…等等。因此我們預期應可利用對泛素鏈具特異性結合之泛素結合域，建構新穎的胜肽辨識分子以應用於解析泛素化修飾的蛋白質。為了驗證這個概念，我們選擇對直線形泛素鏈具特異性辨識的UBD  NF-kappaB essential modulator (NEMO)中的coiled-coil 2 and leucine zipper (CoZi)區域來做為起始胜肽進行序列串接與優化。同時應用石英晶體微天平(Quartz crystal microbalance, QCM)與HaloTag-based assay來確認所建構之新型辨識分子與特定泛素鍵結之專一性。此一可針對直線型泛素鏈進行偵測之新型”類抗體”胜肽辨識分子”A Novel Linear Ubiquitin peptide Binder Opted by tailored Tandem Iteration (ANLUBOTI)”，相較於一般傳統抗體，可用來辨識高度泛素化具高分子量之蛋白質。此新型胜肽辨識分子的建構對於研究泛素化修飾蛋白質體學提供了一嶄新的工具，並能應用於發現與解析未知的直線型泛素鏈修飾蛋白質。

In cell signaling pathway, ubiquitin binding domains (UBDs) of various proteins specifically bind with different Ub linkages and trigger a vast array of cellular events including protein degradation, protein trafficking, transcription, DNA repair, cell-cycle progression and apoptosis…etc. We thus proposed that by creating Ub-linkage specific UBDs, these molecules may serve as novel recognition molecules for ubiquitination profiling tools and applications. A proof-of-principle study was performed, in which the coiled-coil 2 and leucine zipper (CoZi) region of NF  kappaB essential modulator (NEMO) has been employed as a specific linkage binder for linear polyubiquitin chains. Both quartz crystal microbalance (QCM) and the HaloTag-based assays have been applied to validate the specificity between the constructed binders and the corresponding linkage. This antibody-like molecular tool “A Novel Linear Ubiquitin peptide Binder Opted by tailored Tandem Iteration” (ANLUBOTI) enabled the recognition of highly ubiquitinated large molecules. The construction of these novel peptide binders shall facilitate the ubiquitinated proteome discovery, providing a tool readily for specific ubiquitin linkages profiling.