可追蹤的光調控蛋白酶體抑制劑用以實現對蛋白酶體活性的時空調控

Abstract

蛋白酶體是真核生物中主要負責降解蛋白質的蛋白質複合體，細胞中不需要或錯誤折疊的異常蛋白質會經由酵素標記上多個泛素分子形成多泛素鏈，接著被蛋白酶體辨識並降解成短胜肽鏈。過去研究顯示細胞週期、基因表現及細胞凋亡等重要細胞過程都與蛋白酶體的降解途徑息息相關，因此若蛋白酶體功能出現失常會對細胞及生物健康產生多種影響，例如促進癌症和神經退化性疾病的發展。

為了在時空間上更精確地控制蛋白酶體的活性，我們以鄰硝基苯基光保護基修飾蛋白酶體抑制劑MG132的活性位點，並延伸出螢光基團以追蹤抑制劑的細胞中定位。由於鄰硝基苯基光保護基可同時扮演螢光淬滅劑的角色，在照光前我們設計的蛋白酶體抑制劑不會具有抑制活性和螢光；受光照激發後，光保護基會離去並開啟蛋白酶體抑制劑的活性及螢光。經過試管和細胞實驗測試，我們證實合成出來的蛋白酶體抑制劑確實擁有上述的特質，並期待這種分子設計能應用於蛋白酶體的相關研究。

Proteasome is a crucial protein complex in ubiquitin‐dependent pathway of protein degradation in eukaryotes. It recognizes proteins with poly-ubiquitin chains and degrades them into short peptides, which will be further degraded into amino acids for new protein synthesis. Several cellular processes, including cell cycle regulation, gene expression, ER stress, and apoptosis are related to this protein degradation system. Therefore, the dysfunction of proteasomes contributes to the progression of multiple disorders, such as neurodegenerative diseases and cancers.

In this research, we coupled fluorophores and o-nitrobenzyl-based photocages to a well-known proteasome inhibitor, MG132. The photocages protected the active aldehyde group in MG132 and quenched the fluorescence of the fluorophores. Upon irradiation, both the bioactivity and the fluorescence “turned-on” due to the leaving of the photocages. In vitro assays and cellular experiments were performed to support our expectations. This design allowed precise treatment and visualization of proteasome inhibitors with high spatiotemporal resolution. We anticipate that it can be a valuable tool for further research into proteasome and proteasome-related applications.