TOX和Eomes異源蛋白表達及分子特徵研究

Abstract

TOX (thymocyte selection-associated HMG-box protein) 和 Eomes (eomesodermin) 是 參與 T 細胞發育及耗竭重要轉錄因子，亦對免疫反應調節非常重要。透過對 TOX 和 Eomes 蛋白質一級結構的分析，發現分別有 79% 和 55% 區域被預測為本質無序區域 (intrinsically disordered regions，IDRs)。IDRs 特性是缺乏穩定三級結構，因􏰀具有高度 機動性，可促進蛋白質之間交互作用，已知在細胞功能中具有重要作用。然而，TOX 和 Eomes 中 IDRs 具體功能迄今尚未被充分了解。因􏰀，本研究利用大腸桿菌和人類胚 胎腎臟細胞進行重組蛋白 TOX 和 Eomes 的表現。將帶有 His 標記的重組蛋白 His-TOX 和 His-Eomes 表現於大腸桿菌中，藉由西方墨點法觀察到 His-TOX 出現於 55 kDa 和 72 kDa 的兩個色帶，His-Eomes 則在 72 kDa 和 100 kDa 間出現一個色帶。接著，以 HisTrap 管柱和肝素管柱對 His-TOX 進行純化。由於 TOX 的 IDRs 特性，導致純化過程重組蛋 白 His-TOX 易於降解，單一親合管柱純化方法難以獲得高純度蛋白質，故另外採用煮 沸法或增加不同管柱以改善純化效果。􏰀外，質譜分析確認 His-TOX 的二條色帶 55 kDa 和 72 kDa 對人類 TOX 序列覆蓋率均為 37%。72 kDa 色帶亦含有大腸桿菌的伴侶 蛋白 (chaperon protein DnaK)，其序列覆蓋率為 57%。有趣的是，研究發現部分純化的 重組蛋白 His-TOX，若降低其緩衝液鹽濃度會呈現白色混濁，並且該混濁情況仍可恢 復成清澈狀態。差異干涉對比顯微鏡 (differential interference contrast microscope，DIC) 觀察結果顯示，大腸桿菌所表現之部分純化重組蛋白 His-TOX 可能發生液-液相分離 (liquid-liquid phase separation，LLPS)。􏰀外，使用帶有 eGFP (enhance green fluorescent protein) 標記的重組蛋白 eGFP-TOX 和 Eomes-eGFP 在人類胚胎腎臟細胞中的表達，發 現細胞內出現小點 (puncta)，進一􏰁支持其發生 LLPS 的可能性。本研究結果支持重組 蛋白 TOX 和 Eomes 於體外 (in vitro) 條件可能形成 LLPS，本文同時討論 LLPS 的生理 意義及其基於 TOX 和 Eomes LLPS 特性的潛在治療策略。

TOX (thymocyte selection-associated HMG-box protein) and Eomes (eomesodermin) are crucial transcription factors involved in T cell development and exhaustion as well as in the regulation of immune responses. Analysis of the primary structures of TOX and Eomes proteins suggests that 79% and 55% of their regions are predicted to be intrinsically disordered regions (IDRs), respectively. IDRs are characterized by a lack of stable tertiary structure, conferring high mobility that facilitates protein-protein interactions, and are known to play important roles in cellular function. However, the specific functions of the IDRs in TOX and Eomes have not been fully understood. This study utilizes heterologous expression systems, in Escherichia coli (E. coli) and human embryonic kidney cells, to give information about recombinant TOX and Eomes proteins. First, we expressed His-tagged recombinant proteins, His-TOX and His- Eomes, in E. coli. It was observed that recombinant His-TOX was identified in two protein bands corresponding to 55 kDa and 72 kDa by Western blot, while recombinant His-Eomes was identified in a single expression protein band between 72 kDa and 100 kDa. Next, we used HisTrap and heparin columns for purification of His-TOX. Due to the IDR characteristics of TOX, proteins tended to degrade during purification, making it challenging to obtain high- purity protein with a single affinity column. Additional methods such as boiling method or using multiple columns were therefore applied to improve the purification efficiency. We found that both 55 kDa and 72 kDa bands of His-TOX covered 37% of the human TOX sequence under mass spectropetry analysis. The 72 kDa band with a sequence coverage of 57% contained chaperone protein DnaK. It is interesting to note that the partially purified recombinant His- TOX exhibited reversible white particles upon decreasing buffer salt concentration. Differential interference contrast microscopy (DIC) indicated that partially purified His-TOX expressed in E. coli might undergo liquid-liquid phase separation (LLPS). Expressing eGFP- tagged recombinant proteins (eGFP-TOX and Eomes-eGFP) in human embryonic kidney cells also resulted in the formation of puncta which further support the potential occurrence of LLPS. Our results support the presence of the potential formation of LLPS from recombinant TOX and Eomes proteins in vitro. The physiological significance and the implications of novel therapeutic strategy based on the LLPS properties of TOX and Eomes are discussed.