生命科學院: 生化科學研究所指導教授: 張震東黃偉杰Huang, Wei-ChiehWei-ChiehHuang2017-03-022018-07-062017-03-022018-07-062016http://ntur.lib.ntu.edu.tw//handle/246246/272293標的鑑定為理解小分子化合物其生物活性作用之複雜機制所必須，然而現有技術受限於耗時以及特殊儀器已無法滿足與日俱增的需求。本研究發展一項融合疏水性交互作用層析法與親和性沖提之新技術：串聯式疏水性層析法之親和性沖提，提供鑑定小分子與蛋白質交互作用一系統性與效率性之策略。此技術之核心在於利用不同長度烷鏈構成疏水性層析管柱之基質，提供廣泛疏水性作用力與絕大多數蛋白質結合。樣本在進入串聯管柱之後，標的蛋白隨即選擇性地以一選定小分子沖提。作為首次方法學之驗證，我們針對小鼠腦部組織之三磷酸腺苷結合蛋白進行調查，並證實利用此技術能顯著提升三磷酸腺苷結合蛋白的鑑定率，其中約百分之十九為已知標的。本研究鑑定出RKIP為三磷酸腺苷結合蛋白，RKIP與三磷酸腺苷之結合導致減弱其與Raf-1激酶之親和力。更進一步在HEK293細胞中的研究發現，在短期能量耗竭的狀態下，RKIP與Raf-1的結合增強進而抑制下游ERK訊息傳遞路徑之活化。藉由此功能，RKIP對Raf-1激酶之抑制得以受到三磷酸腺苷濃度調節，這些資訊為細胞如何將能量狀態連結訊息傳遞提供新的線索。基於上述成果，我們展望串聯式疏水性層析法之親和性沖提於小分子標的鑑定之應用價值。Target identification is essential to understanding the complicated actions of bioactive small compounds. However, available techniques constrained by processing time and specific equipment do not meet the growing demands. Here, we have developed a novel method combining hydrophobic interaction chromatography and affinity elution, named affinity elution in tandem hydrophobic interaction chromatography (AETHIC) that enables systematic and efficient identification of small molecule-protein interactions. The essence of this strategy is using a series of HIC matrices encompassing aliphatic chains of different length which provide a wide range of hydrophobicity for interactions with most proteins. After samples are applied to the tandem columns, target proteins are selectively eluted with a specific ligand. As our first proof-of-principle of AETHIC, we demonstrated that AETHIC was able to enrich ATP-binding proteins from mouse brain extract as near nineteen percent of total protein identifications are previously assigned ATP binders. In addition, we have revealed that raf kinase inhibitory protein (RKIP) is an ATP binding protein. ATP attenuates the interaction between RKIP and Raf-1. As a result, short-term energy depletion augments interaction between RKIP and Raf-1 resulting in decreased activation of the downstream ERK signaling in cultured HEK293 cells. Therefore, the ATP-binding function renders RKIP’s inhibition on Raf-1 modulated by cellular ATP concentrations. These data shed light on how energy levels link to cellular signaling. In light of these results, we envisaged the potential of AETHIC to be applied in the target identification of bioactive compounds such as metabolites and therapeutic small-molecule drugs.4895392 bytesapplication/pdf論文公開時間: 2026/8/1論文使用權限: 同意有償授權(權利金給回饋本人)標的鑑定小分子化合物三磷酸腺?RKIPRaf-1磷脂質葡萄糖耗竭Target identificationsmall-molecule compoundsATPphospholipidsglucose depletionthesis10.6342/NTU201601648http://ntur.lib.ntu.edu.tw/bitstream/246246/272293/1/ntu-105-D98b46008-1.pdf